Starch in Food: Structure, Function and Applications

Starch in Food

Related Titles Starch, AP (ISBN 978-0-12-746275-2) Yeasts in Food, Woodhead (ISBN 978-1-85573-706-8)

Woodhead Publishing Series in Food Science, Technology and Nutrition

Starch in Food Structure, Function and Applications

Second Edition

Edited by Malin Sjoö¨ Lars Nilsson

Woodhead Publishing is an imprint of Elsevier The Officers’ Mess Business Centre, Royston Road, Duxford, CB22 4QH, United Kingdom 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, OX5 1GB, United Kingdom Copyright Ó 2018 Elsevier Ltd. 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: 978-0-08-100868-3 (print) ISBN: 978-0-08-100896-6 (online) For information on all Woodhead Publishing publications visit our website at https://www.elsevier.com/books-and-journals

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Contents List of Contributors

xxi

Part One Analysing and Modifying Starch 1.

Plant Starch Synthesis Jack Preiss 1. Localization of Plant Starch Synthesis in Plants 1.1 Leaf Starch 1.2 Starch in Storage Tissues 2. Starch Synthesis: Enzyme Reactions in Plants and Algae and Glycogen Synthesis in Cyanobacteria 2.1 Enzyme Reactions of Starch Synthesis 3. Properties of Plant a-1,4-Glucan-Synthesizing Enzymes 3.1 ADP-Glucose Pyrophosphorylase: Kinetic and Regulatory Properties of the Enzyme 3.2 ADP-Glucose Pyrophosphorylase: StructureeFunction Relationships: Quaternary Structure 3.3 Relationship Between the Small and Large Subunits 3.4 The Glucose-1-P-Binding Site in Plant ADP-Glucose Pyrophosphorylase 3.5 Phylogenetic Analysis of the Large and Small Subunits 3.6 Crystal Structure of Potato Tuber ADP-Glc PPase 3.7 The Homotetramer Catalytic Subunit Structure of the Potato Tuber ADP-Glucose Pyrophosphorylase 3.8 ATP Binding 3.9 ADP-Glucose Binding 3.10 Implication for Catalysis 3.11 Allosteric Regulation 3.12 Supporting Data for the Physiological Importance of Regulation of ADP-Glucose Pyrophosphorylase 3.13 Mechanism of Activation of Plant ADP-Glc PPases by Thioredoxin

3 3 4 6 6 7 7 9 11 13 16 17 19 22 22 24 26 27 28

v

vi Contents 4. Characterization of ADP-Glc PPases From Different Plant Sources 4.1 Chlamydomonas reinhardtii 4.2 Barley 4.3 Transport of Cytosolic ADP-Glucose into Endosperm in Cereal Grains 4.4 Pea Embryos 4.5 Maize Endosperm 4.6 Tomato Fruit 4.7 Wheat 4.8 Rice Endosperm and Leaf 4.9 Arabidopsis thaliana 5. Differences in Interaction Between 3PGA and Pi in Different ADP-Glc PPases 6. Identification of Important Amino Acid Residues Within the ADP-Glc PPases 7. Characterization of the Regulatory Domain 8. Starch Synthases 8.1 Characterization of the Starch Synthases 8.2 Soluble Starch Synthase I 8.3 Starch Synthase II 8.4 Starch Synthase III 8.5 Starch Synthase IV 8.6 Double Mutants of the Soluble Starch Synthases 8.7 Starch Synthases Bound to the Starch Granule 8.8 Isolation of the Waxy Protein Structural Gene 8.9 Further Studies of GBSS and Isoforms; Their Involvement in Both Amylopectin and Amylose Synthesis 8.10 Branching Enzyme References Further Reading

2.

30 30 30 36 37 37 38 39 40 41 42 44 47 47 48 48 51 54 55 57 58 60 61 64 77 95

Analyzing Starch Molecular Structure Eric Bertoft 1. Introduction: Characterizing Structures of Starch Components 2. Fractionation of Starch 3. Analysis of Amylose 3.1 Amylose Content of Starch 3.2 Structural Analysis of Amylose 4. Analysis of Amylopectin Structure 4.1 Unit Chain Length and Distribution 4.2 External Chain Length and Internal Chain Distribution 4.3 Units of Clusters 4.4 Units of Building Blocks 4.5 Starch Phosphate Esters

97 100 102 102 104 107 109 113 116 121 123

Contents vii

5. Analysis of Intermediate Materials 6. Future Trends References

3.

126 128 130

Understanding Starch Structure and Functionality Yongfeng Ai, Jay-lin Jane 1. Introduction 2. Components of Starch Granules 2.1 Structures and Properties of Amylose 2.2 Structures and Properties of Amylopectin 2.3 Structures and Properties of Intermediate Components 2.4 Structures and Properties of Phytoglycogen 2.5 Lipids and Phospholipids 2.6 Phosphate Monoesters 2.7 Proteins 3. Structures of Starch Granules 3.1 Morphology of Starch Granules 3.2 Internal Structures of Starch Granules 4. Effects of Structures on Starch Properties 4.1 Starch Gelatinization 4.2 Starch Pasting 4.3 Starch Gelling 4.4 Starch Retrogradation and Syneresis 5. Effects of Crop Maturation 6. Conclusions and Future Trends References

4.

151 152 152 155 159 159 160 160 160 161 161 162 166 166 167 169 169 170 171 172

Starch Bioengineering Andreas Blennow 1. Introduction: Improving Starch Functionality Directly in the Crop 1.1 Starch: Importance, Demands and Opportunities 1.2 The Starch Granule 2. Technologies for Starch Bioengineering 2.1 Gene Technology for Starch Bioengineering 2.2 Analytical Technologies for Analyzing Starch Molecular Structure 3. The Metabolic Reactions Influencing Starch Yield and Structure 3.1 Engineering for Increased Starch Yield 3.2 Engineering Amylopectin 3.3 Engineering for High Amylose 3.4 Engineering for Low Amylose 3.5 Engineering Starch Phosphorylation 3.6 Engineering Starch Molecular Mass and Granule Size

179 179 180 182 182 186 187 190 193 195 196 197 198

viii Contents 4. Physical and Chemical Properties of Bioengineered Starches 5. Functionality and Uses in Food Processing of Bioengineered Starches 6. Toward Predictable Biotechnological Modification of Starch 7. Future Prospects References

5.

199 201 204 206 207

Physical Modification of Starch James N. BeMiller 1. Introduction 2. Thermal Treatments 2.1 Pregelatinized Starch 2.2 Granular Cold-Water-Swelling Starch 2.3 Heat-Moisture Treatment 2.4 Annealing 2.5 Heating Dry Starch 2.6 “Osmotic Pressure Treatment” 3. “Nonthermal” Treatments 3.1 Ultrasonic Treatment 3.2 Milling 3.3 High-Pressure Treatment 3.4 Pulsed Electric Field 3.5 Freezing and Thawing and Freeze-Drying 4. Physical Treatments That Produce Chemical Changes References

6.

223 224 224 226 228 233 236 237 237 237 239 240 243 244 244 244

Measuring Starch in Food Miguel Peris-Tortajada 1. Introduction 1.1 Why Analyze for Starch Content? 1.2 Regulations 2. Sample Preparation 3. Methods of Analysis 3.1 Classical Methods 3.2 Modern Methods 4. Recent Developments: Automation and Future Trends 5. Sources of Further Information and Advice References

255 255 255 256 258 259 261 273 276 278

Contents

7.

ix

Chemical Modification of Starch Yu-Fang Chen, Lovedeep Kaur, Jaspreet Singh 1. Introduction 2. Starch Source 3. Common Types of Chemical Modifications 3.1 Acid Hydrolysis 3.2 Bleaching and Oxidation 3.3 Substitution (Stabilization) 3.4 Cross-linking 3.5 Dual Modification 4. Physicochemical Properties and Functionality of Modified Starches 4.1 Physicochemical Properties 4.2 Morphological Properties 4.3 Thermal Characteristics 4.4 Pasting Properties 4.5 Refrigerated Storage and FreezeeThaw Stability 5. Applications in Foods 5.1 Nutritional Value and Health Aspects 5.2 Digestibility In Vitro and In Vivo 5.3 Recent Industry Trends Acknowledgments References

283 285 287 291 293 293 295 295 296 296 299 301 305 307 308 308 308 316 316 316

Part Two Sources of Starch 8.

The Functionality of Wheat Starch Justyna Rosicka-Kaczmarek, Izabella Kwasniewska-Karolak, Ewa Nebesny, Aleksandra Komisarczyk 1. 2. 3. 4. 5. 6.

Introduction Wheat Starch Production and Use for the Food Industry Granular and Molecular Structure of Wheat Starch Interaction of Starch With Minor Components Hydrolysis of Wheat Starch Improving the Functionality of Wheat Starch for Use in the Food Industry 7. Conclusions and Future Trend References

9.

325 326 330 335 341 344 346 347

Potato Starch Cindy Semeijn, Pieter L. Buwalda 1. Introduction 1.1 General Introduction 1.2 Potato Starch Market and Production

353 353 355

x Contents 2. Granular and Molecular Structure of Potato Starches 2.1 Granules 2.2 Amylose 2.3 Amylopectin 2.4 Minor Starch Components 3. Amylopectin Potato Starches 4. Functionality of Potato Starch and Derivatives for the Food Industry 4.1 Physical Processing 4.2 Degraded Starches 4.3 Cross-linked Starches 4.4 Acetylated Starches 4.5 Hydroxypropylated Starch 4.6 Combined Treatments 4.7 Starch Octenyl Succinates 5. Recent Developments 5.1 Amylopectin Potato Starches 5.2 Inhibited Starches 5.3 Small Granular Potato Starch 5.4 Amylomaltase-Treated Starches 6. Outlook References

10.

356 356 359 359 361 361 363 363 364 364 365 365 365 366 366 366 367 367 368 368 369

Rice Flour and Starch Functionality Jinsong Bao, Christine J. Bergman 1. Introduction 2. Rice Flour and Starch as a Food Ingredient 3. Constitutes of Rice Starch 3.1 Minor Constituents 4. Structure and Functionality of Rice Starch 4.1 Granule Shape and Size 4.2 Starch Crystallinity 4.3 Amylose 4.4 Amylopectin 4.5 Swelling Power and Solubility 5. Gelatinization and the Structure of Rice Starch 5.1 Gelatinization: Pasting Properties 5.2 Gelatinization: Rheological Properties 6. Retrogradation and Other Properties of Rice Starch 6.1 Clarity 6.2 FreezeeThaw Stability 6.3 Digestibility 7. Improving Rice Starch Functionality for Food Processing Applications 7.1 Chemical Modification of Rice Starch 7.2 Physical Modification of Rice Starch 7.3 Genetic Modification of Rice Starch

373 375 377 378 379 379 379 381 382 387 389 392 396 397 400 400 401 402 402 405 407

Contents

8. Future Trends 9. Sources of Further Information and Advise 9.1 Organizations 9.2 Industries 9.3 Literature References Further Reading

11.

xi 407 408 408 409 409 409 419

Functionality of Tuber Starches Subramoney N. Moorthy, Moothandassery S. Sajeev, Rajamohanan J. Anish 1. Introduction 1.1 Tropical Root Crops 2. Tuber Crop Starch Production 2.1 Cassava 2.2 Sweet Potato 2.3 Other Tuber Crops 3. Minor Constituents 3.1 Cassava 3.2 Sweet Potato 3.3 Aroids 3.4 Yams 3.5 Other Starches 4. Structure and Functionality of Starches 4.1 Granule Shape and Size 4.2 XRD and Starch Crystallinity 4.3 Content and Structure of Amylose and Amylopectin 4.4 Swelling Pattern and Solubility 4.5 Thermal Properties 4.6 Viscosity and Pasting 4.7 Retrogradation and FreezeeThaw Stability 4.8 Rheological Properties 4.9 Digestibility 5. Food Applications of Tuber Starches 6. Improving Tuber Starch Functionality for Food Applications: Modifying “Tropical” Starches for Use in the Food Industry 6.1 Physical Modifications 6.2 Chemical Modifications 6.3 Sweet Potato 6.4 Modification of Other Starches 6.5 Biotechnological Methods 7. Future Trends References Further Reading

421 421 424 424 428 428 428 429 429 429 430 430 435 435 445 448 453 460 470 480 481 483 484 485 486 488 490 492 493 494 495 508

xii Contents

12.

The Functionality of Pseudocereal Starches Daysi Perez-Rea, Raquel Antezana-Gomez 1. Introduction 2. Quinoa Starch 2.1 Introduction 2.2 Quinoa Starch Production 2.3 Minor Constituents of Quinoa Starch 2.4 Structure and Functionality of Quinoa Starch 2.5 Improving Quinoa Starch Functionality for Food Processing Applications 2.6 Quinoa Starch and Flour in Food Applications 3. Canihua Starch 3.1 Introduction 3.2 Canihua Starch Production 3.3 Minor Constituents of Canihua Starch 3.4 Structure and Functionality of Canihua Starch 4. Amaranth Starch 4.1 Introduction 4.2 Amaranth Starch Production 4.3 Minor Constituents of Amaranth Starch 4.4 Structure and Functionality of Amaranth Starch 4.5 Improving Amaranth Starch Functionality for Food Processing Applications 4.6 Amaranth Flour in Food Applications 5. Buckwheat Starch 5.1 Introduction 5.2 Buckwheat Starch Production 5.3 Minor Constituents of Buckwheat Starch 5.4 Structure and Functionality of Buckwheat Starch 5.5 Improving Buckwheat Starch Functionality for Food Processing Applications 5.6 Buckwheat Starch and Flour in Food Applications 6. Future Trend References

509 509 509 510 511 511 520 520 521 521 521 522 522 524 524 525 525 525 530 530 531 531 531 531 532 534 535 536 536

Part Three Applications 13.

Utilizing Starches in Product Development Thomas Luallen 1. Introduction 1.1 Starch Source, Structure, Characteristics, and Properties 2. Components of Starch 2.1 Amylose 2.2 Amylopectin

545 546 546 547 547

Contents

2.3 Minor Constituents 3. Food Applications for Natural and Modified Starches 3.1 Native or Common Starches (Natural) 3.2 Modified Starches 4. Methods of Starch Selection 4.1 What Is the Desired Function of the Starch You Are Adding? 4.2 What Is the Method of Processing You Anticipate Using? 4.3 What Is the Food System pH? 4.4 Does the Process Contribute High Shear? 4.5 What Percent of Water-Soluble Materials Will Be Present? 4.6 Is One or More of the Following Used: Fat(s), Salt(s), and Gums? 4.7 Is the Finished Product Subjected to Postprocessing? 4.8 How Will the Product Be Stored? 5. Factors Affecting Starch in Food Products 5.1 Temperature 5.2 Shear 5.3 Packaging 5.4 Storage 5.5 Water 5.6 Sweeteners 5.7 Salts 5.8 Other Food Ingredients (Spices, Fruits, Flavors, etc.) 5.9 Proteins and Other Starches 6. Using the Functional Properties of Starch to Enhance Food Products 6.1 Thermal Processing (Canned-Jarred, Retorted-SterilizedHot Filled) 6.2 Frozen 6.3 Instant Products (Soups, Sauces, and Gravies) 6.4 Functional Differences Based on Process of Production 6.5 Snack Foods 6.6 Dressings, Sauces, Gravies, and Other Condiments 6.7 Bakery Products 6.8 Pet Products 6.9 Meat Products 6.10 Cereals, Pasta, Bars, and Related Products 6.11 Confections (Candy) 6.12 Dairy and Related Products 6.13 Fat Replacement, Substitution, or Mimetics 6.14 Emulsification and EncapsulationdBeverages References

xiii 547 549 549 551 552 552 552 553 553 553 554 554 554 558 558 558 559 559 559 560 561 561 562 563 563 564 565 566 566 567 568 570 570 571 572 573 574 575 578

xiv Contents

14.

Modified Starches and the Stability of Frozen Foods Pei Wang, Xueming Xu 1. Introduction 2. Deterioration Mechanism of Frozen Foods 2.1 Main Characters in the Quality Loss of Frozen Food 2.2 Water Crystallization 2.3 Deterioration of Food Components in Frozen Food 3. Improving Frozen Food Quality Through Modified Starches 3.1 Genetical Modification 3.2 Physical Modification 3.3 Chemical Modification 4. Conclusion and Future Perspective References

15.

581 581 581 582 583 586 586 587 588 590 590

Starch in Baked Products Patricia Le-Bail, Nesrin Hesso, Alain Le-Bail Part I: Starch and Its Interaction With Ingredients in Baked Products 1. Composition and Primary Structure of Starch 1.1 Glucidic Fraction 1.2 Amylopectin 1.3 Amylose 1.4 Nonglucidic Fraction 1.5 Lipidic Fraction 1.6 Nitrogen Fraction 1.7 Mineral Fraction 2. Morphology and Ultrastructure of Starch Grain 2.1 Morphology of Starch Grain 2.2 Ultrastructure of Starch Grain 2.3 Crystalline and Helical Structures 2.4 Crystallographic Approach and Molecular Models 2.5 Crystalline Lattice 2.6 Effect of Hydration 2.7 Crystallinity of Native Starches A and B 3. Hydrothermic Transitions 3.1 Glass Transition of Starches 3.2 Gelatinization 3.3 Retrogradation and Gelation 3.4 Amylose Gelation 3.5 Amylopectin Gelation 4. V Amylose and Inclusion Complexes 4.1 Specific Complexes 4.2 Aspecific V6 Complexes

595 595 595 595 596 597 597 597 597 598 598 598 599 599 600 601 602 603 603 603 604 605 605 605 605 607

Contents xv

Part 5. 6. 7. 8. 9. 10.

2: Cases of Study Introduction: Starch in Baked Products Starch in the Case of Pan Bread Starch in the Case of Cakes Starch in the Case of Cookies Starch in the Case of Biscuits Starch at Surface of Bakery Products and Impact on Crust Properties 11. Conclusion References

16.

609 609 612 614 618 619 621 623 624

Starch in Brewing Applications Glen Fox 1. Introduction 2. History of Brewing Beer 3. Starch-Degrading Enzymes 3.1 Limit Dextrinase 3.2 Beta-Amylase 3.3 Alpha-Amylase 3.4 Alpha-Glucosidase 3.5 Types of Beers 3.6 Lagers 3.7 Ales 3.8 Dry Beers 3.9 Other Nonbarley Beer Types 4. Important Factors for Mashing 5. Better Understanding of the SubstrateeEnzyme System 6. Other Sources for Fermentable Sugars 7. Fermentation 8. Wild Yeasts and Specialty Beers 9. Fermentable Sugars 10. Starch Impact on Whiskey and Other Distilled Products 10.1 Other Types of Whisky 11. Starch Gelatinization 12. The Barley Grain and Starch Structure 13. Variation in Starch Structure in Other Brewing Cereals 14. Conclusion References

17.

633 634 635 635 636 636 637 637 637 637 637 638 640 642 642 643 645 646 647 648 648 649 650 651 652

Starch-Based Microencapsulation Yangyang Jin, Jason Z. Li, Amir Malaki Nik 1. Introduction 2. The Nature of Starch 3. Starch Modifications

661 662 665

xvi Contents 3.1 Starch Hydrolysis 3.2 Chemical Modifications 3.3 Physical Modifications 4. Modified Starches for Microencapsulation 4.1 Modified Starches in Spray-Drying Process 4.2 Case Studies 5. Conclusion References Further Reading

18.

665 666 669 670 670 677 686 687 690

Starch Nanoparticles Qingjie Sun 1. Introduction 2. Preparation of Starch Nanoparticles 2.1 Acid Hydrolysis 2.2 Physical Treatments 2.3 Nanoprecipitation 2.4 Enzymatic Debranching and Recrystallization 2.5 Emulsion 2.6 Polyelectrolyte Complex Formation 2.7 Electrospinning 2.8 Electrospray 2.9 Self-Assembly 3. Characterization of Starch Nanoparticles 3.1 Morphology and Size 3.2 Molecular Composition 3.3 Crystallinity 3.4 Fourier Transform Infrared Spectroscopy 3.5 Thermal Properties 3.6 Rheological Properties 3.7 Digestibility Properties 3.8 Swelling Properties 3.9 Toxicological Experiments 4. Surface Functionalization of Starch Nanoparticles 4.1 Chemical Modification 4.2 Physical Modification 5. Interaction of Starch Nanoparticles With Protein 6. Application of Starch Nanoparticles 6.1 Nanocomposites 6.2 Pickering Emulsion 6.3 Food Bioactive Ingredients and Drug Delivery Carriers 6.4 Fluorescent Agent 6.5 Adsorbent Agents 6.6 Purification of Targeted Enzyme 7. Conclusion and Prospectives References

691 692 693 693 699 701 705 705 706 706 707 707 707 713 713 716 717 719 721 721 723 725 725 726 726 727 727 728 731 733 734 735 735 736

Contents xvii

19.

Starch-Based Films Kristine Koch 1. 2. 3. 4. 5. 6. 7. 8.

Introduction Definitions Global Production Regulations Governing Food Contact Materials Conversion Techniques Impact of Coating Technique Challenges in Full-Scale Processes Material Properties 8.1 General 8.2 Tensile Properties 8.3 Barrier Properties 9. Future Materials and Concluding Remarks References

20.

747 748 750 750 751 752 752 753 753 755 757 760 761

Starch Interactions With Native and Added Food Components Carmen C. Quiroga Ledezma 1. Introduction 2. Starch Composition and Structure 2.1 Primary Components 2.2 Secondary Components 2.3 Added Components 3. Starch Phase Transitions 3.1 Gelatinization 3.2 Annealing 3.3 Retrogradation 4. Starch Interactions With Other Food Constituents 4.1 Interactions With Lipids 4.2 Interactions With Proteins 4.3 Interactions With Carbohydrates 4.4 Interactions With Salts 4.5 Interactions With Surfactants 4.6 Interactions With Polyols, Polyphenols, and Carboxylic Acids 4.7 Interactions With Aroma and Flavor Compounds 5. Future Trends 6. Conclusions References

769 770 770 771 772 772 772 773 774 775 775 778 782 786 788 789 790 791 791 792

xviii Contents

Part Four Starch and Health 21.

Starch Digestion and Applications of Slowly Available Starch Marion G. Priebe, Coby Eelderink, Renate E. Wachters-Hagedoorn, Roel J. Vonk 1. Introduction 2. Prevention of Postprandial Hyperglycemia: the Role of Starch 2.1 Regulation of Postprandial Plasma Glucose 2.2 Hyperglycemia and Persons at Risk 3. Targets to Influence Postprandial Glycemia 3.1 Intestinal Starch Digestion 3.2 Absorption in the Small Intestine 3.3 Transit Time 3.4 Gastrointestinal Hormones 3.5 Food Characteristics Influencing Postprandial Glycemia 4. Techniques for Monitoring Starch Digestion 4.1 In Vitro Approaches 4.2 In Vivo Approaches 5. Current (Nutritional) Strategies to Prevent Postprandial Hyperglycemia 5.1 Administration of a-Glucosidase Inhibitors 5.2 Use of the Glycemic Index 5.3 The Glycemic Index and Slowly Available Starch 5.4 Addition of Dietary Fiber 6. Future Trends 6.1 Extending the Choice of Foods With Slowly Available Starch 6.2 Characterization of Slowly Available Starch 6.3 Stimulating Secretion of Gastrointestinal Hormones References

22.

805 806 806 806 808 808 809 809 810 811 813 813 813 816 816 817 818 819 820 820 821 821 821

Development of Foods High in Slowly Digestible and Resistant Starch Luis A. Bello-Perez, Javier D. Hoyos-Leyva 1. Introduction 2. Molecular and Physicochemical Characteristics of Functional Starches 3. Slowly Digestible and Resistant Starch Production as Ingredient for Food Industry 3.1 Starch Modifications With Nutritional Purpose 3.2 Commercial Functional Starches 3.3 Functional Properties of RS-Rich Powders

827 828 829 830 837 839

Contents

4. Development of Foods With Functional Starch Ingredients 4.1 Chemical and Physical Changes That Induce SDS and RS Formation in Different Food Matrixes 4.2 Raw or Cooked Starchy Foods Preparation and Changes in Starch Digestion 4.3 SDS and RS in Gluten-Free Products 5. Future Trends 6. Conclusions References

23.

xix 840 841 845 847 847 848 848

Starch: Physical and Mental Performance, and Potential Health Problems Suzanne Hendrich 1. Introduction 1.1 Types of Starch, Including Digestion-Resistant Starches 1.2 Starch Digestion and Glucose Bioavailability, Starch Residue Fermentation and Products 1.3 Major Theories or Hypotheses Regarding Effects of Starches on Physical Performance 1.4 Major Theories or Hypotheses Regarding Effects of Starches on Mental Performance 1.5 Major Theories or Hypotheses Regarding High Starch DietsdAcute and Long-Term Adverse Effects 2. Dietary Starches and Physical Performance 2.1 Effects on Physical PerformancedAnimal Studies 2.2 Effects on Physical PerformancedHuman Studies 2.3 Summary 3. Dietary Starches and Mental Performance 3.1 Effects of Starches on Mental PerformancedAnimal Studies 3.2 Effects of Starches on Mental PerformancedHuman Studies 3.3 Summary 4. Dietary StarchesdAdverse Effects 4.1 Animal Models 4.2 Human Studies 4.3 Summary 5. Conclusions References

Index

855 855 855 856 857 857 857 857 858 860 861 861 861 864 864 864 865 867 867 868 873

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List of Contributors Yongfeng Ai, University of Saskatchewan, Saskatoon, SK, Canada Rajamohanan J. Anish, Kerala University, Trivandrum, India Raquel Antezana-Gomez, San Simon University, Cochabamba, Bolivia Jinsong Bao, Zhejiang University, Hangzhou, China Luis A. Bello-Perez, Instituto Politećnico Nacional, Yautepec, Mexico James N. BeMiller, Purdue University, West Lafayette, IN, United States Christine J. Bergman, University of Nevada Las Vegas, Las Vegas, NV, United States Eric Bertoft, Bertoft Solutions, Turku, Finland Andreas Blennow, University of Copenhagen, Frederiksberg, Denmark Pieter L. Buwalda, Wageningen University and Research, Wageningen, The Netherlands; Coo¨peratie AVEBE U.A., Veendam, The Netherlands Yu-Fang Chen, Massey University, Palmerston North, New Zealand Coby Eelderink, University of Groningen, Groningen, The Netherlands Glen Fox, The University of Queensland, St Lucia, QLD, Australia; Stellenbosch University, Stellenbosch, South Africa Suzanne Hendrich, Iowa State University, Ames, IA, United States Nesrin Hesso, ONIRIS, UMR GEPEA CNRS 6144, Nantes, France Javier D. Hoyos-Leyva, Instituto Politećnico Nacional, Yautepec, Mexico Jay-lin Jane, Iowa State University, Ames, IA, United States Yangyang Jin, Ingredion Incorporated, Bridgewater, NJ, United States Lovedeep Kaur, Massey University, Palmerston North, New Zealand Kristine Koch, Swedish University of Agricultural Sciences, Uppsala, Sweden Aleksandra Komisarczyk, Lodz University of Technology, Lodz, Poland Izabella Kwasniewska-Karolak, Lodz University of Technology, Lodz, Poland Patricia Le-Bail, INRA, UMR BIA, Nantes, France Alain Le-Bail, ONIRIS, UMR GEPEA CNRS 6144, Nantes, France Jason Z. Li, Ingredion Incorporated, Bridgewater, NJ, United States Thomas Luallen, Starquest F.O.O.D. Consulting, Clinton, IL, United States

xxi

xxii List of Contributors Amir Malaki Nik, International Flavors & Fragrances Incorporated, Union Beach, NJ, United States Subramoney N. Moorthy, CTCRI, Trivandrum, India Ewa Nebesny, Lodz University of Technology, Lodz, Poland Daysi Perez-Rea, San Simon University, Cochabamba, Bolivia Miguel Peris-Tortajada, Universitat Polite`cnica de Vale`ncia, Valencia, Spain Jack Preiss, Michigan State University, East Lansing, MI, United States Marion G. Priebe, University of Groningen, Groningen, The Netherlands Carmen C. Quiroga Ledezma, Universidad Privada Boliviana e UPB, Cochabamba, Bolivia Justyna Rosicka-Kaczmarek, Lodz University of Technology, Lodz, Poland Moothandassery S. Sajeev, CTCRI, Trivandrum, India Cindy Semeijn, Coo¨peratie AVEBE U.A., Veendam, The Netherlands Jaspreet Singh, Massey University, Palmerston North, New Zealand Qingjie Sun, Qingdao Agricultural University, Qingdao, China Roel J. Vonk, University of Groningen, Groningen, The Netherlands Renate E. Wachters-Hagedoorn, University of Groningen, Groningen, The Netherlands Pei Wang, Nanjing Agricultural University, Nanjing, China Xueming Xu, Jiangnan University, Wuxi, People’s Republic of China

Part One

Analysing and Modifying Starch


Chapter 1

Plant Starch Synthesis Jack Preiss

Michigan State University, East Lansing, MI, United States

This chapter reviews enzymic reactions involved in starch synthesis in higher plants and algae. Existing information on the properties of various starch biosynthetic enzymes in the plant, algal, and cyanobacterial systems will be described and compared. Alteration of starch structure due to the mutational effects on these biosynthetic enzymes allows postulation for some specific functions for starch synthases (SSs) and branching enzymes (BEs). Finally regulation of starch synthesis at the enzymatic level will be discussed and in relation to this regulation, recent results indicating how starch content has been increased in certain plants will be indicated. A previous chapter (Shannon and Garwood, l984) in the second edition of Starch Chemistry and Technology discusses the various maize endosperm mutants or mutant combinations (26 of them) that shows an effect on the quantity or the nature of the starch formed and is still of interest. This information remains of interest and the reader is referred to that review. This review will deal with mutants where the biochemical process affecting the mutation has been elucidated. Pe´rez and Bertoft (2010) have published a recent and detailed informative review on starch structure based on recent instrumentation applications. That article presents the current view on the general features of the starch granules, and amylose and amylopectin structures. There is a number of recently published review articles (Preiss, 2009; Zeeman et al., 2010; Ko¨tting et al., 2010; Preiss, 2010; Geigenberger, 2011; Stitt and Zeeman, 2012; Schwarte et al., 2015; Streb and Zeeman, 2012; Nakamura, 2015) on starch biosynthesis covering many of the areas presented in this chapter.

1. LOCALIZATION OF PLANT STARCH SYNTHESIS IN PLANTS 1.1 Leaf Starch Starch is deposited in granules in almost all green plants and in various types of plant tissues and organs, e.g., leaves, roots, shoots, fruits, grains, and Starch in Food. http://dx.doi.org/10.1016/B978-0-08-100868-3.00001-9 Copyright © 2018 Elsevier Ltd. All rights reserved.

3

4 PART j ONE Analyzing and Modifying Starch

stems. Illumination of the leaf in bright light causes the formation of starch granules in the chloroplast organelle (Pe´rez and Bertoft, 2010). Disappearance of starch occurs either by exposure of the observed by iodine staining of the tissue or by light or electron microscopy. Starch accumulates due to carbon fixation during photosynthesis and the starch formed in the light is degraded in the dark to products that are in most cases utilized for sucrose synthesis. Mutants of Arabidopsis thaliana unable to synthesize starch, grow at the same rate as the wild type (WT) in a continuous light regime because they are able to synthesize sucrose (Caspar et al., 1986), but their growth rate is drastically reduced if grown in a dayenight regime. The reason for this is that the accumulated starch is required for sucrose synthesis at night; the sucrose is transported from the leaf to the sink tissues. Biosynthesis and degradation of starch in the leaf is therefore a dynamic process having diurnal level fluctuations in its storage (Stitt and Zeeman, 2012; Streb and Zeeman, 2012). Starch also plays an important role in the operation of stomatal guard cells, where it is degraded during the day. In the late afternoon or evening while the stomata are open, the starch is resynthesized. Leaf starch is lower in amylose content than what is observed in storage tissues (Matheson, 1996). The amylose structure is also of a smaller molecular size.

1.2 Starch in Storage Tissues In storage organs, fruit or seed, during the development and maturation of the tissue, synthesis of starch occurs. At the time of sprouting or germination of the seed or tuber, or ripening of the fruit, starch degradation in these tissues can occur and the derived metabolites are used as a source both for carbon and energy. The degradation and biosynthetic processes in the storage tissues can therefore be temporally separated. However, there is a possibility that during each phase of starch metabolism some turnover of the starch molecule can occur. The main site of starch synthesis and accumulation in the cereals is the endosperm, with starch granules that are located within the amyloplasts. Starch content in potato tuber, maize endosperm, and in roots of yam, cassava, and sweet potato can range between 65% and 90% of the total dry matter (Pe´rez and Bertoft, 2010). Patterns of starch accumulation during development of the tissue are specific to the species and are related to the unique pattern of differentiation of the organ (Pe´rez and Bertoft, 2010). Starch granules in storage tissues can vary in shape, size, and composition (Pe´rez and Bertoft, 2010). They can be spherical, oval, polygonal, lenticular elongated, or kidney shaped. The dimensions of the starch granules can vary from 2 to 3 mm as seen in small wheat granules to up to 100 mm as observed in canna starch. Starch granules as isolated in plants such as amaranth or taro, however, can have dimensions in the submicron level. Thus the shape and size of the granules depends on the source. But in each tissue there is a range of

Plant Starch Synthesis Chapter j 1

5

sizes and shapes. The diameter of the starch granule changes during the development of the reserve tissue. There are also some fine features, characteristic of each species, e.g., the “growth rings,” spaced 4e7 mm apart, and the fibrillar organization seen in potato starch, that allows one to identify the botanical source of the starch by microscopic examination (Pe´rez and Bertoft, 2010). Two polymers are distinguished in the starch granule. Amylose is essentially a linear polymer and amylopectin, a highly branched polymer. Amylose is mainly found as linear chains of about 840 to 22,000 units of a-Dglucopyranosyl residues linked by a-(1/4) bonds (molecular weight around 136,000 to 3.5 l06). The number of anhydroglucose units, however, varies quite widely with plant species and stage of development. Some of the amylose molecules are branched to a small extent (a-1/6-D glucopyranose; one per l70 to 500 glucosyl units). Amylopectin, in contrast, which usually comprises about 70% of the starch granule, is more highly branched with about 4%e5% of the glucosidic linkages being a-1/6. Amylopectin molecules are large flattened disks consisting of a-(1,4)-glucan chains joined by frequent a-(1,6)-branch points. The satisfactory models of amylopectin structure proposed fitting the experimental data available are those proposed by Robin et al. (1974), Manners and Matheson (1981), and by Hizukuri (1986). These are known as cluster models. The chemical and physical aspects of the starch granule and components amylose and amylopectin are discussed in reviews by Morrison and Karkalis (1990) and Hizukuri (1996) and recently by Pe´rez and Bertroft (2010). Fig. 1.1 shows the proposed cluster model of amylopectin.

A-chain

B2-chain (long B)

Internal segments

External segments

B1-chain (short B)

C-chain

FIGURE 1.1 Proposed basic structure of amylopectin (Pe´rez and Bertoft, 2010). The circles are glucose residues and lines represent a-1 / 4 linkages. The bent arrows represent a-1 / 6 linkages. The reducing end is seen as ø.


2. STARCH SYNTHESIS: ENZYME REACTIONS IN PLANTS AND ALGAE AND GLYCOGEN SYNTHESIS IN CYANOBACTERIA 2.1 Enzyme Reactions of Starch Synthesis The specific sugar nucleotide utilized for synthesis of the a-1,4-glucosidic linkages in amylose and amylopectin is adenosine diphosphate-glucose (ADP-Glc). ADP-Glc synthesis is catalyzed by ADP-Glc pyrophosphorylase (Reaction (1.i), E.C. 2.7.7.27; ATP: a-D-glucose-1-phosphate adenylyltransferase). ATP þ a-glucose-1-P 4 ADP-glucose þ PPi

(1.i)

Reaction (1.ii) is catalyzed by SS (E.C. 2.4.1.21; ADP-glucose; 1,4-a-Dglucan 4-a-glucosyl-transferase). A similar reaction is noted for glycogen synthesis in cyanobacteria and other bacteria (Preiss and Romeo, 1989; Preiss, 1996, 2014; Ball and Morell, 2003; Ballicora et al., 2003) where the reaction is referred to as glycogen synthase (E.C. 2.4.1.21). ADP-glucose þ a-1,4-glucan / a-1,4-glucosyl-a-1,4-glucan þ ADP (1.ii) Reaction (1.iii) shows catalysis by BE (E.C. 2.4.1.18; 1,4-a-D-glucan6a-(1,4-a-glucano)-transferase). a-1,4-oligosaccharide chain / a-1,4, a-1,6-branched chain

(1.iii)

The branch chains in amylopectin are longer than in glycogen and are about 20e24 glucose units long. There is less branching in amylopectin. About 5% of the glucosidic linkages are a-1,6. In glycogen, the chain lengths (CLs) are about 10e13 glucose units long and 10% of linkages are a-1,6. Thus, the (SBEs) may have different properties with respect to the size of chain transferred, or placement of branch point, than the enzyme that branches glycogen. Alternatively, the interaction of the SBEs with the SSs may be different from the interaction of the bacterial BEs with their respective glycogen synthases. The chain-elongating properties of the SSs could be different from those observed for the bacterial glycogen synthases and may account for some of the differences observed in the amylopectin structure. The differences in the catalytic properties of the SSs and BEs isolated from different plant sources and their interactions may also account for the differences observed in the various plant starch structures (Pe´rez and Bertoft, 2010). Isozymic forms of plant SSs and BEs have been reported and the different properties of these isozymes will be discussed. They seem to play different roles in the synthesis of the two polymers of starch, amylose and amylopectin, and are products from different genes. In many different plants as well as in Chlamydomonas reinhardtii, a granule-bound starch synthase (GBSS)


7

involved in the catalysis of Reaction (1.ii) has been shown to be involved in the synthesis of amylose.

3. PROPERTIES OF PLANT a-1,4-GLUCAN-SYNTHESIZING ENZYMES 3.1 ADP-Glucose Pyrophosphorylase: Kinetic and Regulatory Properties of the Enzyme In bacteria and in plants there is only one physiological function for ADP-Glc and that is to be a donor of glucose for synthesis of a-1,4-glucosyl linkages of plant or algal starch. It would therefore be advantageous to conserve the ATP utilized for synthesis of the sugar nucleotide by regulating a-1,4-glucan synthesis at the level of ADP-Glc formation. Over 50 ADP-glucose pyrophosphorylases (ADP-Glc PPases), bacterial, algal, and plant, have been studied with respect to their regulatory properties (Preiss, 1996, 2009, 2010, 2014; Preiss and Romeo, 1989; Ball and Morell, 2003; Ballicora et al., 2003, 2004; Smith-White and Preiss, 1992). In almost all cases, glycolytic intermediates activate ADP-Glc synthesis, while AMP, ADP, and/or Pi are inhibitors (Preiss, 1996, 2010, 2014; Preiss and Romeo, 1989; Ball and Morell, 2003; Ballicora et al., 2003, 2004; Smith-White and Preiss, 1992). Glycolytic intermediates in the cell may be considered as a signal of carbon excess; therefore, under conditions of limited growth with available excess carbon in the environment, accumulation of glycolytic intermediates would be the signal for activation of ADP-Glc synthesis. In plants and algae, with active CO2 fixation either via the Calvin or Hatch-Slack pathway and ATP formation during photosynthesis, the enzyme ADP-Glc PPase would be affected by the availability of ATP in the cell as well as the carbon dioxide fixation product, 3-phospho-glycerate (3PGA). The ADP-Glc PPase of higher plants, green algae, and the cyanobacteria are allosterically activated by 3PGA and inhibited by inorganic phosphate (Pi) (Preiss, 2009, 2010; Ballicora et al., 2004; Smith-White and Preiss, 1992). The 3PGA activation can be anywhere from 3- to 60-fold. Of importance is that increasing concentrations of Pi can reverse activation by 3PGA. These effects are important in the regulation of starch synthesis. Spinach leaf (Ghosh and Preiss, 1966; Preiss et al., 1967; Copeland and Preiss, 1981) and other plant leaves (Sanwal and Preiss, 1967), Chlorella (Sanwal et al., 1968), and potato tuber ADP-Glc PPases (Iglesias et al., 1993; Ballicora et al., 1995, 1998) have been studied in the most detail with respect to kinetic properties and structure. The results obtained with potato tuber and spinach leaf enzyme are summarized in Table 1.1. 3PGA activates the spinach leaf ADP-Glc PPase 20-fold and activates the potato tuber enzyme 30-fold. Pi can inhibit both enzymes about 50% at 40e45 mM. However, in the presence of 1 mM 3PGA, the enzymes are less sensitive to inhibition by Pi and 50%


TABLE 1.1 Kinetic Constants of ADP-Glucose Pyrophosphorylase From Spinach Leaf and Potato Tuber

Spinach leaf

Potato tuber

Effector/Substrate

Kinetic Constant (mM)

n

Fold-Activation Source

3PGA

0.051

1.0

20

Pi (3PGA)

0.045

1.1

Pi (þ1 mM 3PGA)

0.97

3.7

Glc-1-P (3PGA)

0.12

0.9

Glc-1-P (þ3PGA)

0.035

0.9

ATP (3PGA)

0.38

0.9

ATP (þ3PGA)

0.062

1.0

3PGA

0.16

1.0

Pi (3PGA)

0.04

NR

Pi (þ3 mM 3PGA)

0.63

NR

Glc-1-P (þ3PGA)

0.057

1.1

ATP (þ3PGA)

0.076

1.6

30

A0.5 is the activation constant; the concentration of activator required for 50% of maximal velocity. I0.5 is the inhibitor concentration required for 50% inhibition in the presence or absence of the activator, 3PGA. S0.5 is the substrate concentration required for 50% of maximal activity. NR stands for not reported.

inhibition occurs at 0.97 mM for the spinach enzyme. In the presence 3 mM PGA, 0.63 mM Pi is required for 50% inhibition. Thus the synthetic rate of ADP-Glc synthesis will be dependent on the ratio of 3PGA/Pi concentrations. It is expected that during photosynthesis that Pi concentrations would be lower due to higher rate of ATP synthesis and greater rate of synthesis and accumulation of phosphorylated glycolytic intermediates including 3PGA. Of interest is that the apparent affinity of the substrates, ATP and glucose-1-P (Glc-1-P) for the ADP-Glc PPase is higher in the presence of the activator, 3PGA. The spinach leaf enzyme requires about four- to sixfold less substrate concentration for 50% maximal velocity and the constant is denoted as S0.5. 3PGA lowers the S0.5 of the substrates for the potato tuber enzyme about 3.5- to 6-fold. The kinetic and regulatory properties of the ADP-Glc PPases from the leaf extracts of spinach, barley, butter lettuce, kidney bean, maize, peanut, rice, sorghum, sugar beet, tobacco, and tomato have been studied in detail and are quite similar (Sanwal et al., l968).


9

3.2 ADP-Glucose Pyrophosphorylase: StructureeFunction Relationships: Quaternary Structure Bacterial ADP-Glc PPases are homotetrameric in structure (Preiss, 1996, 2014; Ballicora et al., 2003). The catalytic and allosteric sites are on the same and each subunit. In plants and in green algae, however, the ADP-Glc PPases have been shown to be heterotetramers with two homologous subunits, a2b2 (Preiss, 2009, 2010; Ballicora et al., 2004; Smith-White and Preiss, 1992) having different molecular sizes. The small subunit is about 50e54 kDa and has catalytic activity. The large subunit, about 51e60 kDa, is generally the regulatory subunit. The large subunit modulates the sensitivity of the small subunit toward allosteric effectors, via large subunit/small subunit interactions (Preiss, 2009, 2010; Ballicora et al., 2004). However, recent results indicate that some large subunits, particularly those in leaf (Ventriglia et al., 2008), also have catalytic activity. ADP-Glc PPase from potato tuber is composed of two different subunits, 50 and 51 kDa, with a a2b2 heterotetrameric subunit structure (Ballicora et al., 1995, 1998, 2004; Iglesias et al., 1993). The small subunit of many higher plant ADP-Glc PPases is highly conserved among plants with 85%e95% identity (Smith-White and Preiss, 1992; Ballicora et al., 1995, 1998). The homotetrameric potato enzyme composed exclusively of small subunits has a lower apparent affinity (A0.5 ¼ 2.4 mM) for the activator 3PGA than the heterotetramer (A0.5 ¼ 0.16 mM) and is more sensitive to the inhibitor Pi (I0.5 ¼ 0.08 mM in the presence of 3 mM 3PGA) as compared with the heterotetramer (I0.5 ¼ 0.63 mM) (Ballicora et al., 1995, 1998). The potato large subunit greatly increases the affinity of the small (catalytic) subunit for 3PGA and lowers the affinity for the inhibitor Pi (Ballicora et al., 1995, 1998). In plants there may be only one conserved small (catalytic) subunit and several large (regulatory) subunits that are distributed in different parts of the plant (Ventriglia et al., 2008; Crevilleń et al., 2003, 2005). This is of physiological significance as expression of different large subunits in different plant tissues may confer distinct allosteric properties to the ADP-Glc PPase needed for the different parts of the plant’s distinct need for starch (Crevilleń et al., 2003, 2005). It has been shown with Arabidopsis ADP-Glc PPase that coexpression of its small subunit, APS1, with the different Arabidopsis large subunits, ApL1, ApL2, ApL3, and ApL4, resulted in heterotetramers with different regulatory and kinetic properties (Tables 1.2 and 1.3). Homotetramer APS1 had low affinity for 3PGA while the heterotetramers of APS and the large subunits had much greater affinity for 3PGA (Crevilleń et al., 2003; Table 1.2). The heterotetramer of the small subunit APS1 with ApL1, the predominant leaf large subunit (Crevilleń et al., 2003), had the highest sensitivity to the allosteric effectors, 3PGA and Pi, as well as the highest apparent affinity for the substrates ATP and Glc-1-P (Crevilleń et al., 2003). The heterotetrameric pairs of


TABLE 1.2 Kinetic Parameters for 3PGA of Arabidopsis thaliana Recombinant ADP-Glc PPase in the Synthesis Direction (Crevilleń et al., 2003) Control

0.2 mM Pi

2 mM Pi

3PGA

A0.5 (mM)

n

A0.5 (mM)

n

A0.5 (mM)

n

APS1

5.7

1.6

13.9

2.3

N.D.

APS1/ApL1

0.018

0.8

0.094

1.4

1.6

2.9

APS1/ApL2

0.87

0.9

1.54

1.3

10.5

1.5

APS1/ApL3

0.34

0.8

0.70

1.4

2.7

2.7

APS1/ApL4

0.16

0.8

0.40

1.2

1.7

1.5

APS1 is the small subunit 1, ApL1, ApL2, ApL3, and ApL4 are large subunits 1, 2, 3, and 4, respectively, from A. thaliana. N.D. indicates not determined. The kinetic parameters were calculated without inhibitor Pi, or in the presence of inhibitor Pi at 0.2 mmol or at 2 mM. A0.5 is the activator concentration required for 50% of maximal activation.

TABLE 1.3 Kinetic Parameters for the Substrates of Arabidopsis thaliana Recombinant ADP-Glc PPase in the Synthesis Direction (Crevilleń et al., 2003). ATP

Glucose-1-P

Tetramer Complex

S0.5 (mM)

n

S0.5 (mM)

n

APS1

0.40

1.5

0.076

0.9

APS1/ApL1

0.067

1.0

0.019

1.0

APS1/ApL2

0.575

1.6

0.085

0.9

APS1/ApL3

0.094

1.4

0.052

1.0

APS1/ApL4

0.118

1.2

0.06

0.9

The activator 3PGA concentrations used for each enzyme tetramer subunit combination was the concentration required for maximal velocity. S0.5 is the concentration of substrate required for 50% of maximal activity.

APS1 with either ApL3 or ApL4, large subunits, prevalent in sink or storage tissues (Crevilleń et al., 2005), had intermediate sensitivity to the allosteric effectors and intermediate affinity for the substrates ATP and Glc-1-P (Table 1.3; Crevilleń et al., 2003). ApL2 also present mainly in sink tissues had low affinity for either 3PGA or Pi (Ventriglia et al., 2008; Crevilleń et al., 2003). Thus, differences on the regulatory properties conferred by the Arabidopsis large subunits were found in vitro. Differences noted for source


11

and sink large subunit proteins strongly suggests that starch synthesis is modulated in a tissue-specific manner in response to 3PGA and Pi, as well as to the substrate levels. APS1 and ApL1 would be finely regulated in source tissues by both effectors and substrates while in sink tissues the heterotetramers of APS1 with ApL2, ApL3, or ApL4 with lower sensitivity to effectors and substrates would be controlled more by the supply of substrates. Based on mRNA expression, APS1 is the main small subunit or catalytic isoform responsible for ADP-Glc PPase activity in all tissues of the plant. ApL1 is the main large subunit in source tissues, whereas ApL3 and ApL4 are the main isoforms present in sink tissues (Crevilleń et al., 2005). It was also found that sugar regulation of ADP-Glc PPase genes was restricted to ApL3 and ApL4 in leaves (Crevilleń et al., 2005). Sucrose induction of ApL3 and ApL4 transcription in leaves allowed formation of heterotetramers that are less sensitive to the allosteric effectors, resembling the situation in sink tissues.

3.3 Relationship Between the Small and Large Subunits The amino acid sequence similarity between the small and large subunits (w50%e60% identity) suggests a common origin (Ballicora et al., 1995, 1998). In both sink and source tissues the small subunit has catalytic activity while catalytic activity is only observed for the large subunits that may reside in the leaf and not in the sink large subunits (Ventriglia et al., 2008). Most probably gene duplication and divergence has led to different and functional roles catalytic and regulatory for the subunits. The ancestor of small and large subunits possibly is a bacterial subunit having both catalytic as well as regulatory function in the same subunit. This is supported by the similarity between the two plant subunits with many active bacterial ADP-Glc PPases (Ballicora et al., 2004; Smith-White and Preiss, 1992). The large subunit from the potato (Solanum tuberosum L.) tuber ADP-Glc PPase was shown to bind substrates (Fu et al., 1998a). The plant heterotetramer therefore, as well as bacterial homotetramers, binds four ADP-[14C] glucose molecules (Fu et al., 1998a; Haugen and Preiss, 1979). It can be postulated that the large subunit maintained its structure needed for binding of substrate, but catalytic ability was eliminated by mutations of essential residues. To test this hypothesis, it was attempted to create a large subunit with significant catalytic activity by the mutation of amino acid residues involved in substrate binding as well as in catalysis (Frueauf et al., 2003). Thus, sequence alignments of ADP-Glc PPase large and small subunits with reported activity were compared to identify critical missing residues for catalytic activity in the large subunit (Frueauf et al., 2003). The subset of the ones absent in the large subunit was of particular interest. Lys44 and Thr54 in the large subunit of potato tuber were selected as the best candidates to study because the homologous residues, Arg33 and Lys43 in the small (catalytic) subunit, were completely conserved in the active bacterial and plant catalytic


TABLE 1.4 Sequence Comparison of ADP-Glc PPase Subunits in a Conserved Region With Critical Amino Acids for Catalysis (Frueauf et al., 2003) Escherichia coli

26 LAGGRGTRLKDLTNKRAKPAVH

47

PSS

26 LGGGAGTRLYPLTKKRAKPAVP

47

APS1

96 LGGGAGTRLYPLTKKRAKPAVP

117

ApL1

96 LGGGAGTRLFPLTKRRAKPAVP

117

ApL2

91 LGGGAGTRLFPLTSKRAKPAVP

112

ApL3

95 LGGGDGAKLFPLTKRAATPAVP

116

ApL4

97 LGGGNGAKLFPLTMRAATPAVP

118

PLS

38 LGGGEGTKLFPLTSRTATPAVP

59

Sequence comparison of the catalytic site of ADP-Glc PPases from E. coli, Arabidopsis, and potato. Sequences and their accession numbers are E. coli, P00584; PSS, potato tuber small subunit CAA88449; APS1, P55228; ApL1, P55229; ApL2, P55230; ApL3, P55231; ApL4, Q9SIK1; PLS, potato tuber large subunit Q00081. The conserved arginine and lysine (or threonine) residues are indicated in bold.

subunits. Moreover, Lys44 and Thr54 are in a highly conserved region of both bacterial and plant ADP-Glc PPases (Table 1.4). The potato large subunits Lys44 and Thr54 were mutagenized to Arg44 and Lys54, respectively. The mutant, LargeK44R/T54K, expressed in the absence of the small subunit had no activity. Possibly the large subunit cannot form a stable tetramer in the absence of the small subunit as seen earlier with Arabidopsis enzyme (Ventriglia et al., 2008). Because WT small subunit has intrinsic activity, the activity of the large subunit mutants cannot be tested when coexpressed. Thus, the large subunit mutants were coexpressed with inactive small subunit D145N, in which the catalytic residue Asp145 was mutated (Frueauf et al., 2003) reducing by more than three orders of magnitude small subunit activity (Table 1.5). Coexpression of the large subunit double-mutant K44R/T54K with Small D145N generated an enzyme having 10% and 18% of the WT enzyme in the ADP-Glc synthetic direction, respectively (Table 1.5). Single mutations K44R or T54K generated enzymes with no significant activity. The combination of both mutations in the large subunit (Small D145N/LargeK44R/T54K) gave the most dramatic effect (Table 1.5). Therefore, it was concluded that the two residues Arg44 and Lys54 are needed for restoring catalytic activity to the large subunit. Replacement of the homologous two residues with Lys and Thr in the small subunit (by mutations R33K and K43T) decreased the activity one and two orders of magnitude, respectively, in either the ADP-Glc synthetic or pyrophosphorolytic directions, confirming the hypothesis (Table 1.5; Frueauf et al., 2003).


13

TABLE 1.5 Activity Catalytic (Small) and Regulatory (Large) Subunits of Potato Tuber ADP-Glc PPase Mutants Subunits Catalytic (Small)

Regulatory (Large)

ADP-Glc Synthesis (mmol/min/mg)

WT

WT

32 1

D145N

WT

0.017 0.001

D145N

K44R

0.031 0.001

D145N

T54K

0.92 0.08

D145N

K44R/T54K

3.2 0.2

R33K

WT

3.6 0.2

K43T

WT

0.32 0.1

The enzymes activities of purified coexpressed small and large subunits were measured for ADPglucose (ADP-Glc) synthetic activity. For ADP-Glc synthesis, 4 mM of 3PGA (activator), 2 mM of ATP, and 0.5 mM of Glc-1-P were used.

The mutant enzymes were still activated by 3PGA and inhibited by phosphate (Pi). The WT enzyme and Small D145N/LargeK44R/T54K had very similar kinetic properties indicating that the substrate site domain has been conserved. The apparent affinities for the substrates and the allosteric properties of small subunit D145N/LargeK44R/T54K resembled those of the WT enzyme (Ballicora et al., 2005). The new form has a similar sensitivity to Pi inhibition and the activatoreinhibitor interactions were the same as WT enzyme. That the large subunit restored enzyme activity to the inactive small subunit heterotetramer due to only two mutations is evidence that the large and small subunits are derived from the same ancestor.

3.4 The Glucose-1-P-Binding Site in Plant ADP-Glucose Pyrophosphorylase The heterotetramer Small D145N mutant and LargeK44R/T54K mutant was disrupted in each subunit at their Glc1P site and their kinetic properties compared. As indicated above, catalysis occurs in the large subunit of Small D145N/LargeK44R/T54K heterotetramer. As seen in Table 1.6, the Glc-1-P substrate-binding residue in the small subunit is Lys198 (Fu et al., 1998a). Substitution of Lys with Arg lowers the affinity of the enzyme for Glc-1-P about 150-fold and substitution of the Lys with Ala or Glu lowers the affinity 440- or 546-fold, respectively (Table 1.6). Amino acid substitution of the homologous residue, Lys213, in the large subunit does not have the same effect. In Small D145N/LargeK44R/T54K enzyme, the mutation, K213R of the large subunit severely decreased the apparent affinity for G1P, whereas mutation of K198R on the small subunit did not (Table 1.6). This indicated

S0.5 for Glc-1-P (mM)

Vmax (Units/mg)

S0.5 for ADP-Glc (mM)

Vmax (Units/mg)

Wild type

0.057 0.003 (1.1)

48 1

0.20 0.01 (1.3)

55 1

SK198RLwt

7.7 0.1 (1.3)

24 1

0.49 0.02 (1.1)

16 1

SK198ALwt

22.0 2.5 (1.5)

46 3

1.3 0.1 (1.0)

20 1

SK198ELwt

31.1 2.7 (1.8)

1.7 0.1

2.1 0.2 (1.0)

1.4 0.1

SwtLK213R

0.044 0.002 (1.1)

27 1

0.36 0.03 (1.8)

37 1

SwtLK213A

0.037 0.001 (1.0)

25 1

0.46 0.02 (1.7)

31 1

SwtLK213E

0.036 0.001 (0.9)

31 1

0.68 0.01 (1.8)

45 1

SK198RLK213

5.6 0.1 (1.5)

24 1

0.72 0.01 (1.9)

14 1


TABLE 1.6 Apparent Affinity of Glc-1-P and ADP-Glc of Potato Tuber Wild-Type and Mutant ADP-Glc PPases


15

that the large subunit double mutant, and not Small D145N, was the catalytic subunit. In the WT enzyme, Lys213 does not seem to play any important role, but in Small D145N/LargeK44R/T54K it recovered its ancestral ability to confer to the enzyme a high-apparent affinity for G1P. Previous results showed that Asp145 in the small subunit of the WT is essential for catalysis (Frueauf et al., 2003), and in the WT enzyme, replacement of Lys198 in the small subunit of the WT enzyme, decreased the Glc-1P affinity (Fu et al., 1998a). Disruption of the homologous residue Lys213 in the large subunit has much less effect. In Small D145N/LargeK44R/T54K where the large subunit is now the catalytic subunit, the K213R mutation of the large subunit severely decreased the apparent affinity for Glc1P, whereas the K198R mutation of the small subunit did not indicate that the large subunit double mutant, and not Small D145N, was the catalytic subunit. In the WT enzyme, Lys213 does not seem to play any important role, but Small D145N/LargeK44R/T54K recovered its ancestral ability for the enzyme to have a low physiological Km for Glc1P. Previous results showed that in the WT ADP-Glc PPase, Asp145 of the small subunit is essential for catalysis, but homologous Asp160 in the large subunit is not 39. Also mutation of D160 to N or E in the active large subunit LK44R/T54K abolished activity. This confirms that catalysis of Small D145N/ LargeK44R/T54K does occur in the large subunit. A comparative model of LK44R/T54K shows the predicted role of Arg44 and Lys54 (Fig. 1.2). In the model, Asp160, which is homologous to the catalytic Asp145 in the small subunit and catalytic Asp142 in the Escherichia coli ADP-Glc PPase (Frueauf et al., 2001, 2003), interacts with Lys54. This type of interaction (Lys54eAsp160) has also been observed in crystal structures of enzymes catalyzing similar reactions, such as dTDP-glucose pyrophosphorylase (dTDP-Glc PPase) (Blankenfeldt et al., 2000) and UDPN-acetyl-glucosamine pyrophosphorylase (UDP-GlcNAc PPase) (Brown et al., 1999), and postulated to be important for catalysis by correctly orienting the aspartate residue (Blankenfeldt et al., 2000; Brown et al., 1999; Sivaraman et al., 2002). Lys54 interacts with the oxygen bridging the a- and b-phosphates as it has been observed in the crystal structure of E. coli dTDP-Glc PPase (Sivaraman et al., 2002). The interaction may neutralize a negative charge density stabilizing the transition state and making PPi a better leaving group. Arg44 interacts in the model with the b- and g-phosphates of ATP, which correspond to the PPi by-product (Fig. 1.2). Likewise, Arg15 in the E. coli dTDP-Glc PPase was postulated to contribute to the departure of PPi42 and kinetic data agreed with interaction of PPi with Arg44 in the model. A Lys44, in both the catalytic large subunit mutant and the small subunit, of the potato ADP-Glc PPase decreased the apparent affinity for PPi at least 20-fold (Ballicora et al., 2005). In WT large subunit, Lys44 and Thr54 cannot interact as Arg44 and Lys54 (Fig. 1.2).

16 PART j ONE Analyzing and Modifying Starch Evolution Lys44

Arg44

Thr54

Lys54

Asp160

Asp160 Gly131

Gly131

Cys73

Cys73 Asp166

Asp166

Asp31 LK44R/T54K

R44, K54 (catalytic)

Asp31 LWT

K44, Thr 54 (noncatalytic)

FIGURE 1.2 Involvement of the large (regulatory) subunit of mutant K44R/T54K in enzyme catalysis. The WT and double-mutant large subunits were modeled based on the dTDP-Glc PPase and UDP-GlcNAc PPases as indicated. Portions of residues 31e73 and 131e136 are shown. The deoxyribose triphosphate portion common to dTTP and ATP is modeled with Mg2þ as a blue sphere. The nitrogen atom of the adenylyl group attached to the ribosyl unit is also in blue. The dotted green lines depict hydrogen bonds.

3.5 Phylogenetic Analysis of the Large and Small Subunits A phylogenetic tree of the ADP-Glc PPases present in photosynthetic eukaryotes may also shed information about the origin of the two subunits. The tree shows that plant small and large subunits can be divided into two and four distinct groups, respectively (Ballicora et al., 2005). The two main groups of S subunits are from dicot and monocot plants, whereas large subunit groups correlate better with their documented tissue expression. The first Largesubunit group, group I, is generally expressed in photosynthetic tissues (Ballicora et al., 2005) and comprises large subunits from dicots and monocots. These subunits recently have been shown to have catalytic activity and have in their sequences Arg and Lys in the equivalent residues of 102 and 112 of A. thaliana large subunit, ApL1. Group II displays a broader expression pattern, whereas groups III and IV are expressed in storage organs (roots, stems, tubers, seeds). Subunits from group III are only from dicot plants, whereas group IV are seed-specific subunits from monocots. These last two groups stem from the same branch of the phylogenetic tree and split before monocot and dicot separation. These subunits are probably inactive in catalytic activity as they are lacking Arg and Lys in the homologous residues seen in A. thaliana ApLI and ApL234.


17

3.6 Crystal Structure of Potato Tuber ADP-Glc PPase The crystal structure of potato tuber homotetrameric small (catalytic) subunit ˚ resolution (Fig. 1.3(a); Jin et al., ADP-Glc PPase has been determined to 2.1 A 2005). The structures of the enzyme in complex with ATP and ADP-Glc were ˚ resolution, respectively. Ammonium sulfate was determined to 2.6 and 2.2 A used in the crystallization process and was found tightly bound to the crystalline enzyme. It was also shown that the small subunit homotetrameric potato tuber ADP-Glc PPase was also inhibited by inorganic sulfate with the I0.5 value of 2.8 mM in the presence of 6 mM 3PGA. Sulfate is considered as an analog of phosphate, the allosteric inhibitor of plant ADP-Glc PPases. Thus the atomic resolution structure of the ADP-Glc PPase probably presents a conformation of the allosteric enzyme in its inhibited state. The crystal structure of the potato tuber ADP-Glc PPase (Jin et al., 2005) allows one to determine the location of the activator and substrate sites in the threedimensional structure and their relation to the catalytic residue, Asp145. The structure also provides insights into the mechanism of allosteric regulation and these aspects will be discussed later. The overall fold of the potato tuber ADP-Glc PPase small subunit catalytic domain is quite similar to that of two other pyrophosphorylases, viz., N-acetylglucosamine 1-phosphate uridylyl-transferase (GlmU) from E. coli 43 (Fig. 1.3(b)) and S. pneumoniae (Sulzenbacher et al., 2001; Kostrewa et al., 2001) and glucose 1-phosphate thymidylyl-transferase (Rffh) from P. aeruginosa (Blankenfeldt et al., 2000) and E. coli (Sivaraman et al., 2002), although their primary sequences have only very low sequence similarities. The catalytic domain is composed of a seven-stranded b sheet covered by a helices, a fold reminiscent of the dinucleotide-binding Rossmann fold (Raetz and Roderick, 1995). At one of its ends, the central b-sheet is topped by a twostranded b-sheet. The catalytic domain makes strong hydrophobic interactions with the C-terminal domain through an a-helix that encompasses residues 285e297 (Fig. 1.3(c)). The catalytic domain is connected to the C-terminal b-helix domain by a long loop containing residues 300e320. This loop makes numerous interactions with the equivalent region of another monomer. The C-terminal domain comprises residues 321e451 and adopts a lefthanded b-helix fold composed of six complete or partial coils with two insertions, one of that encompasses residues 368e390. The other encompasses residues 401e431. This type of left-handed b-helix domain fold has been found in the structures of bacterial acetyl-transferases, including E. coli UDPN-acetylglucosamine 3-O-acyl-transferase (Raetz and Roderick, 1995), Methanosarcina thermophila carbonic anhydrase (Kisker et al., 1996), Mycobacterium bovis tetrahydrodipicolinate N-succinyl-transferase (Beaman et al., 1997) and GlmU (Sivaraman et al., 2002), and in other proteins such as T4 bacteriophage gp5 (Kanamaru et al., 2000). However, the b-helix domain seen in the other structures is an acetyl-transferase or succinyl-transferase


FIGURE 1.3 (a) Crystal structure of potato tuber ADP-glucose pyrophosphorylase (ADP-Glc PPase) small (catalytic) subunit monomer. The catalytic domain is in yellow and the b-helix subunit is in pink. ADP-Glc is shown in atom type where the carbon atoms are in green, the oxygen atoms red, nitrogen atoms blue, the phosphorus atoms magenta, and the sulfate groups ˚ ) (magenta)) and GlmU (gold) orange. (b) Overlay of ADP-Glc PPase (cyan, RmlA (r.m.s.d. 1.9 A ˚ ). (c) ADP-Glc PPase tetramer. The disulfide bond is boxed. (d) Interactions between (r.m.s.d. 2.5 A the monomers in the tetramer.


19

domain. In the present structure of ADP-Glc PPase, the b-helix domain is involved in cooperative allosteric regulation with the N-terminal catalytic region and interactions with the N-terminal region within each monomer, and contributes to oligomerization.

3.7 The Homotetramer Catalytic Subunit Structure of the Potato Tuber ADP-Glucose Pyrophosphorylase (Jin et al., 2005) The crystalline potato tuber ADPGlc PPase small subunit is a tetramer with ˚3 approximate 222 symmetry and approximate dimensions of 80 90 110 A 0 (Fig. 1.3(c)). It can be viewed as a dimer of dimers, labeled A, A , B, and B0 . Monomers A and B interact predominantly by end-to-end stacking of their b-helix domains, although there is also a significant interface between the linker loop connecting the two domains (Fig. 1.3(d)). This interface buries ˚ 2 of surface area. The catalytic domains of A and B0 (and B and A0 ) also 2544 A make an extensive interface. Several hydrogen bond and hydrophobic interactions stabilize the interface between A and B0 , burying a surface area of ˚ . All residues defining dimerization interfaces are identical or similar in 1400 A the large subunit. Fig. 1.3(d) delineates all oligomerization interactions seen within the tetramer in the asymmetric unit. Cys12 of monomer A and the equivalent cysteine residue of monomer A0 make a disulfide bond, as do equivalent cysteine residues of monomers B and B0 . The intersubunit disulfide bond between the small (catalytic) subunit is preserved in the heterotetramer. However, there is no disulfide bond between the large (regulatory) subunits, as Cys12 is not conserved. This disulfide bond establishes the relative orientation of the small subunits in the heterotetramer to be like A and A0 in the a4-homotetramer structure. The disulfide bond is the only interaction made between A and A0 (or B and B0 subunits). Potato tuber ADP-Glc PPase is redox-regulated by reduction and oxidation of the intermolecular disulfide bond between the two small subunits (Fu et al., 1998b; Ballicora et al., 1999, 2000). This covalent regulatory modification is discussed later. The N-terminal catalytic domain resembles a dinucleotide-binding Rossmann fold and the C-terminal domain comprising residues 321e451 adopts a left-handed parallel b helix that is involved in cooperative allosteric regulation and a unique oligomerization. The structures of the enzyme in an ATP and ADP-Glc complex are also observed. Communication between the regulatorbinding sites and the active site involves several distinct regions of the enzyme including the N-terminus, the Glc-1-P-binding site, and the ATPbinding site are proposed. These structures provide insights into the mechanism for catalysis and allosteric regulation of the enzyme. Sulfate is an inhibitor of potato tuber ADP-Glc PPase small subunit homotetramer with I0.5 ¼ 2.8 mM in the presence of 6 mM 3PGA (Jin et al., 2005). The electron density map for potato tuber ADP-Glc PPase small subunit suggests that there are three sulfate ions tightly bound to the enzyme


(a) A Catalytic

beta helix

B’ (b)

R316 Q314 H84 R53 K404

R83

K69

D370

H134 T135

R41 D403 K441

FIGURE 1.4 ADP-Glc PPase monomer showing (a) the sulfate-binding region between the catalytic and beta-helix domains and (b) the amino acid residues interacting with sulfate. The sulfate residues are yellow and the interacting residues are green in one subunit. The neighboring subunit and its residues are purple.

(Fig. 1.4). Most probably, this is due to the high-sulfate concentration ˚ (150 mM) in the crystallization solution. Two sulfate ions bind within 7.5 A of each other in a crevice located between the N- and C-terminal domains of the enzyme (Jin et al., 2005). A third sulfate ion binds between the two subunits of the enzyme. The sulfate ions make numerous interactions with residues shown to be involved in the allosteric activator-binding site, as demonstrated by chemical modification (Morell et al., 1988; Greene et al., 1996a) and site-directed mutagenesis studies (Fu et al., 1998b). The structures contain 12 sulfate ions within a tetramer in the asymmetric unit (three per monomer) and are all, therefore, representative of the inhibited conformation of the enzyme. Sulfate 1 makes hydrogen bond interactions with R41, R53, K404, and K441 (Fig. 1.4; Jin et al., 2005). The side-chain nitrogen atom of R41 makes hydrogen bond interactions with one of the sulfate ion oxygen atoms, and


21

D403 makes a salt bridge interaction with R41 to facilitate the binding. D413 in the potato tuber enzyme large subunit (D403 in the small subunit) was identified as important for activation by 3PGA (Greene et al., 1996a; Ballicora et al., 1998). All these residues are conserved in virtually all plant ADP-Glc PPases, and four of the five (all but K441) are strongly conserved in bacterial ADP-Glc PPases (Fig. 1.4). Site-directed mutagenesis studies have identified residues K441 and K404 in the small subunit of potato tuber as important for 3PGA activation (Greene et al., 1996a; Ballicora et al., 1998). The enzyme’s affinity for 3PGA was lowered and the inhibition by Pi diminished when mutations at these residues were Ala (neutral) or Glu (negative). The kinetic parameters for the substrates, ADP-Glc, PPi and the cofactor Mg2þ were not affected. Mutations on the homologous residues in the large subunit showed lesser or no effects on regulation of the enzyme (Ballicora et al., 1998). Therefore, it was concluded that K404 and K441 in potato tuber ADPGlc PPase small subunits are important for the binding of 3PGA and Pi, and the main role of the large subunit is to interact with the small subunit and modulate its activation mechanism (Ballicora et al., 1998). These studies indicate that the activator 3PGA binds at or near the inhibitor-binding site defined in the structure by sulfate 1. Sulfate 2 makes similar interactions with surrounding positively charged residues, R53, R83, H84, Q314, and R316 (Fig. 1.4). Site-directed mutagenesis studies have shown that H83 of the E. coli enzyme (H84 in the potato tuber enzyme small subunit) is involved in activator binding (Hill and Preiss, 1998). Chemical modification with phenylglyoxal has identified R294 in the Anabaena sp. enzyme (R316 in the potato tuber enzyme small subunit) as an important residue for inhibition by Pi, as mutations of this residue lowered the apparent affinity for Pi more than 100-fold (Sheng and Preiss, 1997). Mutations of this Arg residue to Ala, Gln, or Lys caused a change in inhibitor selectivity such that these mutants were inhibited by NADPH or FBP (Frueauf et al., 2002). Taken together, these studies confirm the importance of the sulfate ion-binding site in the allosteric regulation of the enzyme and indicate that 3PGA may also bind near the sulfate 2-binding site. Sulfate 3 is located between two subunits, viz., A and B0 . This sulfate ion interacts with R83 of one monomer and K69, H134, and T135 of the other subunit. K69 and R83 are conserved in both the small and large subunits of all plant ADP-Glc PPases. H134 is conserved in all small subunits, and residue T135 is conservatively replaced by Asn in other plant small subunits. The precise role of this location is not yet clear. Sulfate binding may be nonspecific or it may interfere with the dimerization of the subunits, thus causing the R / T equilibrium to be more toward the T (inhibited) state. The current structural results strongly support previous data on the allosteric regulation of this enzyme and provide some insights on how the binding of allosteric effectors could affect catalysis.


3.8 ATP Binding When ATP binds to the enzyme, both A and A0 monomers undergo almost identical conformational changes. Several regions move significantly, viz., a loop region from residue 27 to 34, another loop region from residue 106 to 119, and residues K40, R41, Q75, and F76 (Fig. 1.4 in reference Jin et al. (2005)). Both loop regions make direct interactions with the adenine portion of the nucleotide. Specifically, the main chain nitrogen atom of G28 makes a hydrogen bond with N3 of the adenine ring; several hydrophobic interactions are established between the adenine ring and L26 and G29; the side chain of Q118 makes a hydrogen bond with N6. These residues all undergo correlated conformational change upon ATP binding. Interactions of Q75 with both G30 and W116, and interactions of the K40 side chain with P111 couple the motions of the Q75, G30, and 106e119 regions (Jin et al., 2005). Furthermore, ATP binding in the A and A0 subunits drives conformational change in the B and B0 subunits, as P111 of A and A0 is packed snugly against W129 in B0 and B, respectively (Jin et al., 2005). Motion of P111 accompanying ATP binding leads directly to motion of W129 and, in fact, the entire region from 165 to 231 in the B/B0 subunits.

3.9 ADP-Glucose Binding Three of the four subunits (A, A0 , and B), bind ADP-Glc in the ADP-Glc PPase/ADP-Glc complex (Fig. 1.3(a)). The B0 subunit binds neither ATP nor ADP-Glc, and is conformationally more rigid than the other three subunits. A and A0 bind ADP-Glc identically, and ADP-Glc binding produces conformational changes in A and A0 identical to that which occurs when ATP binds (described above). The adenyl and ribosyl units of ADP-Glc in A and A0 are positioned identically to the adenyl unit of ATP, and the interactions between the enzyme and ADP-Glc are also identical to those seen in the ATP complex (Jin et al., 2005). No electron density is seen for the glucosyl moiety of ADP-Glc in the A and A0 active sites, indicating it to be disordered. This indicates that the conformational changes seen in A and A0 on ATP or ADPGlc binding are due almost exclusively to the adenyl and ribosyl moieties. Both phosphate groups are also ordered. In contrast, the entire ADP-Glc molecule is well ordered in the B subunit active site. The adenyl and ribosyl positions are very similar to those seen in the A and A0 subunits through the region 112e117, which undergoes conformational change upon ATP or ADPGlc binding in A and A0 and is disordered in B and B0 both with and without ADP-Glc in the active site. The two phosphate groups and the glucosyl units are very well ordered in the B active site and adopt positions and conformations similar to that seen in other sugar nucleotide pyrophosphorylase complex structures (Fig. 1.5). There are several direct interactions between the enzyme and the glucosyl unit of ADP-Glc. These include hydrogen bonds between


23

H1 S1

H2

S6

S2

S7

S3

H3

S8

H6

H1

H9

H5

S10 S11

H5

S4

H4

H7

H2

H8

H5

S5

S9

H3

H4 H10

H6

ADPGlc interaction ATP interaction

FIGURE 1.5 Sequence alignment of ADP-Glc PPase from different species. Secondary structure of the potato tuber enzyme is shown above the sequence. Cylinders, helices; straight block arrows, beta strands; curved block arrows, turns in the beta-helix domain. Green stars are residues interacting with ADP-Glc and red stars are residues interacting with ATP. Residues that are identical are shaded in purple. Ana_glgC, Anabaena ADP-Glc PPase; Ath_S, APS1, Arabidopsis thaliana small subunit; Atu_glgC, Agrobacterium tumefaciens ADP-Glc PPase; Bst_glgC, Bacillus stearothermophilus ADP-Glc PPase; Cre_S, Chlamydomonas reinhardtii small subunit; Eco_glgC, Escherichia coli ADP-Glc PPase; Hvu_S_endosp, Hydra vulgaris small subunit; Rsp_glgC, Rhodopseudomonas spheroides ADP-Glc PPase; Stu_L, potato tuber large subunit; Stu_s, potato tuber small subunit; Zma_S_maize endosp, maize small subunit.

E197, S229, D280, and the glucosyl unit (Fig. 1.6). In addition K198 makes a salt bridge with the phosphate group attached to the glucosyl unit. The B subunit undergoes a very large subdomain movement in response to ADP-Glc binding. Two residues, E197 and K198, are critical for binding the glucosyl and phosphate moieties of ADP-Glc; K198 has been characterized as a Glc1P binding residue by site-directed mutagenesis (Fu et al., 1998a) and both are part of a motif present in many sugar nucleotide pyrophosphorylases. These two residues are shifted out of the binding pocket in the B subunit of the

24 PART j ONE Analyzing and Modifying Starch D145 D280

K43

G28 F181

E197 K198 Q148 FIGURE 1.6 Hydrogen bond interactions between ADP-glucose (ADP-Glc) and the ADPglucose pyrophosphorylase (ADP-Glc PPase) catalytic subunit. Protein carbon bonds are green. ADP-Glc carbon bonds are yellow, oxygen atoms are red, nitrogen atoms are blue, and phosphate atoms are purple.

ATP-bound structure, while they are pulled more inward in the unbound B subunit and are pulled in significantly in the ADP-Glc-bound molecule (Fig. 1.6).

3.10 Implication for Catalysis Detailed kinetic studies on ADP-Glc PPase have shown that a sequential biebi mechanism fits the data with ATP binding first (Haugen and Preiss, 1979; Kleczkowski et al., 1993a; Paule and Preiss, 1971). Structural data from two related enzymes, GlmU46 and Rffh44, indicate the presence of a metal ion in the active site. A Co2þ ion (in GlmU) and an Mg2þ ion (in GlmU and Rffh) are located in almost identical locations in the two distinct enzymes when the active sites are aligned. In GlmU, both the Mg2þ and the Co2þ are chelated to two conserved residues (Asp105 and Asn227) and to the two phosphate groups of the product (UDP-N-acetylglucosamine). In Rffh, the Mg2þ is also bound to two conserved carboxylate residues (Asp223 and Asp108) and to the a-phosphate group of TTP. When the ADP-Glc PPase active site is aligned with these active sites, two acidic residues, Asp145 and Asp280, are spatially close to the metal-chelating residues in Rffh and GlmU. Mutation of Asp145 residue to Asn in the potato tuber enzyme and the equivalent Asp142 in the E. coli enzyme results in a reduction in catalytic activity by four orders of magnitude (Frueauf et al., 2001; Jin et al., 2005). Taken together, it is concluded that the metal-mediated catalytic mechanism proposed for RffH and GlmU is also used by ADP-Glc PPase. Also concluded is that the metal ion is chelated by the residues equivalent to D145 and D280 in all ADP-Glc PPases


25

and that the mutational sensitivity of D145 is due to the requirement for metal ion in the reaction. The absolute requirement for a metal ion has been biochemically demonstrated for ADP-Glc PPase from several organisms (Preiss, l996, 2014; Ballicora et al., 2003, 2004; Gomez-Casati et al., 2001). Structural data from GlmU, RmlA, and RffH have shed considerable light on the mechanism of sugar nucleotide pyrophosphorylases, and the similarity between the active site of a-4-ADP-Glc PPase and these enzymes indicates a similar mechanism for all of these enzymes. The structure of the RffH/dTTP complex is particularly informative, because it identifies the GXGXRL loop, which is strongly conserved in all of these enzymes, to be the site of the triphosphate moiety of ATP and shows that the residue equivalent to R33 in the a subunit of ADP-Glc PPase is critical for triphosphate binding. Conformational change of this loop will therefore have profound effects on the activity of the enzyme. In addition to R33, D145, D280, K43, E197, and K198 (potato tuber a4 numbering, Fig. 1.6) are also conserved in the other sugar nucleotide pyrophosphorylases of known structures, are in similar locations in the active sites, and make similar interactions with the sugar nucleotide. Based on results with other sugar nucleotide pyrophosphorylase structures (Brown et al., 1999; Sivaraman et al., 2002), the following is postulated for catalysis. The b- and g-phosphate groups of ATP bind to the conserved loop around R33, folding back over the nucleotide and leaving the space opposite the pyrophosphate entity free for G1P binding. The location of the phosphate group in Glc1P and that of the a-phosphate group of ATP are close to the same positions seen for the phosphate groups in the B subunit ADP-Glc complex. K198 is an absolutely conserved amino acid in ADP-Glc PPases and in other sugar nucleotidyl-transferases. K198 stabilizes the negative charge on the phosphate group of G1P in this model, increasing the nucleophilicity of the O1P atom. Electrostatic repulsions between the negative charges on the phosphate group of G1P and the phosphate groups of ATP are compensated for by chelation between the phosphate groups of ATP, G1P, and Mg2þ. A number of conserved basic side chains also surround the phosphate groups at the active site (R33, K43, K198). Additional counterbalancing charges come from the N-terminal dipole of the helical turn at R33 and the main chain amide nitrogen pocket formed by residues G27 to T32. In fact, R33 makes a close hydrogen bond with a phosphate oxygen atom of ADP-Glc in the ADP-Glc-bound structure. In mutagenesis studies of GlmU (Johnson et al., 1993), mutation of R15 (equivalent to R33 of ADP-Glc PPase) reduced kcat almost 6000-fold, while Km was doubled, confirming that this residue has an important role in orienting and charge compensation of the pyrophosphate group. P36 within this flexible loop adopts a cis-peptide bond. The loop is located at the interface between the N-terminal catalytic domain and the C-terminal b-helix domain within the immediate vicinity of the regulator-binding site, suggesting a possible route for cross-talk between the active site and the allosteric regulation site. While most of the residues in the active site are conserved in the catalytically


inactive large subunit, there are two changes: R33 is a lysine residue and K43 is a threonine residue. Since K43 interacts with ADP-Glc and R33 is critical for proper ATP triphosphate binding, both are likely to be important in catalytically deactivating the large subunit.

3.11 Allosteric Regulation Crystal structure analysis identified three allosteric inhibitor-binding sites, sulfate 1, sulfate 2, and sulfate 3, two of which (sulfate 1 and sulfate 2) have been shown by numerous biochemical experiments to be involved in allosteric regulation of the enzyme. The high sequence homology of the residues in the sulfate 3-binding site suggests that it too may represent an important allostericbinding site. The major question is how these allosteric-binding sites communicate with the active site to affect catalytic activity. Part of the answer to this question comes from careful study of the conformational changes that occur in the GXGXXG loop encompassing amino acids 27e34 on active site binding of either ATP or ADP-Glc. This loop has a similar conformation in all unbound subunits, where the loop is flipped into the active site. A hydrogen bond between K43 and the main chain oxygen atom of either G29 or A30 contributes to the stability of this conformation. Upon binding, however, the loop is forced to move out of the active site to accommodate ATP or ADP-Glc binding and the hydrogen bond is severed. Given that K41 contacts sulfate 1 in the allosteric-binding site, conformational change of K41 on replacement of the inhibitor sulfate or phosphate ion with the activator (3PGA) could lead to conformational change of K43, destabilizing the catalytically incompetent, flipped-in conformation of this loop. K43 is also pointing directly into the active site and makes a hydrogen bond with the adenyl unit in the ADP-Glcbound B subunit (Fig. 1.6). Therefore, conformational change of K43 will also directly affect the active site. Conformational change of this loop could also come directly from movement of the K41 region, since it is only six amino acids away from the 27e34 loop. As discussed above, the motion of the 27e34 loop is correlated with the motion of the 108e116 loop, which also undergoes conformational change on ATP binding. Conformational change in the K41 region upon activator binding could, therefore, be correlated with conformational change of the 108e116 loop, preorganizing the active site for ATP binding. In support of this model, the P52L potato tuber large subunit mutant (equivalent to P36 in the small (catalytic) subunit) is substantially less sensitive to 3PGA activation (Greene et al., 1996b). P36 is a cis-proline residue, and mutation of it is likely to significantly alter communication between the allosteric-binding site and the 27e33 GXGXGRL loop. Several pieces of evidence indicate that intersubunit motion may also play a role in allosteric regulation of the enzyme. Removal of the C12 disulfide bond, either by mutation or by reduction, results in an enzyme that is less activated by 3PGA (Ballicora et al., 1999, 2000) indicating that intersubunit interaction


27

between the catalytic subunits is an important part of the allosteric mechanism. Release of this constraint could increase flexibility and result in more intersubunit motion. Sulfate 3, which bridges two subunits, may inhibit intersubunit motion, resulting in a more inhibited enzyme. The binding of ADP-Glc to the B subunit and concomitant motion of the 168e231 subdomain, which is required for interaction of E197 and K198 with ADP-Glc, results in differences in the subunit interface. Intersubunit motion may, therefore, drive the motion of this subdomain upon activation, preorganizing the active site for binding of G1P. The complete mechanism of the allosteric regulation of ADP-Glc PPase cannot be deduced until the conformation of the enzyme in its activated state is elucidated. Nonetheless, current structural results strongly support previous data on allosteric regulation of this enzyme, and provide some insights into how the binding of allosteric effectors could affect catalysis. The crystal structure of the homotetrameric bacterial ADP-Glc PPase from Agrobacterium tumefaciens has also been reported (Cupp-Vickery et al., 2008). Similarities and differences of the bacterial enzyme structure to the potato ADPGlc PPase catalytic small subunit were noted (Cupp-Vickery et al., 2008). Recent data have indicated other highly conserved amino acids in the small (catalytic) subunit of the potato ADP-Glc PPase in being important in eliciting the 3PGA activation (Figueroa et al., 2013). Gln74 and Trp116 were shown to be in critical loop regions adjacent to the ATP-binding site and important for allosteric activation in E. coli ADP-Glc PPase. Mutation of residues Gln74 and/or Trp113 of the E. coli enzyme to Ala abolished fructose-1,6-bis-P activation of the enzyme (Ballicora et al., 2007; Figueroa et al., 2011). The Gln74 and Trp113 residues are highly conserved in the ADP-Glc PPases from many sources and the equivalent amino acids in the small subunit of the potato tuber enzyme are Gln75 and Trp116 (Figueroa et al., 2013). Potato tuber small subunits mutant Q75A or W116A had reduced activation by 3PGA about fivefold (Figueroa et al., 2013). In Anabaena, Q58A and W96A ADP-Glc PPase mutants reduced 3PGA activation about 4.5-fold. The apparent affinity increase of the substrates, S0.5, induced by the activator was also affected by the mutation of Q75A. S0.5 of the substrates and Mg2þ were not lowered by the presence of activator in the mutated enzymes Q75 or W116A. Pi inhibition was also affected. The I0.5 values in the mutated enzymes were about five- to eightfold higher than the WT enzyme in the presence of 3PGA. In other words, sensitivity to inhibition was less in the presence of an activator. Gln75 and Trp116 of the potato tuber enzyme were thus important residues with respect to the function of 3PGA activation.

3.12 Supporting Data for the Physiological Importance of Regulation of ADP-Glucose Pyrophosphorylase The reaction catalyzed by plant ADP-Glc PPases is activated by 3PGA and inhibited by orthophosphate and is an important step for regulation of starch


synthesis in higher plants as well as glycogen synthesis in the cyanobacteria (see reviews Preiss and Romeo, l989; Preiss, l996, 2014; Ball and Morell, 2003). In addition it has been shown that in vitro the potato tuber ADP-Glc PPase activity can also be regulated by its redox state (Fu et al., 1998b; Ballicora et al., 1999, 2000) as well as in vivo (Geigenberger, 2011; Tiessen et al., 2002, 2003). Considerable experimental evidence is available to support the concept that ADP-Glc PPase is an important regulatory enzyme for the synthesis of plant starch. A class of C. reinhardtii starch-deficient mutants have been isolated and shown to contain an ADP-Glc PPase not activated by 3PGA (Ball et al., 1991; Iglesias et al., 1994). Evidence for the allosteric regulation by ADP-Glc PPase being pertinent in vivo has also been obtained with A. thaliana (Lin et al., 1988a,b). An A. thaliana mutant, TL25, lacked both subunits and accumulated less than 2% the limits of detection of starch seen in the normal plant (Lin et al., 1988a), which would indicate that starch synthesis is almost completely dependent on the synthesis of ADP-Glc. Mutant, TL46, also starch-deficient, lacked the regulatory 54 kDa subunit (Lin et al., 1988b). The mutant had only 7% of the WT ADP-Glc PPase activity and it was shown that in optimal photosynthesis the starch synthetic rate of the mutant, TL46, was only 9% of that of the WT (Neuhaus and Stitt, 1990). At low light, starch synthesis in TL46 was only 26% of the WT rate (Neuhaus and Stitt, 1990). These observations support the idea that regulation of ADP-Glc PPase by 3PGA and Pi is physiologically pertinent for the adjustment of leaf starch synthesis. In addition, a maize endosperm mutant ADP-Glc PPase that is less sensitive to inhibition by Pi than the WT enzyme, had 15% more dry weight and more starch than the normal endosperm (Giroux et al., 1996). Also, genetic manipulation of ADP-Glc PPase activity in potato tuber (Stark et al., 1992) and wheat endosperm (Smidansky et al., 2002), leads to increase of starch. This also has been shown for rice (Smidansky et al., 2003) as well as for cassava root (Uzoma et al., 2006). These results confirm that ADP-Glc synthesis is rate limiting for starch synthesis. Thus, data seen with the allosteric mutant ADP-Glc PPases of C. reinhardtii (Ball et al., 1991; Iglesias et al., 1994), maize endosperm (Giroux et al., 1996), and Arabidopsis (Lin et al., 1988a,b) present strong evidence that in vitro allosteric effects are functional in vivo. Other data also agree with the view that the allosteric regulatory properties of the Arabidopsis leaf ADP-Glc PPase are important for adjustment of leaf starch synthesis in different photoperiods (Mugford et al., 2014).

3.13 Mechanism of Activation of Plant ADP-Glc PPases by Thioredoxin ADP-Glc PPase from potato tuber has an intermolecular disulfide bridge linking the two small subunits by the Cys12 residue and can be activated by


29

reduction of the Cys12 disulfide linkage (Geigenberger, 2011; Ballicora et al., 1999, 2000; Tiessen et al., 2002, 2003). At low concentrations (10 mM) of 3PGA, spinach leaf reduced both thioredoxin f and m and activated the enzyme. Fifty percent activation was seen for 4.5 and 8.7 mM reduced thioredoxin f and m (Ballicora et al., 2000). The activation was reversed by oxidized thioredoxin. Cys12 is conserved in the ADP-Glc PPases from plant leaves and other tissues except for the monocot endosperm enzymes. In photosynthetic tissues, this reduction may also be physiologically pertinent in the fine regulation of the ADP-Glc PPase. Both potato tuber and potato leaf ADP-Glc PPases are plastidic; the leaf enzyme in the chloroplast and the tuber enzyme in the amyloplast (Kim et al., 1989). The ferredoxinethioredoxin system is located in the chloroplast and thus, with photosynthesis, reduced thioredoxin is formed and would activate the leaf ADP-Glc PPase. At night, oxidized thioredoxin is formed and would oxidize and inactivate the ADP-Glc PPase. This activation/inactivation process during the light/dark cycle allows a fine tuning and dynamic regulation of starch synthesis in the chloroplasts. Thioredoxin isoforms are present in many different subcellular locations of plant tissues: cytosol, mitochondria, chloroplasts, and even nuclei (Balmer et al., 2006; Jacquot et al., 1997) and are also present in amyloplasts (Balmer et al., 2006). It has been shown that in potato tubers from growing plants, starch synthesis is inhibited within 24 h after detachment (Tiessen et al., 2002, 2003) despite having high in vitro ADP-Glc PPase activity and high levels of substrates, ATP and Glc 1-P and an increased 3PGA/Pi ratio. In the detached tubers, the small subunit in nonreducing SDSPAGE is solely in dimeric form and relatively inactive in contrast to the enzyme form of growing tubers where it was composed as a mixture of monomers and dimers. The detached tuber enzyme had a great decrease in affinity for the substrates as well as for the activator. Treatment of tuber slices with either DTT or sucrose reduced the dimerization of the ADP-Glc PPase small subunit and stimulated starch synthesis in vivo. A strong correlation between sucrose content in the tuber and the reduced/activated ADP-Glc PPase was noted. These results indicate that reductive activation, observed in vitro of the tuber ADP-Glc PPase is important or regulating starch synthesis (Fu et al., 1998b; Ballicora et al., 1999, 2000). Redox regulation of ADP-Glc PPase has also been observed for the rice endosperm ADP-Glc PPase (Tuncel et al., 2014a). Of interest is that the Cys residues of the cereal cytosolic ADP-Glc PPase subunits are not conserved. The redox regulation occurs with the Cys residues present in the large subunits of the rice endosperm ADP-Glc PPase (Tuncel et al., 2014a). Site-directed mutagenesis of conserved Cys residues, C47 and C58 to Ala at the N-terminal of the large subunit showed that 3PGA activation affinity was decreased about 3.5- to 4-fold (Tuncel et al., 2014a). The Cys47A and Cys58A mutants did not significantly alter the rice enzyme sensitivity to inhibitor, Pi, or the apparent affinities for the substrates. These results are similar to those


observed with the reduced potato ADP-Glc PPase except that the apparent affinity (S0.5 value) for ATP of the reduced potato enzyme was twofold higher than the oxidized form (Fu et al., 1998b; Ballicora et al., 1999, 2000).

4. CHARACTERIZATION OF ADP-GLC PPASES FROM DIFFERENT PLANT SOURCES Table 1.1 summarized the kinetic and regulatory properties of purified potato tuber and spinach leaf ADP-Glc PPases. As reviewed (Preiss, 2010; Ballicora et al., 2003) the properties of many other plant, algal, and cyanobacterial ADP-Glc PPases are similar. However, some ADP-Glc PPases in plant reserve tissue show some differences with respect to allosteric properties and their nonplastidic location. These are summarized below in the text as well as in Table 1.7.

4.1 Chlamydomonas reinhardtii The ADP-Glc PPase from different green algae was characterized as being highly regulated by the 3PGA/Pi ratio (Sanwal and Preiss, l967; Ball et al., 1991; Iglesias et al., 1994). The enzyme from C. reinhardtii has been purified to apparent homogeneity (81 mmol/min/mg) and characterized as a heterotetramer (a2b2), typical of the ADP-Glc PPase from plants (Iglesias et al., 1994). A starch-deficient mutant of C. reinhardtii was shown to contain an ADP-Glc PPase that could not be activated by 3PGA, but which exhibited sensitivity to Pi inhibition similar to that of the WT enzyme (Ball et al., 1991).

4.2 Barley The barley leaf ADP-Glc PPase has been purified to homogeneity (69.3 mmol/ min/mg) and it shows high sensitivity toward activation by 3PGA and inhibition by phosphate (Sanwal et al., l968; Kleczkowski et al., 1993a). Substrate kinetics and product inhibition studies in the synthesis direction suggested a sequential isoordered biebi kinetic mechanism (Kleczkowski et al., 1993a). ATP or ADP-Glc bind first to the enzyme in the synthesis or pyrophosphorolysis direction, respectively, similar to the E. coli enzyme (Haugen and Preiss, 1979). However, partially purified barley endosperm ADP-Glc PPase was shown to have low sensitivity to the regulators 3PGA and Pi (Kleczkowski et al., 1993b). The enzyme was activated only 1.3-fold in direction of ADP-Glc synthesis. Pi at high concentrations (20 mM) inhibited only 16% and 3PGA did not reverse the inhibition. However, 3PGA lowered the S0.5 for ATP about 1.6-fold. Also in the absence of 3PGA the ATP saturation curve was sigmoidal with a Hill coefficient n of 2.1. In the presence of 3PGA the ATP saturation curve changed from a sigmoidal to a hyperbolic form (Kleczkowski et al., 1993b). At 0.1 mM

TABLE 1.7 Kinetic Constants of ADP-Glc PPases of Cyanobacteria, Green Algae, and Higher Plants ADP-Glc PPase (Source)

Effector/Substrate

Kinetic Constant A0.5/I0.5 (mM)

n

Fold-Activation

Chlamydomonas (Ball et al., 1991; Iglesias et al., 1994)

3PGA

0.23

1.3

15

Pi (3PGA)

0.054

1.0

Pi (þ2.5 mM 3PGA)

0.53

1.7

Glc-1-P (3PGA)

0.22

1.7

Glc-1-P (þ3PGA) 0.03

0.03

1.2

ATP (3PGA)

0.48

1.2

ATP (þ3PGA)

0.08

1.3

ATP (3PGA)

0.48

1.2

ATP (þ3PGA)

0.08

1.3

Glc-1-P (3PGA)

0.22

1.7

Glc-1-P (þ3PGA)

0.03

1.2

3PGA

0.005

1.0

Pi (3PGA)

0.025

1.0

Pi (þ2.5 mM 3PGA)

0.53

1.7

Glc-1-P (3PGA)

0.33

1.0

Glc-1-P (þ3PGA)

0.11

1.0

ATP (3PGA)

1.00

1.0

ATP (þ3PGA)

0.08

1.0

Barley endosperm (Kleczkowski et al., 1993b) (none)


Barley leaves (Sanwal et al., l968; Kleczkowski et al., 1993a)

1.0

>20

31 Continued

TABLE 1.7 Kinetic Constants of ADP-Glc PPases of Cyanobacteria, Green Algae, and Higher Plantsdcont’d Effector/Substrate


n

Fold-Activation

Maize endosperm (Plaxton and Preiss, 1987)

3PGA

0.12

1.0

27.5

3PGA (þ1 mM Pi)

1.2

1.5

3PGA (þ8 mM Pi)

3.6

2.6

Pi (3PGA)

Insensitive to Pi inhibition

Pi (þ1.0 mM 3PGA)

0.44

1.7

Pi (þ10 mM 3PGA)

9.8

1.7

Glc-1-P (3PGA)

0.67

0.9

Glc-1-P (þ3PGA)

0.03

1.0

ATP (3PGA)

0.84

1.3

ATP (þ1 mM 3PGA)

0.11

1.0

3PGA

0.2

NR

Pi (þ0.5 mM 3PGA)

0.7

NR

Glc-1-P (þ3PGA)

0.086

NR

ATP (þ3PGA)

0.12

NR

3PGA

0.09

NR

Pi (3PGA)

0.08

NR

Pi (þ2.5 mM 3PGA)

0.88

NR

Glc-1-P (þ3PGA)

0.10

NR

ATP (þ3PGA)

0.11

NR

Tomato fruit (Chen and Janes, 1997; Petreikov et al., 2010)

Tomato leaf (Sanwal et al., l968)

>100

5.4


ADP-Glc PPase (Source)

Wheat endosperm (Gomez-Casati and Iglesias, 2002)

Wheat leaf (Gomez-Casati and Iglesias, 2002)

Pi

0.7

1.3

3PGA (0.7 mM Pi)

0.81

1.0

3PGA (1.5 mM Pi)

1.51

1.4

3PGA (5 mM)

3.33

2.5

Glc-1-P

0.092

1.0

ATP

0.12

NR

Fru-6-P

No effect

Fru-6-P (þ0.7 mM Pi)

2.5

NR

3PGA

0.01

1.0

3PGA (þ2.0 mM Pi)

1.9

2.3

Pi (þ3PGA)

0.2

1.2

Glc-1-P

0.45

1.1

Glc-1-P (þ3PGA)

0.08

1.0

ATP

0.73

1.2

ATP (þ3PGA)

0.22

1.1

3PGA

0.65

NR

Pi (0.5 mM 3PGA)

0.19

NR

Pi (0.5 mM 3PGA)

0.40

NR

Glc-1-P (þ5 mM 3PGA)

0.17

NR

ATP (þ5 mM 3PGA)

0.18

NR

11

43

33

No effect


Rice endosperm (Sikka et al., 2001)

3PGA

Continued

TABLE 1.7 Kinetic Constants of ADP-Glc PPases of Cyanobacteria, Green Algae, and Higher Plantsdcont’d Effector/Substrate


n

Fold-Activation

Rice leaf (Sanwal et al., l968)

3PGA

0.18

NR

23

Pi (3PGA)

0.06

NR

Pi (þ3PGA)

0.27

NR

3PGA

0.59

25

Pi (0.6 mM 3PGA)

1.48

Pi (6.0 mM 3PGA)

9.47

ATP (10 mM 3PGA)

0.58

Glc-1-P (10 mM 3PGA)

0.41

3PGA

2.54

Pi (0.6 mM 3PGA)

24.7

Pi (6.0 mM 3PGA)

16.0

ATP (10 mM 3PGA)

0.5

Glc-1-P (10 mM 3PGA)

1.32

3PGA

0.81

2.0

Pi (3PGA)

0.095

1.0

Pi (þ2.5 mM 3PGA)

0.57

2.2

Glc-1-P (3PGA)

0.18

1.1

Glc-1-P (þ3PGA)

0.05

1.1

ATP (3PGA)

3.2

2.2

ATP (þ3PGA)

0.80

1.0

Rice endosperm (Lee et al., 2007; Tuncel et al., 2014b; recombinant WTS2bL2)

WT S2b homo-tetramer

Synechocystis (Iglesias et al., 1991; Levi and Preiss, 1976)

13


ADP-Glc PPase (Source)

Anabaena (Iglesias et al., 1991)

Synechococcus (Levi and Preiss, 1976) 6301

0.12

1.0

Pi (3PGA)

0.044

1.0

Pi (þ2.5 mM 3PGA)

0.46

1.7

Glc-1-P (3PGA)

0.13

1.2

Glc-1-P (þ3PGA)

0.08

1.0

ATP (3PGA)

1.55

2.2

3PGA

0.11

1.0

Pi (3PGA)

0.072

1.0

Pi (þ1.0 mM 3PGA)

1.0

3.9

Glc-1-P (3PGA)

0.48

1.0

Glc-1-P (þ1 mM 3PGA)

0.14

1.0

ATP (3PGA)

1.35

1.0

ATP (þ1 mM 3PGA)

0.30

1.0

17

25


3PGA

35


ATP the activation by 3PGA was about fourfold (Kleczkowski et al., 1993b) and phosphate 6.0 mM reversed the effect. A recombinant enzyme with a (His) 6-tag from barley endosperm was expressed using the baculovirus insect cell system (Rudi et al., 1997). It shows no sensitivity to regulation by 3PGA and Pi. However, the enzyme was assayed at saturating concentration of substrates and only in the pyrophosphorolysis direction. For ADP-Glc PPases the synthetic direction is more sensitive to activation. When the recombinant enzyme without the (His)6 tag is expressed in insect cells, the heterotetrameric form still was not activated by 3PGA nor inhibited by Pi at saturating levels of substrates (Rudi et al., 1997). Whether 3PGA had any effect on the affinity for the substrates as shown in the enzyme purified from the endosperm was not reported. Of interest is that the small (catalytic) subunit when expressed alone is very responsive to the allosteric effectors, 3PGA and Pi (Doan et al., 1999). This would suggest that the large subunit in barley endosperm desensitizes the small subunit to activation by 3PGA and inhibition by Pi and that is opposite seen for large subunits of potato tuber (Ballicora et al., 1995, 1998) and Arabidopsis (Kavakli et al., 2002). The small subunit from barley is encoded by a single gene that can be transcribed to form two transcripts. One is found highly expressed only in endosperm while the other is expressed both in endosperm and in leaves (Thorbjørnsen et al., 1996a). Moreover, the barley ADP-Glc PPase large and small subunits have been expressed in E. coli to yield active enzyme with catalytic and regulatory properties similar to the native barley enzyme (Luo and Kleczkowski, 1999). Of interest is that the starch-deficient barley mutant Risø16 is deficient in ADP-Glc PPase due to a large deletion in the catalytic small subunit (Johnson et al., 1993). It only contained 20% of the normal ADP-Glc PPase activity. The other starch synthetic enzymes, SS and BE, were close to normal. The ADP-Glc PPase activity in normal barley, Bomi, was divided in content as 17.6% in the plastid with the rest of activity present in the cytosol. Mutant Risø16, however, had all of its ADP-Glc PPase activity in the plastid. The mutant is therefore greatly reduced in, or lacks entirely, the cytosolic form of ADP-Glc PPase activity. The cytosolic ADP-Glc PPase is obviously the major source of glucose for starch in barley endosperm. The cytosolic and plastidial ADP-Glc PPase small subunits are encoded in separate genes.

4.3 Transport of Cytosolic ADP-Glucose into Endosperm in Cereal Grains In green algae and in leaf cells of higher plants ADP-Glc PPase was first shown to reside in the chloroplast (Preiss, 2009; Zeeman et al., 2010; Streb and Zeeman, 2012). However later, in plastids isolated from barley (Johnson et al., 1993; Thorbjørnsen et al., 1996b) and maize endosperm (Denyer et al., 1996a), the existence of two ADP-Glc PPases, a plastidial form and a major cytosolic


37

form were found. Indeed, 95% of the maize endosperm ADP-Glc PPase activity is reported to be extraplastidial (Denyer et al., 1996a). Cytosolic forms of ADP-Glc PPase have also been found in wheat (GomezCasati and Iglesias, 2002; Tetlow et al., 2003) and rice (Sikka et al., 2001). Since starch synthesis occurs in plastids, it was further proposed that in cereal endosperms, synthesis of ADP-Glc in the cytosol requires transport of the ADP-Glc across the amyloplast envelope (Johnson et al., 1993). An ADP-Glc transporter known as BT1 protein encoded by the BT1 gene was isolated from endosperms of maize in 1991 (Sullivan et al., 1991). The protein ranges from 39 to 44 kD and is located in the amyloplast membrane (Sullivan et al., 1991; Cao et al., 1995; Shannon et al., 1998; Kirchberger et al., 2007). The protein has been also isolated from barley (Patron et al., 2004) wheat (Bowsher et al., 2007) and rice (Cakir et al., 2016). Mutants of the BT1 proteins in wheat, maize, and rice endosperms caused high reductions in their starch contents. Transport/exchange studies of maize BT1 showed ADP-Glc counterexchanged with ADP (Kirchberger et al., 2007) while in wheat the counterexchange of ATP was either with ADP or AMP (Bowsher et al., 2007). Rice was similar to wheat in that ADP-Glc exchange could be with either ADP or AMP (Cakir et al., 2016).

4.4 Pea Embryos ADP-Glc PPase from developing pea embryos was purified to apparent homogeneity (56.5 U/mg) and it was found to be activated up to 2.4-fold by 1 mM 3PGA in the ADP-Glc synthesis direction (Hylton and Smith, 1992). In pyrophosphorolysis, 1 mM Pi inhibited the enzyme 50% and 3PGA reversed this effect. The effect of 3PGA or Pi on the S0.5 for ATP was not analyzed. A mutation of the pea rb locus reduces the starch content about 8.5-fold in developing pea embryos (Smith et al., 1989). The ADP-Glc PPase activity was at least 10-fold lower in the mutant rbrb embryo peas than in the RbRb embryo peas (Smith et al., 1989). The activity of the other starch biosynthetic enzymes, SS and BE, were similar in the mutant embryo peas. Thus the decrease in starch content was attributed to the deficiency in ADP-Glc PPase activity (Smith et al., 1989).

4.5 Maize Endosperm Partially purified maize endosperm ADP-Glc PPase (34 U/mg) was found to be activated by 3PGA and Fru-6-P (25- and 17-fold, respectively) and inhibited by Pi (Plaxton and Preiss, 1987). Of interest is that the enzyme in the absence of 3PGA is relatively insensitive to Pi inhibition. However, in the presence of 1 mM 3PGA, the enzyme is 50% inhibited by 0.44 mM Pi (Plaxton and Preiss, 1987). In presence of 10 mM 3PGA 50% inhibition requires 9.8 mM Pi. Thus maize endosperm ADP-Glc PPase activity can be regulated by the ratio 3PGA/Pi concentrations.


The heterotetrameric endosperm enzyme has been cloned and expressed in E. coli and its regulatory properties were compared to an isolated allosteric mutant having an ADP-Glc PPase less sensitive to Pi inhibition (Giroux et al., 1996). Increased seed weight of 11%e18% was noted in the mutant (Giroux et al., 1996). As indicated above, the increase of seed weight (starch) noted in the mutant maize endosperm ADP-Glc PPase insensitive to Pi inhibition would support the importance of the allosteric effects of 3PGA and Pi in vivo. Also as indicated above, it is believed that the major endosperm ADP-Glc PPase isoform is located in the cytosol (Thorbjørnsen et al., 1996b; Denyer et al., 1996a). The interactions between the maize endosperm large subunit, SH2, and the small subunit BT2 have been studied (Greene and Hannah, 1998). In the null large Sh2 mutant, the BT2 subunit remains as a monomer in the endosperm while the SH2 subunits are in a 100 kD complex (dimer?) in the bt2 null mutant. Interactions between SH2 and BT2 were observed when the subunits were expressed in the yeast two-hybrid system (Greene and Hannah, 1998). Truncation of the N or C termini of either subunit eliminated the subunit interactions (Greene and Hannah, 1998). The cereal ADP-Glc PPases of maize and wheat endosperms are heat labile (Boehlin et al., 2014). Attempts have been made to improve heat stability of the maize endosperm enzyme by mutations of selected amino acids in the maize ADP-Glc large subunit using a phylogenetic approach. Sites affecting heat stability with enhanced activity at 37 C and 55 C as well as increasing activator affinity or enhancing activity in the absence of the activator, 3PGA, were identified and combined into one large subunit gene (Park and Chung, 1998). This gene labeled Sh2-E coupled with the small catalytic subunit gene resulted in an enzyme with exceptional heat stability with high-catalytic rates at increased temperatures and decreased Km values for both substrates in absence of the activator (Boehlin et al., 2014).

4.6 Tomato Fruit Three clones encoding different ADP-Glc PPase isoforms were isolated from a cDNA library from tomato plants (Park and Chung, 1998). Sequence comparison and phylogenetic analysis revealed that all of them represent different types of the large subunits of the enzyme. The three isoforms are suggested to be organ-specific in their expressions. In another study, four cDNA clones were isolated by PCR, three corresponding to large subunits and one to a small subunit (Chen et al., 1998a). Four tomato ADP-Glc PPase transcripts were detected in fruit and leaves. The intracellular location of ADP-Glc PPase in developing pericarp of tomato was investigated by immunolocalization with highly specific antitomato fruit ADP-Glc PPase antibody and the enzyme was localized in cytoplasm as well as in plastids (Chen et al., 1998b). In tomato leaf cells, the ADP-Glc PPase immunolocalization was restricted to the chloroplast (Chen and Janes, 1997).


39

When the enzyme from tomato fruit was purified to apparent homogeneity (45 U/mg), multiple forms were detected by two-dimensional electrophoresis and immunological studies. Three polypeptides corresponded to large subunits, and two corresponded to small subunits. The purified tomato fruit enzyme was highly sensitive to 3PGA/Pi regulation (Chen and Janes, 1997). The tomato (Solanum lycopersicon) genome contains a single gene encoding for the small subunit and three genes for large subunit proteins. Large subunit L1 is expressed in sink tissue while L3 is expressed in source tissue. They are expressed in both a tissue and temporally specific manner. The allosteric contributions of the different large subunits were compared by expressing each one in E. coli, in conjunction with the small subunit and individually, and the resulting enzyme activity was determined (Petreikov et al., 2010). Results indicate different kinetic characteristics of the tomato L1/ S and L3/S heterotetramers. L1/S had lower affinity for the activator 3PGA and was less sensitive to Pi inhibition than L3/S. The recombinant L3 protein was also active when expressed alone. Size exclusion and immunoblotting showed that it functioned as a monomer. L3, however, was less sensitive to 3PGA activation or Pi inhibition. Subunit interaction modeling pointed to two amino acids potentially affecting subunit interactions. However, directed mutations did not impact on subunit tetramerization. The results indicate a hitherto unknown active role for the large subunit in tomato in synthesis of ADP-Glc. The nature of the catalytic and regulatory sites in L3 remains to be determined.

4.7 Wheat Ainsworth et al. (1993, 1995) isolated two cDNA clones that encode a large and a small subunit from wheat endosperm ADP-Glc PPase. The deduced amino acid sequence has high degree of homology with the small subunits of ADP-Glc PPases from rice (about 90% identical amino acids), potato (86% of identical amino acids) and had conserved sequence elements thought to represent the substrate binding and allosteric activator sites. Transcripts from both gene sets accumulate to high levels in the endosperm during grain development with the majority of the expression in the endosperm (Burton et al., 2002). The results are consistent with data from other graminaceous endosperms, indicating distinct plastidial and cytosolic isoforms of ADP-Glc PPase composed of different subunits. In another study, the ADP-Glc PPase from wheat endosperm has been purified and characterized (Gomez-Casati and Iglesias, 2002). It showed novel regulation (Table 1.7). The enzyme was insensitive to activation by 3PGA. However, it was inhibited by Pi with an I0.5 of 0.75 mM. ADP (I0.5 ¼ 3.2 mM) and fructose-1,6-bis-P (I0.5 ¼ 1.5 mM) also were inhibitors. All these inhibitions were reversed by 3PGA (Gomez-Casati and Iglesias, 2002). The purified wheat leaf ADP-Glc PPase, however, was characterized as a typical


3PGA/Pi-regulated enzyme with an A0.5 of 10 mM and I0.5 of 20 mM (Gomez-Casati and Iglesias, 2002). Two isoforms of ADP-Glc PPase have been reported in extracts of wheat endosperm (Tetlow et al., 2003). One extract from the amyloplastidial fraction was activated twofold by 3PGA and inhibited by Pi.

4.8 Rice Endosperm and Leaf Rice endosperm ADP-Glc PPase has been purified to apparent homogeneity (42.9 U/mg) (Nakamura and Kawaguchi, 1992). Electrophoretic analyses detected multiple isoforms, and no kinetic characterization was reported. Another report shows that the purified endosperm ADP-Glc PPase is activated by 3PGA (>40-fold) and inhibited by Pi (Sikka et al., 2001). The inhibition was reversed by 3PGA. The allosteric kinetic constants (Lee et al., 2007; Tuncel et al., 2014b) are given in Table 1.7. As indicated previously 90% of the ADP-Glc PPase is extraplastidic (cytosolic) while 10% was associated with the amyloplast (Sikka et al., 2001; Cakir et al., 2016). The rice leaf ADP-Glc PPase is highly activated by 3PGA (14.5-fold) and the A0.5 was 0.18 mM (Sanwal et al., l968). Pi was a strong inhibitor as 50% inhibition in the absence of 3PGA occurred with 0.06 mM Pi. In the presence of 1 mM 3PGA, Pi inhibition was with 0.27 mM Pi (Sanwal et al., l968). The rice endosperm enzyme as other plant ADP-Glc PPases is also a heterotetramer composed of two regulatory (large) subunits as well as two catalytic (small subunits) (Tuncel et al., 2014b). Lee et al. (Tuncel et al., 2014b) reported that the rice gene ADP-Glc PPase family consisted of two genes encoding two small subunits and four genes encoding large subunits. Also determined is that the leaf-preferential products of the small and large subunit genes were plastid-targeted proteins while two of the genes, encoded a small subunit and a large subunit that were localized in the cytosol. Mutation or lesion of either of the cytosolic gene products resulted in the rice having a shrunken size endosperm due to decreased starch amounts of about 69% e78%, respectively (Tuncel et al., 2014b). This is in agreement with the view that the cytosolic ADP-Glc PPase is responsible for the major part of starch synthesize in the endosperm. Lee et al. (Tuncel et al., 2014b) propose on the basis of their finding an interesting model suggesting the roles and function for the different rice ADP-Glc PPase small and large subunits in the rice leaf and endosperm tissues during different stages of rice endosperm development. In leaf, the small subunit, labeled OsAGPS2a, and large subunit, labeled OsAGPL3, is the main heterotetramer ADP-Glc PPase complex largely functional in synthesis of leaf starch in chloroplasts. In the early stage of developing rice endosperm (1e4 days after flowering (DAF)) the major ADP-Glc PPase subunits, OsAGPS1 (small subunit) and OsAGPL1 (large subunit) is the major ADP-Glc PPase complex (and in the amyloplast) involved in starch synthesis while in


41

the cytosol the ADP-Glc PPase complex composed of OsAGPS2b (small subunit) and OsAGPL2 plays a minor role. During the middle (4e10 DAF) to late stages (10e20 DAF) of the developing endosperm, the cytosolic OSAGPS2b/OsAGPSL2 complex is now the major ADP-Glc PPase functioning in ADP-Glc synthesis while the endosperm OSAGPS1/OsAGPSL1 complex has a minor role. Since other cereals have ADP-Glc PPases isozymes in both the cytosol and endosperm it would be of interest to know whether a similar change in sequence of ADP-Glc PPases isozyme function occurs in their development. Tuncel et al. (2104b) also report the ADP-Glc PPase activity of various mutants and WT enzyme. The small subunit mutant enzyme activity was only 5% of the WT enzyme. The null large subunit (L2 null) mutant had 40% activity of WT while a missense mutant (T139I) had 60%. The seed weights (indicative of starch content) of the two large mutant subunit rice seeds were decreased 30%e73%. These results do indicate an important in vivo function for the cytosolic ADP-Glc PPase and large subunits in starch synthesis. The kinetics and regulatory properties of the recombinant cytosolic rice ADP-Glc PPase, isoforms OSAGPS2b (small subunit)/OsAGPL2 (large subunit) have been studied (Tuncel et al., 2014b; Hwang et al., 2005). As seen in Table 1.7 the kinetic values of the recombinant enzyme are in good agreement with those of the native enzyme partially purified from seed extracts (Sikka et al., 2001). Of interest is that the S2bWT homotetramer also showed about fourfold lower affinity for 3PGA and threefold lower affinity for the substrate Glc 1-P (Salamone et al., 2000). In addition, the S2bWT is extremely resistant to Pi inhibition at both low and high 3PGA concentrations. These results show that the L2 subunit is essential for optimal allosteric regulation and catalytic activity of the rice endosperm ADP-Glc PPase.

4.9 Arabidopsis thaliana The A. thaliana ADP-Glc PPase is discussed in Section 3.2. Table 1.2 presents the regulatory properties (3PGA activation and interaction with Pi) inhibition of the recombinant ADP-Glc PPases having complexes of the small catalytic enzyme in complex with four different large subunits. Table 1.3 describes the kinetic parameters for the substrates, ATP and Glc-1-P for the different ADP-Glc heterotetrameric complexes of the small subunit and four different Arabidopsis large subunits. As indicated, there is only one conserved small (catalytic) subunit and several large (regulatory) subunits that are distributed in different parts of the plant (Ventriglia et al., 2008; Crevilleń et al., 2003, 2005). As indicated this is of physiological importance. Expression of different large subunits in different plant tissues confers distinct allosteric properties to the ADP-Glc PPase required for the specific accumulation of starch in different parts of the plant (Crevilleń et al., 2003, 2005).


Table 1.7 also summarizes the properties of the ADP-Glc PPases of the cyanobacteria, Synechocystis (Iglesias et al., 1991) and Anabaena (Iglesias et al., 1991) and the blue-green bacterium, Synechococcus 6301 (Levi and Preiss, 1976).

5. DIFFERENCES IN INTERACTION BETWEEN 3PGA AND PI IN DIFFERENT ADP-GLC PPASES As indicated above, almost all plant ADP-Glc PPases are activated by 3PGA and inhibited by Pi. The nature of interaction between the allosteric activator and inhibitor effectors may vary for the different ADP-Glc PPases. As previously reviewed (Ballicora et al., 2003), four patterns of interactions can be distinguished between 3PGA and Pi. The four patterns are summarized in Table 1.8. Pi and 3PGA affect the enzyme separately, and increasing concentrations of 3PGA can reverse or antagonize Pi inhibition for most ADP-Glc PPases in Group A. A second pattern, exhibiting distinctive regulatory properties, is seen in Group B ADP-Glc PPases, which are found in the reserve tissues of some cereals. The enzymes from pea embryos (Hylton and Smith, 1992), barley endosperm (Kleczkowski et al., 1993b; Rudi et al., 1997), and wheat endosperm (Gomez-Casati and Iglesias, 2002) may be considered as relatively insensitive to regulation; mainly, they exhibit no activation by 3PGA. However, full characterization of purified ADP-Glc PPase of wheat endosperm shows that the enzyme is under regulation by the coordinate action of various metabolites (Gomez-Casati and Iglesias, 2002). The wheat endosperm enzyme is allosterically inhibited by Pi, ADP, and Fru-1,6-bis-P (Table 1.7). In all cases, inhibition is reversed by 3PGA and fructose-6-P which individually, in the absence of the inhibitors, have no effect on the enzyme’s activity (Gomez-Casati and Iglesias, 2002). However, activity is affected by a specific 3PGA/Pi ratio in a unique manner (Table 1.8). Indeed, Pi inhibition is the prime signal. The importance of Pi inhibition on the wheat endosperm ADP-Glc PPase and its significance on in vivo starch accumulation and seed yield has been shown via plant genetic transformation (Smidansky et al., 2002). The reported in vitro properties of the wheat endosperm enzyme (Gomez-Casati and Iglesias, 2002) are congruent with the fact that Pi limits starch biosynthesis in crop plants and suggest that levels of several metabolites can alter the biosynthetic pathway patterns in the endosperm tissue (Plaxton and Preiss, 1987). Another variation of 3PGA activation interaction with Pi inhibition is seen in Group C enzymes, represented by CAM plant leaf ADP-Glc PPases from Hoya carnosa and Xerosicyos danguyi (Singh et al., l984) and from maize endosperm (Plaxton and Preiss, 1987; Table 1.7). The enzymes can be activated about 10- to 25-fold by 2 mM 3PGA, but in the absence of 3PGA they are insensitive to Pi inhibition. In the absence of 3PGA, the maize endosperm enzyme is only inhibited 20% by 10 mM Pi, and the CAM plant leaf enzymes

TABLE 1.8 Different Interaction Patterns 3PGA Activation and Pi Inhibition of Plant ADP-Glc PPases Group

Principle Effector

Secondary Effector

A

3PGA þ Pi

Pi þ 3PGA

Vmax

Ultrasensitive interaction between effectors

Cyanobacteria green algae spinach leaf potato tuber

B

Pi

3PGA

Vmax

3PGA reverses inhibition caused by Pi

Wheat endosperm

C

3PGA

Pi

Vmax

Pi only inhibits enzyme activated by 3PGA

CAM plants maize endosperm

D

3PGA

Pi

Km

3PGA increases affinity for substrate, ATP and Pi reverses the effect

Barley endosperm

Effect On

Source


43


50% by 2 mM Pi. Further addition of Pi does not increase inhibition. However, with low and subsaturating concentrations of 3PGA (0.15e0.25 mM), these enzymes become more sensitive to Pi inhibition and become totally inhibited at 0.5e2 mM Pi. As seen with other ADP-Glc PPases, 3PGA concentrations can reverse the Pi inhibition and decrease the affinity of the enzymes for Pi. A fourth pattern of interaction (enzymes of group D) between allosteric activator and inhibitor is seen with barley endosperm (Kleczkowski et al., 1993b). The ADP-Glc PPase, which is poorly activated by 3PGA, is inhibited by Pi. However, 3PGA lowers (up to threefold) the S0.5 for ATP (i.e., the apparent affinity of ATP is increased) and the Hill coefficient. At 0.1 mM ATP, activation by 3PGA is about fourfold; 2.5 mM Pi reverses the effect. Thus, in barley endosperm, the prime effect of 3PGA or Pi may be to either increase or decrease the apparent affinity of the enzyme for the substrate, ATP.

6. IDENTIFICATION OF IMPORTANT AMINO ACID RESIDUES WITHIN THE ADP-GLC PPASES Amino acid residues playing important roles in the binding of substrates and allosteric regulators have been identified in the ADP-Glc PPases by chemical modification and site-directed mutagenesis studies. Photoaffinity analogs of ATP and ADP-Glc, 8-azido-ATP and 8-azido-ADP-Glc, were used and identified Tyr114 as an important residue in the enzyme from E. coli (Lee and Preiss, 1986; Lee et al., 1986). Site-directed mutagenesis of this residue rendered a mutant enzyme exhibiting a marked increase in S0.5 for ATP but also a lower apparent affinity for Glc-1P and the activator fructose-1,6-bis-P. The Tyr residue must be close to the adenine ring of ATP or ADP-Glc but probably also near the Glc 1-P and the activator regulatory sites. The homologous Tyr114 in the enzyme from plants is a Phe residue suggesting that the functionality is not given by the specific residue but by its hydrophobicity. Chemical modification studies on the E. coli ADP-Glc PPase, that showed involvement of Lys195 in the binding of Glc 1-P (Parsons and Preiss, 1978a,b), were confirmed by site-directed mutagenesis (Hill et al., 1991). Site-directed mutagenesis was also used to determine the role of this conserved residue in the small subunit Lys198 and large subunit Lys213 of the potato tuber ADPGlc PPase (Fu et al., 1998a). Mutation of Lys198 of the small subunit with Arg, Ala, or Glu decreased the apparent affinity for Glc 1-P 135- to 550-fold (Fu et al., 1998a). There is little effect on kinetic constants for ATP, Mg2þ, 3PGA, and Pi. The results show that the Lys198 in the small subunit is directly involved in the binding of Glc 1-P (Fu et al., 1998a). On the other hand, the homologous site residue, Lys213 in the large subunit does not seem to be involved since similar mutations on Lys213 had little effect on the affinity for Glc 1-P (Fu et al., 1998a). This is consistent with the view that the potato tuber large subunit is a modulatory subunit and does not have a catalytic role (Ballicora et al., 2005).


45

Asp142 in E. coli ADP-Glc PPase in modeling studies was predicted to be close to the substrate site and this amino acid was identified as mainly involved in catalysis (Frueauf et al., 2001). Site-directed mutagenesis of D142 to D142A and D142N confirmed that the main role of Asp142 is catalytic for a decrease in the specific activity of 10,000-fold was observed with no other kinetic parameters showing any significant changes (Frueauf et al., 2001). This residue is highly conserved throughout ADP-Glc PPases from different sources, as well as throughout the superfamily of nucleotide-sugar pyrophosphorylases (Frueauf et al., 2003). The role of this Asp residue was also investigated by site-directed mutagenesis in the heterotetrameric potato tuber ADP-Glc PPase. The homologous residues of the small subunit Asp145 and large subunit Asp160 were separately replaced by either Asn or Glu residues (Frueauf et al., 2003). Mutation of the Asp145 of the small subunit rendered the enzymes almost completely inactive. D145N mutant had a four-order of magnitude in decrease of activity while D145E, a more conservative mutation, was two-orders of magnitude decrease in specific activity. The homologous mutations in the large subunit alone (D160) did not alter the specific activity but did affect the apparent affinity for 3PGA (Frueauf et al., 2003). Thus, these results agree with the view that each subunit in potato tuber ADP-Glc PPase plays a particular role, catalysis for the small subunit and a regulatory role for the large subunit. Pyridoxal-5-phosphate (PLP) can be considered to have some structural analogy to 3PGA, and it was found to activate the ADP-Glc PPases from spinach leaf (Morell et al., 1988; Ball and Preiss, 1994) and Anabaena. In spinach ADP-Glc PPase, PLP bound at Lys440, very close to the C-terminal of the small subunit, as well as binding to three Lys residues in the large subunit. Binding to these sites was prevented by the allosteric effector 3PGA, which indicated that they are close or directly involved to the binding of this activator. With Anabaena ADP-Glc PPase, PLP modified Lys419 and Lys382 (Charng et al., 1994). That these residues were regulatory-binding sites was confirmed by site-directed mutagenesis of the Anabaena ADP-Glc PPase (Sheng et al., 1996). Mutation of the homologous Lys residues in the potato tuber enzyme small subunit Lys441 and Lys404 indicated that they were also part of the 3PGA site in heterotetrameric ADP-Glc PPases and that they contribute additively to the binding of the activator (Ballicora et al., 1998). Moreover, mutation on the small subunit yielded enzymes with lesser affinity to 3PGA, 3090- and 54-fold, respectively, than the homologous mutants on the large subunit. Results indicate that Lys404 and Lys441 on the potato tuber small subunit are more important than their homologous counterparts on the large subunit, Lys417 and Lys455. It seems that the large subunit seems to contribute to the enzyme activation by making more efficient the activator sites already present in the small subunit, rather than providing more effective allosteric sites (Fu et al., 1998a). Arginine residues were found in ADP-Glc PPases from cyanobacteria to be functionally important as shown by chemical modification with phenylglyoxal


(Sheng and Preiss, 1997; Iglesias et al., 1992). The role played by Arg294 in the inhibition by Pi of the enzyme from Anabaena PCC 7120 was shown by Ala scanning mutagenesis studies (Sheng and Preiss, 1997) and later, further research indicated that replacement of Arg with Ala or Gln produced mutant enzymes with a changed pattern of inhibitor specificity, as having NADPH rather than Pi as the main inhibitor (Frueauf et al., 2002). All of these results suggest that the positive charge of Arg294 may play a key role in determining inhibitor selectivity, rather than being specifically involved in Pi binding. However, studies on the role of Arg residues located in the N-terminal of the enzyme from Agrobacterium tumefaciens demonstrated the presence of separate subsites for the activators Fru-6-P and pyruvate as well as a desensitization of R33A and R45A mutants to Pi inhibition (Gomez-Casati et al., 2001). Random mutagenesis experiments performed on the potato tuber ADP-Glc PPase have been useful to identify residues that are important for the enzyme. Mutation of Asp253 on the small subunit showed a specific effect on the Km for Glc 1-P, which increased 10-fold respect to the WT enzyme (Laughlin et al., 1998a). The small magnitude in the increase (only one, rather than three to four orders of magnitude) suggests that the Asp253 residue is not directly involved in Glc 1-P binding. This residue, however, is conserved in the NDPsugar PPases that have been crystallized and the structure solved (Blankenfeldt et al., 2000; Brown et al., 1999; Sivaraman et al., 2002). Alignment of Asp253 in the structure according to the secondary structure prediction (Singh et al., 1984) places the residue close to the substrate site without direct interaction with Glc-1-P suggesting that substitution of the Asp253 causes an indirect effect on the Glc-1-P by alteration of the Glc-1-P binding domain. Another random mutagenesis study (Greene et al., 1996a) concerns Asp416 (described in the article as Asp413) of the potato tuber ADP-Glc PPase small subunit and its effect on 3PGA activation. This residue is adjacent to Lys417, shown to be a site for PLP binding and 3PGA activation (Ball and Preiss, 1994). Also, several modifications on the C-terminus caused modifications on the regulation of different plant ADP-Glc PPases (Giroux et al., 1996; Salamone et al., 2002). The finding that Lys and Arg residues are important in allosteric effector binding and situated at the C-terminus in ADP-Glc PPases of plants and cyanobacteria is different with what is observed for the bacterial ADP-Glc PPases. Lys39 (E. coli) and Arg residues in the N-terminal of the A. tumefaciens enzyme were shown to be important for the interaction of the activators and inhibitors (Gomez-Casati et al., 2001; Parsons and Preiss, 1978a,b; Gardiol and Preiss, 1990). These results suggest that the regulatory domains may be at different sites in the bacterial and plant enzymes. Other studies, however, with chimeric ADP-Glc PPases constructed from E. coli and A. tumefaciens have shown that the C-terminus in the bacterial ADP-Glc PPases are also functional in determining effector specificity and affinity


47

(Ballicora et al., 2002). Regulation of ADP-Glc PPases most likely is determined by interactions occurring between the N- and C-termini in the enzyme.

7. CHARACTERIZATION OF THE REGULATORY DOMAIN Truncation of 10 amino acids in the small subunit of potato tuber ADP-Glc PPase affected its regulatory properties by increasing the apparent affinity for the activator 3PGA and decreasing the inhibitor Pi affinity (Ballicora et al., 1995). When the large (regulatory) subunit was truncated 17 amino acids at the N-terminal, similar results were observed (Laughlin et al., 1998b). The E. coli enzyme is also affected in its the regulatory properties when the N-terminal is shortened by 11 amino acids (Wu and Preiss, 1998, 2001). The N-terminal region of the ADP-Glc PPase is predicted to be a loop and the data suggest that it regulates enzyme activity in being the “allosteric switch,” involved in the transition between the activated and nonactivated conformations of the enzyme. A shorter N-terminus allows the enzyme to be in an “activated” conformation.

8. STARCH SYNTHASES SS catalyzes the transfer of the glucosyl moiety of the sugar nucleotide ADPGlu, either to a maltooligosaccharide or glycogen or the growing starch polymers (amylose and amylopectin), forming a new (1/4)-a-glucosidic linkage (Reaction (1.ii)). Since the glucosyl unit in ADP-Glc is in the form of an a-D-glucopyranosyl unit, and the newly formed glucosidic bond also has the a-D configuration, the SS is a retaining GT-B glycosyl-transferase, according to the nomenclature of Henrissat et al. (2001). In bacteria, where the final product is glycogen, there is a similar reaction for synthesis of the (1/4)a-glucosidic linkage, and the enzyme is referred to as glycogen synthase. There are a number of differences between the bacterial and plant enzymes. One, in bacteria, e.g., E. coli, there is usually only one glycogen synthase, encoded for by one glycogen synthase gene (Kumar and Preiss, 1986). However, in every plant tissue studied, more than one form of SS can be identified. This has been summarized in a number of reviews (Zeeman et al., 2010; Preiss, 2010; Stitt and Zeeman, 2012; Schwarte et al., 2015; Ball and Morell, 2003). The plant SSs are, therefore, encoded for by more than one gene. Some SSs are retained within the starch granule and are designated as GBSS. Some have been solubilized by a-amylase digestion of the granule, while others, designated as soluble SSs, can be found in the soluble portion of the plastid fraction. SS or glycogen synthase activity can be measured by transfer of (14C)glucose from ADP-Glc into an appropriate primer, such as amylopectin or rabbit glycogen, followed by precipitation of the labeled polymer (MacDonald and Preiss, 1983).


8.1 Characterization of the Starch Synthases A phylogenetic tree based on the various deduced amino acid sequences of SSs from crop plants and the green alga, Chlamydomonas, has identified five subfamilies of SSs (Ball and Morell, 2003; Henrissat et al., 2001). These synthases are designated as GBSS, starch synthase I (SSI), starch synthase II (SSII), starch synthase III (SSIII), and starch synthase IV (SSIV) (Henrissat et al., 2001). There is also evidence that in monocots the SSII class may have diverged further to classes SSIIa and SSIIb (Harn et al., 1998). Indeed, the GBSS family may have also diverged into other classes, viz., GBSSI, GBSSIb, or GBSSII (Edwards et al., 2002; Fujita and Taira, 1998; Vrinten and Nakamura, 2000). Also, in rice plants a genome analysis has revealed the presence of nine genes for the soluble SSs, one SSI, three SSII, two SSIII, two SSIV, and even an SSV (Hirose and Terao, 2004; Ohdan et al., 2005). A. thaliana has a single gene for each soluble SS (Henrissat et al., 2001) and GBSS (Henrissat et al., 2001). Multiple forms of soluble SSs are present in rice endosperm (Hirose and Terao, 2004; Dian et al., 2005), barley endosperm (Morell et al., 2003), pea seeds (Craig et al., 1998; Edwards et al., 1996), wheat endosperm (Hawker and Jenner, 1993; Denyer et al., 1995; Li et al., 2000), maize endosperm (Harn et al., 1998; Gao et al., 1998; Cao et al., 1999; Zhang et al., 2004), Arabidopsis leaves (Delvalle´ et al., 2005; Zhang et al., 2005, 2008; Roldań et al., 2007; Tenorio et al., 2003), potato leaf and tuber (Edwards et al., 1995; Marshall et al., 1996; Kossmann et al., 1999), and C. reinhardtii (Fontaine et al., 1993). Fig. 1.7 shows alignments of conserved residues in the SSs with the wellstudied E. coli glycogen synthase (Preiss, l996; Yep et al., 2004a,b, 2006; Sheng et al., 2009). Almost all residues present at the E. coli glycogen synthase active site have identical counterparts in the SSs. The KTGGL sequence motif is important in binding of the ribose and phosphate moiety of the ADP-Glc. Glu 377 a part of FEPCGL region and has been shown to be the catalytic residue (Yep et al., 2004a). Along with Lys305 is proposed to bind to the phosphate and glucosyl moieties of ADP-Glc (Yep et al., 2006; Sheng et al., 2009).

8.2 Soluble Starch Synthase I Owing to the multiplicity of SS isozymes, it is unclear what specific functions these isoforms would have in starch granule synthesis. Considerable effort to isolate mutant plants specifically deficient in one of the isoforms of the SS has been done to determine their individual functions and some insight has been provided into the possible functions of the SSs, either soluble or granule bound, in the synthesis of both amylose and amylopectin. In the case of SSI, a mutant of Arabidopsis has been isolated by T-DNA insertion that is deficient in SSI activity and having an altered amylopectin structure (Cao et al., 1999).


49

E. coli

9E

15

KTGGLADV 95Y137 DWH161 HN 246NGL

300RL 304QKG 355Y 373PSRFEPCGLTQL

Anabaena sp.

9E

15

KVGGMGDV 96Y129 DW 152 HN 262NGI

280RL 284QKG 336Y 334PSRFEPCGISQM

Maize SSI

140E 146

KTGGLGDV 32Y277 DWH307 HN 401NGL

455RL 459QKG 510F 528PSRFEPCGLNQL

potato SS1

139E 145

KTGGLGDV232Y277 DWH307 HN 401NGL


Wheat SS1

139E 145

KTGGLGDV232Y277 DWH307 HN 401NGL


Wheat SSIIa

316E 322

KTGGLGDV403Y446 DWH476 HN562 NGL 621RL 625QKG 676F 694PSRFEPCGLNQL

Maize SSIIa

249E 255

KTGGLGDV333Y 376DWH 406HN 492NGL 551RL 555QKG 606F 624PSRFEPCGLNQL

Potato SSII

284E 290

KTGGLGDV371Y 414DWH 444HN 530NGL 589RL 593QKG 644F 662PSRFEPCGLNQL

Potato SSIII

788E 794

KTGGLGDV 875Y909DWS 938 HN 986NGL1040RL1044QKG1100Y1118PSIFEPCGLTQL

Wheat SSIII

1188E 1194 KTGGLGDV1275Y1308DWS1337HN1385NGL1439RL1443QKG1499Y1517PSIFEPCGLTQL

C.reinhardii GBSS 156E 162

KTGGLGDV 259Y 294DWH 323HN 416NGL 470RL 474QKG 526Y 544PSMFEPCGLTQL

Barley GBSS

84E

90

KTGGLGDV 176Y 227DWH 257HN 346NGL 401RL 405QKG 457F 475TSRFEPCGLIQL

Maize GBSS

85E

91

KTGGLGDV 177Y 228DWH 258HN 347NGL 402RL 406QKG 459F 477TSRFEPCGLIQL

Potato GBSS

89E 95

KTGGLGDV 181Y 232DWH 262HN 351NGL 406RL 410QKG 461F 480PSRFEPCGLIQL

Rice GBSS

91E 97

KTGGLGDV 183Y 234DWH 264HN 353NGL 408RL 412QKG 463F 481PSRFEPCGLIQL

FIGURE 1.7 Alignment of residues of plant and algal starch synthases with Escherichia coli glycogen synthase. Conserved critical residues are shown in bold lettering. The sequences were obtained online from NCBI Sequence Viewer version 2.0.

Other starch metabolizing enzymes, ADP-Glc PPase, BE, a- or b-amylase, maltase, a-glucanotransferase, pullulanase, phosphorylase, or GBSSI were unaffected by the mutation. The soluble SS activity was reduced by 56%e72% in crude extracts and the remaining activity presumably was associated with the other soluble SSs than SSI. The starch content in the mutant was reduced to 77% of the normal and had slightly more amylose. The mutant amylopectin fraction was completely debranched with isoamylase and pullulanase and the CL distribution compared to the WT Arabidopsis leaf starch. Whereas the WT starch showed the typical polymodal distribution of CL with the maximum CL of glucose units being 11 and 12, the mutant, labeled as Atss-1-1, had a unimodal CL distribution with the most abundant CLs being 13e15 glucose units long. Further comparisons showed a marked reduction of CLs of 8e12 glucose units in the mutant and a significant increase in CLs of 17e20 glucose units (Delvalle´ et al., 2005). Debranching of the b-amylase limit digest of the mutant and WT starches showed that the mutant starch product had more chains having less than 10 glucose units and less chains containing 11e24 glucose units. The conclusion was that SSI is involved in incorporating glucose units into small chains “filling up the cluster structure” but is not involved in making longer interior chains (Delvalle´ et al., 2005). These results


are consistent with earlier results (Commuri and Keeling, 2001) showing that recombinant maize SSI had a reduced catalytic activity of three- to fourfold when the primer glycogen side chains had been lengthened by Glc-1-P and phosphorylase action before subjection to SS action. Also, most of the label incorporated into glycogen in vitro by SSI was into chains less than 10 glucose residues in length. Based on these, it was concluded that SSI has a role in extending the shorter A and B chains to a length of about 14 glucose units or less. Further extension or synthesis of larger chains would have to be done by the other SSs. Thus, SSI may be mainly involved in the synthesis of the exterior A and B chains of amylopectin. These conclusions are also consistent with the finding that the maximal velocity of maize SSI is about 1.5- to 6.5-fold greater with glycogen than with amylopectin (Commuri and Keeling, 2001) and is due to the shorter chains present in glycogen than in amylopectin. However, the Km for amylopectin was 13-fold lower than for glycogen suggesting a greater affinity of maize SSI for amylopectin. The Km for ADP-Glc was 0.1 mmol and of interest was that citrate at 0.5 mol/L stimulated SSI activity to form a polymer in the absence of primer. This has been confirmed in earlier studies (Pollock and Preiss, 1980). Another study also showed that maize SSI was a 77 kDa monomer (Mu et al., 1994). The mature Arabidopsis SSI enzyme subunit was determined to be 67 kDa (Cao et al., 1999) and the rice-soluble SSI was estimated to be 55e57 kDa (Baba et al., 1993). These molecular sizes are similar to what has been found for pea embryo SSI (77 kDa) (Denyer and Smith, 1992), wheat endosperm (77 kDa) (Denyer et al., 1995), potato tuber (79 kDa) (Edwards et al., 1995), and potato leaf (70.6 kDa) (Kossmann et al., 1999). The expression levels of SSI in potato tubers was different from those found for other SSs, GBSSI, SSII, and SSIII (Kossmann et al., 1999), having drastically lower expression levels during development. The expression level was appreciably higher in sink and source leaves suggesting a minor role in starch synthesis in storage tissue. Moreover, application of antisense technique could reduce the SSI activity to nondetectable levels in tubers. There was no great effect in the starch content or in the structure of amylopectin. Potato tuber SSI activity is minor compared to the activities seen for SSII and SSIII and thus may play a minor role in tuber starch synthesis while playing a more important role in leaves. The gene coding for full-length maize SSI (SSI-1) and the genes coding for N-terminally truncated SSI (SSI-2 and SSI-3) have been individually expressed in E. coli (Imparl-Radosevich et al., 1998) and purified to homogeneity. The expressed enzymes had essentially the same properties noted for other SSI. In rice SSI mutants, the amylopectin had reduced chains of degree of polymerization (DP) 8e12 and greater chains of 6e7 and 16e19 (Fujita et al., 2006). CL analysis of amylopectin showed that SSI preferentially synthesized DP 7e11 chains by elongating DP 4e7 short chains of either glycogen or amylopectin. These results indicated that SSI distinctly generates DP 8e12 chains from short DP 6e7 chains emerging from the branch point in


51

the A or B1 chain of amylopectin. However, despite being a major SS isozyme in the developing endosperm, absence of SSI had no effects on either size of seed shape and starch granules and endosperm starch crystallinity, suggesting that other SS enzymes may partly compensate for the absence of SSI, suggesting that amylopectin chains are synthesized by the coordinated actions of the different SS isoforms. In wheat endosperm, McMaugh et.al. (2014) reported that reduction of SSI expression also caused similar results as seen and indicated above in rice (Fujita et al., 2006). There were reduced chains of DP 8e12 and greater chains of 6e7 and 16e19. The results suggest SSI is involved in the synthesis of DP 8e12 chains from A chains of DP 6e7 and the external segments of amylopectin chains.

8.3 Starch Synthase II SSs II have been characterized in several plants including Chlamydomonas. A maize endosperm mutant termed dull1(du1), due to the dull appearance of its mature kernels (Mangelsdorf, 1947), has about 15% of the starch as a slightly altered amylopectin structure known as “intermediate material,” distinguished between amylopectin and amylose on the basis of its starcheI2 complex (Wang et al., 1993a,b; Nelson and Pan, 1995). The du1 starch had the highest degree of branching among many normal and mutant kernel starches studied (Mangelsdorf, 1947; Wang et al., 1993a). Of interest was that if the mutant du1 allele was combined with other starch mutants, alterations in starch structure were seen more severe than those seen in the single mutants (Shannon and Garwood, l984; Nelson and Pan, 1995). The first enzymatic studies done on the du1 mutant were done in 1981 and both SSII and SBEIIa were found to have reduced activity in the endosperm compared to the normal maize endosperm (Boyer and Preiss, 1981). SSII was shown to be different from SSI (Boyer and Preiss, 1981; MacDonald and Preiss, 1985). SSII requires a primer for activity and could not catalyze an unprimed reaction even in the presence of 0.5 M citrate and has less affinity for amylopectin than SSI. However, 0.5 M citrate lowered the Km for amylopectin, 17-fold. The activity with glycogen as a primer is half observed with amylopectin. So glycogen is not as effective as a primer as is amylopectin and that is different from what was observed for SSI. Both maize endosperm SSI and SSII had a Km for ADP-Glc of 0.1 mM (Pollock and Preiss, 1980; Ozbun et al., 1971). The maize du1 gene has been extensively characterized (Gao et al., 1998). A part of the du1 locus was cloned by transposon tagging and an almost complete DU1 cDNA sequence was determined. It was found that the gene coded for a predicted 1674 amino acid peptide with two regions 51% and 73% similar, respectively, with corresponding regions in SSIII of potato tuber (Marshall et al., 1996; Gernot et al., 1996). The deduced amino acid sequence of the DU1 cDNA codes for a putative SS with a predicted molecular size of


188 kDa. It is the C-terminal portion beyond amino acid residue 1226 that is most probably where the glucosyl-transferase activity resides as the 450 residues of DU1 is similar to the well-known sequences of bacterial glycogen and plant SSs. Gao et al. (1998) conclude that the SS coded by DU1 may account for the soluble isoform identified earlier as SSII (Boyer and Preiss, 1981; Ozbun et al., 1971). The only discrepancy is that the deduced molecular size of the DU1 is 188 kDa while the SSII studied by Ozbun et al. (1971) was 95 kDa. However, the deduced molecular size does match the 180 kDa molecular mass maize of SSII as reported by Mu et al. (1994). The DU1 gene product contains two repeated regions in its unique amino terminus and one of the regions has a sequence identical to a conserved segment of SBEs (Gao et al., 1998). This may be related to the observation that the du1 mutation also reduces SBEIIa activity. The reduction of SBEIIa activity may be a secondary effect because of lack of interaction between SSII with SBEIIa in the mutant or that expression of SBEIIa is inhibited due to lack of SSII. In other experiments (Cao et al., 1999), it was shown that DU1 was one of two major soluble SSs and when the C-terminal 450 residues of DU1 was expressed in E. coli, it was shown to have SS activity. Of interest was that antisera prepared against DU1 detected a soluble protein in endosperm extracts of molecular size of greater than 200 kDa and this protein was absent in du1 mutants. The antisera reduced SS activity by 20%e30% in kernal extracts. In the same study, antisera prepared on SSI reduced SS activity by 60%. In du1 mutants antisera prepared against SSI reduced the SS activity to essentially zero suggesting that SSI and SSII were the only maize endosperm soluble SSs. Because of the high similarity in the sequence of the DU1 SSII to the potato SSIII and both are exclusively soluble, it is argued that DU1 is the evolutionary counterpart of potato SSIII (Edwards et al., 1995; Marshall et al., 1996; Gernot et al., 1996). It is proposed that DU1 or maize SSII should be known as maize SSIII (Gao et al., 1998). In pea, mutants at the rug5 locus were isolated after chemical mutagenesis (Wang et al., 1990). The mutation caused the peas to become wrinkled seeded, a sign that they were starch deficient and the three mutant alleles were shown to have 30%e40% less starch (Craig et al., 1998). Both mature and developing pea embryos were shown to be devoid of SSII as determined in polyacrylamide gel electrophoresis and immunoblotting with antiserum raised with SSII. Analysis of other enzymes involved in starch synthesis such as ADP-Glc PPase, BE, and SSIII did not reveal any significant changes between WT and rug5. However, there was a 1.7-fold increase in the specific activity in the GBSSI activity. Other enzymes such as P-glucomutase, UDP-Glc PPase, or sucrose synthase were not affected by the mutation. A SSIIa activity has been shown to be lacking in barley sex6 mutants (Morell et al., 2003). The mutation located at the sex6 locus on chromosome 7H and the mutant contained less than 20% of the amylopectin levels seen in WT barley. Analysis showed that a 90 kDa band was missingdattributable to


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SSIIa either in the starch granule or in the cytosol. Also there was an alteration of the distribution of other SSs, SSI as well as the BEs, BEIIa and BEIIb. In the WT barley grain, they are distributed both in the granule as well as in the cytosol. In the mutant, they were only present in the cytosol. However, there was no alteration in their activity or in SSIII activity that was also present in the cytosol (Morell et al., 2003). The mutant had a decrease in amylose content of about 35% but the amylopectin reduction was more drastic, 84%e91%. Thus, this mutant may be considered a high-amylose mutant. Analysis of the mutant amylopectin showed that it had a higher percentage of chains, 6e11 glucose units in length than the WT, and a lower percent of intermediate CL of 12e30 resulting in a starch with a reduced gelatinization temperature. The granule morphology was also altered. It is believed that all these effects are mainly due to the complete loss of SSIIa activity, but the alteration of the distribution of the other SSs and the BEs may also play a role in the synthesis of the altered amylopectin. This mutation also presents another approach in obtaining a high-amylose starch. As will be shown later, mutations of BE can not only lead to the production of more amylose but also amylopectins with lesser and longer chains and having properties similar to amylose. Mutations of SSIIa have been observed in wheat and in rice (Umemoto et al., 2002). In wheat, each of the three wheat genomes were mutated to entirely eliminate expression of the SSIIa gene product, Sgp-1 protein (Yamamori et al., 2000) in one line that had reduced starch amounts and an altered starch structure. In rice, two classes of starch have been found. In indica rice variety, the starch is of the long-chain type while in japonica it is of the short-chain variety (Umemoto et al., 2002). Genetic analysis showed that the mutation in japonica rice led to a loss of SSII. Thus in higher plants, it seems that loss of SSII activity in dicots and SSIIa activity in monocots have the same results with respect to reduced starch content due to lowered amylopectin content and altered amylopectin chain size distribution. Thus these genes may have the same function in starch biosynthesis. A mutant of SSII has also been isolated in C. reinhardtii (Fontaine et al., 1993). This mutant, st-3, accumulated only 20%e40% of amount of starch present in WT. The enzyme lacking in the mutant was SSII, one of the two SS isoforms present in C. reinhardtii (Fontaine et al., 1993). There was also an apparent increase in the amylose content as well as a modified form of amylopectin. The changes noted in the mutant amylopectin were a decrease in the number of intermediate glucose branch chains of 8e50 glucose units and an increase in short-chain glucans (2e7 glucose units). The conclusion made was that this SSII was responsible for synthesis of intermediate size branch chains, a similar conclusion reached as indicated above with the function of SSII and SSIIa in higher plants. The SSII of Chlamydomonas is now referred to as SSIII (Ball and Morell, 2003). Of interest is the limit of chain growth with respect to either SSI or SSII action. As previously indicated Commuri and Keeling (2001) showed that


maize SSI catalytic activity decreased as the amylopectin external CL increased in the DP 6e14 range. It is unknown what may be the limitations for SSII chain elongation. Nakamura et al. (2014) measured the rice endosperm SS isozymes SSI, SSIIa, and SSIIIa. The three isoenzymes showed low activity with maltohexose as a primer. In vitro studies indicate that SSI mainly transferred glucosyl units to A and B chains with a DP of 6 and 7 external segments and elongated them to a DP of 8. SSIIa and SSIIIa isozymes appeared to form wider ranges of intermediate chains and long chains, respectively.

8.4 Starch Synthase III In 1996 it was found that the major isoform of soluble SS in potatoes was of a molecular size of 139e140 kDa and it was labeled as SSIII (Marshall et al., 1996; Gernot et al., 1996). SSIII was expressed at the same level in all developmental stages and in contrast to GBSSI and SSII, SSIII was highly expressed in potato sink and source leaves (Marshall et al., 1996). Of interest was that in leaf discs both GBSSI and SSIII expression was induced by sucrose addition under light (Marshall et al., 1996). Antiserum to SSIII reduced the total SS activity by about 75%, indicating that it was the major activity form of the soluble SSs (Tenorio et al., 2003). A cDNA clone of the SS was isolated (Gernot et al., 1996) and analyzed and its transcript predicted a protein of 1230 amino acids with a molecular size of 139 kDa (Gernot et al., 1996). The N-terminus sequence of about 60 amino acids was suggestive of a transit peptide (Tenorio et al., 2003). The N-terminal extension showed little sequence similarity to that of SSII from either pea or potato (Nakamura et al., 2014). However, from amino acid residue 780 to the C-terminal end the sequence is similar to either soluble or GBSSs (Tenorio et al., 2003) or even bacterial glycogen synthases (Kumar and Preiss, 1986). Indeed, The KTGG motif that is involved in ADP-Glc substrate binding (Yep et al., 2004a,b, 2006; Furakawa et al., l990) is conserved as KVGGL at residue 794, as well as the FEPCGL sequence starting at residue 1121 that has been shown to contain an essential Glu residue (see Fig. 1.7) for catalytic activity of the E. coli glycogen synthase (Yep et al., 2004a). Using the antisense RNA approach to SSIII, it was found that SSIII transcripts were eliminated (Tenorio et al., 2003; Gernot et al., 1996). There was no effect on the levels of the other SSs and there was no effect on the amount of starch produced or in the amylose content in these antisense plants as compared to WT (Tenorio et al., 2003; Gernot et al., 1996). However, what was noted in both reports was a drastic alteration in the starch granule morphology (Tenorio et al., 2003; Gernot et al., 1996). The conclusions made were that SSIII was a major factor for synthesis of starch with normal morphology. But the exact role it played in the synthesis of starch remains obscure. Subsequently, the endosperm of hexaploid wheat, Triticum aestivum [L.], was shown to have a SSIII gene (Li et al., 2000). A cDNA was isolated and


55

contained an open reading frame for a 1629 amino acid polypeptide. The N-terminal region started with the transit peptide region of 67 amino acids and the N-terminal region of 656 residues, the SSIII-specific region of 470 amino acids and then at the C-terminal region, the 436 amino acid catalytic domain. These domains were compared to those seen in SSIII from maize DU1 protein (Zhang et al., 2004), potato (Marshall et al., 1996; Gernot et al., 1996), and cowpea (GenBank accession No AJ225088), and Arabidopsis (GenBank accession No AC007296). Of interest is that A. thaliana SSIII has three copies of a carbohydrate-binding module at its N-terminal (Schwarte et al., 2015).

8.5 Starch Synthase IV As of 2003, a novel class of SS amino acid sequences designated as SSIV was noted in expressed sequence tag (EST) databases from several species including Arabidopsis, Chlamydomonas, wheat, and cow pea (Ball and Morell, 2003). The SSs IV show high similarity with each other. Using A. thaliana sequence SSIV as 100%, the cowpea SSIV is 71% identical (accession number AJ006752), the wheat SSIV has 58% identity (accession number AY044844), the rice SSIV-1 57% identity, and rice SSIV-2 58% identity (accession numbers AY373257 and AY373258, respectively). BLAST analysis of the rice genome using the conserved catalytic C-terminus of SS amino acid sequences showed not only two SSIII homologous genes but also two genes homologous to wheat SSIV (Dian et al., 2005). cDNA clones of SSIV-1 and SSIV-2 were isolated and expressed in E. coli (Dian et al., 2005) and were expressed 2.7- and 2.4-fold, respectively, in activity over the baseline level of the E. coli glycogen synthase suggesting that the SSIV genes encoded SS activity. SSIV-1 was expressed mainly in endosperm and weakly in leaves while SSIV-2 was expressed mainly in leaves and weakly in endosperm. The rice SSIV enzymes contained three distinct regions. A putative transit peptide region of 78 amino acids for SSIV-1 and 33 amino acids for SSIV-2, a region homologous to Smc/myosin tail and then a C-terminal catalytic domain region resembling the bacterial glycogen synthase (Yep et al., 2004a,b, 2006; Sheng et al., 2009). As seen with SSIII, the KTGG motif that is involved in ADP-Glc substrate binding is conserved as KXGGL. The FEPCGL sequence that has been shown to contain the Glu residue important for catalysis for the E. coli glycogen synthase (Yep et al., 2004a) are present in the C-terminal region. The SSIV gene encodes an enzyme with a predicted molecular size of about 118 kDa including the transit peptide. The pattern of expression of SSIV in rice was also studied and compared to expression of the other SSs (Hirose and Terao, 2004). Ten genes were identified to be of the SS gene family (Hirose and Terao, 2004). They were grouped on the basis of sequence analysis, as SSI, SSII, SSIII, SSIV, and GBSS (Hirose and Terao, 2004). Of interest were the different patterns of temporal


expression for the various SSs during seed development. The early expressers were SSII-2, SSIII-1, and GBSSII and were expressed in the early stage of grain filling. Those expressed in the mid- to later stage of grain filling, the late expressers, were SSII-3, SSIII-2, and the third group was GBSSI. SSI, SSII-1, SSIV-1, and SSIV-2 were expressed relatively constantly during the whole stage of grain filling (Dian et al., 2005). Recently, a mutant of A. thaliana deficient in SSIV has been isolated (Roldań et al., 2007). As seen in rice (Dian et al., 2005) the SSIV gene contains 16 exons separated by 15 introns (Roldań et al., 2007). Western blots of WT A. thaliana showed a SS of 112 kDa but the two mutant alleles showed absence of the SSIV in Western blots (Roldań et al., 2007). The mutant alleles showed lower growth rates under a 16-h day/8 h night photoregime when compared to WT. There was also a decrease in leaf starch of 35%e40%. If the SSIV protein was restored by transformation to the mutant, then both growth rate and starch levels were restored indicating that the mutant alterations were due to SSIV deficiency. Normal growth of the mutant was restored by growing with continuous light. Because of lowered starch synthetic rate, the levels of sucrose and fructose and glucose were higher in the mutant. The mutant alleles had no decrease in total SS activity nor did it have any significant decrease in other starch metabolizing enzymes. Upon analysis of the mutant, starch content had no change in the amylose/amylopectin ratio, and there were minor effects on the amylopectin structure with a slight decrease in the number of chains from 7 to 10 glucose units long. However, there were significant alterations in the morphology of the starch granules with respect to size and number of granules in the chloroplast. Whereas normal chloroplasts would have 4e5 starch granules per chloroplast, the SSIV mutants had only one. The mutant starch granule was considerably larger than what was observed in WT. Curiously, not only the starch synthetic rate was reduced but also its degradation rate. This was attributed to having only one granule with a large surface having a chloroplast having less overall starch granule surface so that both the starch synthetic and degradation processes proceed at normal rates (Roldań et al., 2007). It was proposed that the function of SSIV was to establish an initial structure for starch synthesis. The interaction of SSIV with other SS isoforms would be of interest to study in their specific involvement in starch granule synthesis. As will be discussed below, there is evidence that the GBSS may also be involved in amylopectin synthesis in addition to amylose synthesis. Thus the mode of amylopectin synthesis by both soluble and GBSSs remains unknown and is still an active area for research. A study of the cDNA of wheat showed that its SSIV is very similar to rice SSIVb and was preferentially expressed in leaves (Leterrier et al., 2008). A single mutation of either SSIVa or SSIVb in rice endosperm produced little change in the rice starch granule (Toyosawa et al., 2016). But a double mutation in rice that caused deficiency in SS genes, SSIIIa and SSIVb, lowered the starch content about 40% and changed the shape of the starch


57

granule from a polyhedral to a spherical granule (Toyosawa et al., 2016). Leaf starch granules were absent in the leaf of the double mutant. The SSIIIa and SSIVb double-mutant starch also contained fewer long chains with a DP of 42 or greater (chains proposed to be involved in connecting amylopectin clusters) (Toyosawa et al., 2016). A model of granule formation is proposed in that both SSIII and SSIV are involved in synthesizing septum-like oligosaccharide (SLS), a SLS chain involved in binding various amylopectin clusters. The authors postulated that in the presence of a single mutation either SSIII or SSIV can fulfill synthesis of the SLS. In the double SS mutant, the synthesis of SLS is absent and the granular amylopectin structures form a spherical shape (Toyosawa et al., 2016). Starch synthesis requires a primer. The initiation of the synthesis of the primer for starch granule is unknown. Roldań et al. (2007) reported that Arabidopsis SS IV mutants had severe growth defects as well as a great decrease in the number of starch granules within the plastids. The amount of starch decrease was only about 40% as the starch granules in the mutant were larger. The amylopectin structure with respect to CL in the mutant, however, was similar to the WT Arabidopsis. Roldań et al. (2007) speculate that SSIV mutant having a smaller number of starch granules is due to involvement of SSIV in priming of starch granule synthesis. A mechanism was not proposed and remains unknown.

8.6 Double Mutants of the Soluble Starch Synthases Various alterations have been observed in amylopectin structure in the mutants of various SS isoforms. SSI is involved in extending the shorter A and B chains to a length of 14 glucose units or less (Zhang et al., 2005; Commuri and Keeling, 2001). SSII is involved in synthesis of B2 and B3 chains (Morell et al., 2003; Craig et al., 1998; Fontaine et al., 1993; Umemoto et al., 2002; Yamamori et al., 2000). SSIII is a major factor for synthesis of starch with normal morphology. The exact role it plays, however, in the synthesis of starch still remains obscure (Zhang et al., 2005; Umemoto et al., 2002). As indicated above, SSIV is proposed to form an initial structure in starch, that is, initiate starch synthesis. However, the role or sequence or protocol in the synthesis of the starch granule is unknown for these individual SSs. Unknown is their specific contributions to the structure of amylopectin. Their role in starch synthesis may depend on its activity at particular times in vivo, and on the relative activities of other SSs and BE isozymes and their particular interaction with these enzymes and the growing amylopectin structure. Some efforts to understand the SS pathway of amylopectin synthesis was attempted by making transgenic potatoes where the total SSII and SSIII activities were reduced 31%e80% or 36%e91% by the antisense technique (Edwards et al., 1999). The SSII and SSIII constitute greater than 90% of the total soluble SS activity in the tuber (Marshall et al., 1996; Gernot et al., 1996). The GBSS and BE activities were not affected. In one report


(Edwards et al., 1999) the starch granules of double mutant were compared with the WT and the single SS mutants. First, starch granule morphology of the SSII mutants are essentially similar but the SSIII mutant and the SSII/SSSIII double mutants had abnormal morphology (Edwards et al., 1999). The starch granule morphology in the SSII/SSIII mutant differed from the single SSIII mutant in that scanning electron microscopy showed holes through the granule center that was not observed in the single SSIII mutant. An analysis of the amylopectin branch chains of the mutants showed that all had a greater concentration of shorter chains and a lower amount of longer chains than the WT. However, the patterns of long- and short-chain amounts were quite different. SSII and SSIII showed great enrichment of glucose oligosaccharides 9 and 6 units long while the SSII/SSIII had enrichments of glucose units of 7 and 8 and 12 and 13. There was no relationship between the chain pattern observed for SSII/SSIII and the cumulative pattern obtained for the mutant SSII and SSIII chains (Edwards et al., 1999). In addition, the gelatinization behavior of the mutant starches were all different signifying different alterations in structure (Edwards et al., 1999). These results strongly support the early view of Leloir’s group in 1960 that different isozymes of SSs have unique functions in the synthesis of amylopectin (deFekete et al., 1960). As indicated before, the SSII and SSIII mutants do not affect the rate of starch synthesis, but the mutants have different effects on starch structure and the double-mutant SSII/ SSIII act in a synergistic manner in the synthesis of amylopectin and not in an independent manner. This is based on the starch granule seen in the double mutant which is unlike that of the single SSII and SSIII mutants with respect to morphology and amylopectin CLs. Moreover, this can be explained that in the single mutant SSII or SSIII the function observed for the remaining active enzyme is different in WT than in the double mutant (Edwards et al., 1999). This is most probably due to the activity of the SS dependent on the type of growing glucan acceptor it is presented with. Since other SS isoforms are present, the glucan polymer that is synthesized in the absence of one of the SS may be different and would modify the activity of other SSs and even BE activity (Edwards et al., 1999). Thus, the synthesis of amylopectin or starch is not the sum of the independent actions of various SSs and BE isoforms but is due to a complex combination of the activities of various isoforms that vary during seed and leaf development due to varying expression of the different isozymes (Schwarte et al., 2015). Essentially, the same results and conclusions are expressed in another report (Lloyd et al., 1999a).

8.7 Starch Synthases Bound to the Starch Granule Luis Leloir’s group initially discovered SS activity that was associated with the starch granule (deFekete et al., 1960; Recondo et al., 1961). These SSs are designated as GBSS to distinguish them from the SSs mainly in the soluble


59

phase of the chloroplast or amyloplast. The original characterization of GBSS (Nakamura et al., 1996) was made using UDP-Glc as the glucosyl donor. Subsequently ADP-Glc was found to be a superior glucosyl donor reacting at a 10-fold faster rate than UDP-Glc at 12.5 mmol (MacDonald and Preiss, 1985; Recondo et al., 1961). The maize GBSS was released from the starch granule by incubating ground maize starch granules with a-amylase and glucoamylase (MacDonald and Preiss, 1983). The solubilized SS activity was partially purified and two peaks of activity were obtained with 80% of the activity residing with GBSSI that eluted from the DEAE-cellulose column at a lower salt concentration than the GBSSII fraction. The solubilized granule-bound enzymes showed Km values for ADP-Glc about 10-fold lower than that measured before solubilization (0.96 mM for the intact granule activity) (MacDonald and Preiss, 1985). The granule-bound enzyme is also active with UDP-Glc with 1 mM UDP-Glc having about 7% of the activity rate seen with 1 mM ADP-Glc (MacDonald and Preiss, 1983). If the concentration of UDP-Glc is raised to 20 mMol, then the rate of activity is about 73% of that of ADP-Glc. Upon solubilization with a-amylase and glucoamylase, the activity with UDP-Glc essentially vanished. Either the activity observed with UDP-Glc was not solubilized, suggesting that a different enzyme catalyzed the UDP-Glc activity, or became inactive during the amylase procedure. Alternatively, the ability of the starch-bound SS to utilize UDP-Glc is dependent on the close association of the enzyme with the starch granule. In other words, the conformation of the GBSS is altered in the presence of starch to allow UDP-Glc to be catalytically active. The kinetic properties of solubilized GBSS, now starch-free, require a primer for activity (MacDonald and Preiss, 1985). The GBSSII, in contrast to the SSSII, has a higher apparent affinity (lower Km) for amylopectin than the granule type I enzyme. The soluble GBSSI has a higher activity with the rabbit liver glycogen than with amylopectin, whereas the soluble GBSSII has less activity with the rabbit liver glycogen than with amylopectin (MacDonald and Preiss, 1985). The solubilized GBSSI and GBSSII can utilize oligosaccharide primers as do the soluble SSs. Maltose, maltotriose, and other maltosaccharides are effective primers. The products of the reaction observed with maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose, and maltononaose are the maltosaccharides with an additional glucosyl unit. For example, the primer maltotetraose when glucosylated yields maltopentaose (MacDonald and Preiss, 1985). The molecular size of the native enzymes determined by using sucrose density gradients, or size-exclusion chromatography showed that the major granule SSI, has a mass of 60 kDa, while the solubilized GBSSII was about 93 kDa (MacDonald and Preiss, 1985). Antibody prepared against the SSSI effectively neutralized SSSI activity but has no effect on either the activities of GBSSI or GBSSII or even on SSSII. Alternatively, antibody prepared against the starch granule-bound proteins effectively inhibits GBSSI activity but has very little effect on the soluble SSs (MacDonald and Preiss, 1985).


8.8 Isolation of the Waxy Protein Structural Gene Amylose content determines the degree of translucency of the endosperm (hence the name “waxy”), and it affects the cooking and eating qualities of the grains and the industrial properties of the starch extracted from those grains. It is widely accepted that GBSS activity is a function of the protein coded by the waxy gene due to extensive genetic evidence. The waxy locus gene product is a protein of molecular weight 58 kDa associated with the starch granule and similar to that found for the solubilized maize endosperm GBSSI (MacDonald and Preiss, 1985). This protein was extracted by heating the starch with SDS or by incubation at 37 C with urea but SS would become inactive under those conditions. In mutants containing the wx allele there is virtually no amylose, GBSS activity is very low, and the waxy protein is missing (Nelson et al., 1978; Tsai, 1974; Federoff et al., 1983). Shure et al. (1983) prepared cDNA clones homologous to Wx mRNA. Later, restriction endonuclease fragments containing part of the Wx locus were cloned from strains carrying the ac wx-M9, wx-M9, and wx-M6 alleles to further characterize the controlling insertion elements activator (ac) and dissociation (ds) (Federoff et al., 1983). Excision of the ds element from certain wx alleles produces two new alleles (S5 and S9) encoding the wx proteins having altered SS activities (Wessler et al., 1986). Two of these, S9 and S5, had 53% and 32% of the SS activity, respectively, seen in the normal endosperm. Mutant S9, with the higher SS activity, had 36% of the amylose content observed in the nonmutant endosperm, while mutant S5 with an even lower SS activity, 32%, had only 21% of the nonmutant maize amylose content. The correlation between the amount of GBSS activity and amylose amount provided additional evidence that the waxy protein is involved in amylose synthesis. The DNA sequence of the Waxy locus of Zea mays was determined by analysis of both a genomic and an almost full-length cDNA clone (Klosgen et al., 1986). Also, barley Waxy locus has been cloned and its DNA sequenced (Rohde et al., 1988). The deduced amino acid sequences of the maize and barley clones can be compared with the amino acid sequence of the E. coli ADP-Glc-specific glycogen synthase (Kumar and Preiss, 1986). Both clones had the sequence seen in the bacterial enzyme starting at residue Lys15,. KTGGL. As indicated before, the lysine in the bacterial glycogen synthase has been implicated in the binding of the substrate, ADP-Glc (Kumar and Preiss, 1986; Yep et al., 2004a). Moreover, the finding of similarity of sequences between the bacterial glycogen synthase and the putative plant SSs provides more and strong suggestive evidence that the waxy gene is indeed the structural gene for the GBSS. Developing pea embryo starch contains SS activity that is associated with the waxy protein. The molecular weight of the pea SS is about 59 kDa, as determined by ultracentrifugation in sucrose density gradients. The SS activity was solubilized by a-amylase treatment and


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then partially purified (Sivak et al., 1993). The solubilized pea GBSS preparation displayed a relatively high specific activity (over 10 mmol glucose incorporated per minute per milligram protein). This enzyme fraction was subjected to SDS polyacrylamide and gel electrophoresis. Protein staining or immunoblotting with maize Wx antibody showed only the Wx protein (Sivak et al., 1993). Thus, the biochemical examination of SS present in starch granules from two species, maize and pea, strengthens the genetic evidence supporting the role of the Wx protein as a GBSS with a major role in the determination of amylose content of starch.

8.9 Further Studies of GBSS and Isoforms; Their Involvement in Both Amylopectin and Amylose Synthesis GBSSs have been studied in a number of plants and in many cases two isoforms have been identified designated as GBSSI and GBSSII. In pea embryos, there are two forms that are associated with starch, GBSSI and SSII. However, SSII also contributes much of the soluble SS activity in the embryo (Denyer et al., 1993) and its properties were discussed as a soluble SS in Section 4. Clones of the two isoforms of GBSSI and GBSSII from pea embryo (Dry et al., 1992) and potato tubers (Dry et al., 1992; van der Leij et al., 1991) were isolated and characterized. Whereas the GBSSI of both potato and pea embryo were very similar in deduced amino acid sequence of the waxy proteins of maize and barley, the clones of GBSSII from potato and pea were different in sequence but similar to each other. The different feature from GBSSI was the extra N-terminal domain of 203 amino acids that is hydrophilic, having basic amino acids to give it a net positive charge and being serine rich (van der Leij et al., 1991). Both GBSS’s had the KTGGL-ADP-Glc polyphosphate binding domain (Yep et al., 2004a,b, 2006) and the important SRFEPCG-residue (E) domain for activity (Yep et al., 2004a). These clones were used to determine the temporal levels of expression during development. Pea GBSSII is highly expressed earlier in development than GBSSI and the expression is much lower in other organs of pea besides the embryo. In developing potato tubers, GBSSI increased in expression during development similar to patatin while the GBSSII expression was highest in very young tubers and declined in larger potatoes (Denyer et al., 1993). cDNAs of GBSSI and GBSSII from wheat have also been isolated and characterized (Morell et al., 2003). GBSSII was expressed in leaf, culm, and pericarp tissue but not in endosperm where the expression of GBSSI transcripts were high. Thus, expression of the two isoforms maybe tissue- or organ-specific. The wheat GBSSI and GBSSII were 66% identical in sequence. The pea embryo and potato tuber GBSSIs have been studied in vitro to determine the mechanism of how it may synthesize amylose (Denyer et al., 1996b). Amylose was synthesized by the granules, but this was prevented by preincubating the granules with a-glucosidase suggesting the precursor was a


soluble oligosaccharide. Amylose was then synthesized only when maltooligosaccharides were added to reaction mixtures (Denyer et al., 1996b). GBSSI had higher affinity for the maltooligosaccharides than GBSSII (or SSSII) and that transfer of the glucosyl moiety from ADP-Glc was different for the two SSs (Denyer et al., 1999a). A series of maltooligosaccharides were synthesized by GBSSI suggesting that the transfer was not stepwise and more than one glucosyl unit was added before the oligosaccharide dissociated from the enzyme (Denyer et al., 1999a). With GBSSII the product seen was with just one glucose unit added to the maltooligosaccharide substrate. In further studies both potato tuber GBSSI and pea embryo SSII were expressed in E. coli to obtain them in soluble form (Denyer et al., 1999b). It was immediately recognized that GBSSI within the granules had different properties than the soluble form. The affinity for maltotriose to form amylose was less for GBSSI than its soluble form. Whereas the Km value for maltotriose was 0.1 mM for GBSSII, the soluble enzyme form was not even saturated at 1 mmol. The processive order of glucosyl addition was also lost. Of interest was the interaction of amylopectin with potato tuber GBSSI (Denyer et al., 1999b). It acted not only as a substrate but also as an effector. Amylopectin was an effector at lower concentrations where it was a substrate. It increased the affinity for another substrate, maltotriose as well as the rate of reaction with the trisaccharide over threefold. The reaction mode for maltotriose reverted to processive in the presence of amylopectin. It is postulated that the amylopectin induced a conformational change in the GBSSI reverting it to a form it had when it was granule bound (Denyer et al., 1999b). The GBSS enzyme in leaves can be differentiated from those observed in storage tissue. As has been shown in storage tissue, the amylose content of starch can vary from 11% to 37% (Shannon and Garwood, l984). In leaf or transistory starch, the amylose content is less than 15%. In pea, leaf amylose is different from storage amylose with respect to molecular size being of a larger size. This was based on gel filtration chromatography where leaf amylose had a molecular size, based on dextran standards, of 655 kDa as compared to pea embryo amylose of 470 kDa (Edwards et al., 2002). The cDNAs of GBSSIa from embryo and GBSSIb from leaves were isolated and compared. The GBSSIb predicted amino acid sequence was of 613 amino acids with a molecular size of 67.6 kDa with a 68.8% identity and 75.6% similarity with GBSSIa. Mature GBSSIb after transit peptide cleavage is 58.4 kDa while mature GBSSIIa is 58.3 kDa. Both cDNAs were transformed in E. coli and their properties characterized. Unfortunately, GBSSIa from embryo was inactive while GBSSIb was active (Edwards et al., 2002). The GBSSIb activity was compared to potato tuber GBSSI from potatoes also expressed in E. coli and was found to be similar in properties in respect to Km values for ADP-Glc and amylopectin. Both GBSSIa and GBSSIb activities were highly activated when reincorporated into starch granules (Edwards et al., 2002). The two isoforms synthesized distinct isoforms of amylose with the Ia type forming a


63

shorter type of amylose than the Ib form. These results explain why the Ia mutation of pea embryos lacking GBSSIb still synthesizes about 4%e10% of the starch content as amylose and minor amounts of Ib are present in the embryo (Wang et al., 1990). In Arabidopsis leaves, the mechanism of amylose synthesis was also studied with the view of identifying possible primers for amylose synthesis (Zeeman et al., 2002). Maltooligosaccharides such as maltotriose when added to Arabidopsis leaf starch granules and incubated with ADP-[14C] Glc stimulated the synthesis of labeled amylose. These types of experiments had also been done with starch granules from pea and potato (Denyer et al., 1996b) and from C. reinhardtii (Van de Wal et al., 1998). Also a maltooligosaccharide accumulating Arabidopsis mutant, dpe1, synthesized more amylose compared to the WT (Delrue et al., 1992). This strongly suggested that maltooligosaccharides were indeed the primers for amylose synthesis. This raises the question of the mechanism of maltooligosaccharides synthesis for priming of amylose synthesis in plants. Finally, other studies of SSs made in C. reinhardtii should be noted. As indicated before SSII now known as SSIII may be involved in the synthesis of the intermediate size chains of amylopectin (Van de Wal et al., 1998). Also the first evidence that GBSSI was involved in synthesis of amylopectin in addition to amylose synthesis was obtained in C. reinhardtii (Delrue et al., 1992; Maddelein et al., 1994). It was first noted that mutants, sta2, were deficient in GBSSI and had in addition to the loss of amylose, a fraction of altered amylopectin structure with longer chains (Wattebled et al., 2002). It has also been shown in potatoes that GBSSI can elongate amylopectin chains (Denyer et al., 1996b). Other experiments in C. reinhardtii showed that in double mutants lacking both SSIII and GBSSI that an altered starch structure intermediate between glycogen and starch was formed (Maddelein et al., 1994). The cloning of the C. reinhardtii GBSSI has been achieved and it codes for a 69-kDa protein with an extra 11.4 kDa extension at the C-terminus (Wattebled et al., 2002). The C. reinhardtii sta2 mutants transformed with the GBSSI cDNA could now synthesize amylose. An in vitro synthesis of amylose was studied with a starch granule isolated in either WT log phase or in a growth arrested (nitrogen limited) ADP-Glc PPase mutant (Wattebled et al., 2002). The GBSSI-specific activity in this system is 10e50 times higher than what is seen in higher plants and enabled the researchers to increase in vitro, the amount of glucan in the granule by about 1.6-fold. The percentage of amylose increased from 13% to 45% in the granule. X-ray diffraction studies showed an increase in the appearance of crystalline material (Wattebled et al., 2002). A-type crystals remained constant and the total amount of B-type crystals increased from 7% to 33% of the total starch suggesting that GBSSI induces de novo synthesis of B-type crystals. Amylose synthesis depends on the concentration of ADP-Glc, as GBSSI has a high Km for the substrate as compared to the soluble SSs


(Van den Koornhuyse et al., 1996). The phosphoglucomutase (PGM)-deficient mutants do make starch but are deficient in amylose, even though GBSS is present. The PGM-deficient mutants can make amylopectin but not amylose as shown by detailed structure studies of the starch accumulated by the alga mutant (Libessart et al., 1995). A similar structure effect can be seen when the algae has a defective ADP-Glc PPase (Van den Koornhuyse et al., 1996). This indicates that the relatively high Km value seen for ADP-Glc for GBSSI in vitro, most likely is also the in vivo Km value for GBSS. Other studies also show that a diminution of ADP-Glc levels in the cell causes a lowering of amylose to very low or nonexistence levels (Clarke et al., 1999; Lloyd et al., 1999b).

8.10 Branching Enzyme 8.10.1 Branching Enzyme Assays BE can be assayed in numerous ways. The iodine assay is based on the decrease in absorbance of the polysaccharide/I2 complex resulting from the branching of either amylose or amylopectin. Aliquots are taken at intervals during the incubation of the amylose or amylopectin with BE, and iodine reagent is added (Guan et al., 1994a; Boyer and Preiss, 1978; Hizukuri et al., 1981). The decrease of absorbance is measured at 660 nm for amylose and, for amylopectin, at 530 nm. A unit of activity is defined as decrease in absorbance of 1.0 per min at 30 C at the defined wavelength. The phosphorylase stimulation assay (Boyer and Preiss, 1978a,b; Hawker et al., 1974; Takeda et al., 1993) is based on the stimulation of the “unprimed” (in other words, without added glucan) phosphorylase activity of the rabbit muscle phosphorylase a. This is due to the presence of BE in the assay mixture, increasing the number of nonreducing ends available to the phosphorylase for elongation. A unit is defined as 1 mmol transferred from Glc-1-P per min at 30 C. The branch linkage assay (Takeda et al., 1993) is an assay that measures the number of branch chains formed by BE catalysis, rather than an indirect effect of its action as in the two assays described above. The enzyme fraction is incubated with the substrate, NaBH-reduced (Zeeman et al., 2010) amylose. The reaction is then ended by boiling and the product is incubated for debranching, with pure Pseudomonas isoamylase. Finally, the reducing power of the oligosaccharide chains transferred by the enzyme is measured by a reducing sugar assay. Amylose reduced with borohydride is used (rather than amylose itself) to lower the initial reducing power seen with amylose. The branch linkage assay is the most quantitative assay for BE, but the presence of impurities in the enzyme extract such as amylolytic activity, interferes the most with this assay. The phosphorylase stimulation assay is the most sensitive. The I2 assay is not very sensitive but allows the testing of BE specificity with various maltodextrins, providing information on the possible role of the different BE isoforms. It may be best to employ all three assays when studying the properties of the BEs, but, above all, if reliable information is being


65

sought, the BEs must be purified to the extent that all degradative enzymes are eliminated before studying its properties.

8.10.2 Plant and Algal Branching Enzymes; Characterization of Isozymes Maize endosperm has three BE isoforms (Boyer and Preiss, 1978b; Hawker et al., 1974; Takeda et al., 1993; Guan and Preiss, 1993). BEI, BEIIa, and BEIIb from maize kernels (Guan et al., 1994a; Boyer and Preiss, 1978; Singh and Preiss, 1985; Khoshnoodi et al., 1993, 2004) were purified to the point that they no longer contained amylolytic activity (Guan and Preiss, 1993; Singh and Preiss, 1985). Molecular weights were 82 kDa for BEI and 80 kDa for BEIIa and BEIIb (Boyer and Preiss, 1978a,b). Table 1.9 summarizes the properties of the various maize endosperm BE isozymes from the studies of Takeda et al. (1993) and Guan and Preiss (1993). Of the three isoforms, BEI had the highest activity in the iodine assay in branching amylose and its rate of branching amylopectin was about 3% observed with amylose. The BEIIa and BEIIb isozymes branched amylopectin at twice the rate they branched amylose and catalyzed the branching of amylopectin at 2.5 to 6 times the rate observed with BEI. Takeda et al. (1993) analyzed the branched products made in vitro from amylose by the BE isoforms. Isoamylase was used to debranch the products of each reaction followed

TABLE 1.9 Specific Activities (Units/mg Protein) of Maize Endosperm BE Isoforms Branching Enzyme Assay

BEI

BEIIa

BEIIb

Phosphorylase stimulation (A)

1196

795

994

Branching linkage assay (B)

2.8

0.32

0.14

Amylose (C1)

800

29.5

39

Amylopectin (C2)

24

59

63

Ratio of assay (A/B)

460

2484

7100

A/C1

1.5

27

25

A/C2

49.8

13.5

15.8

C2/C1

0.03

2

1.6

Iodine stain assay (C)

Activities

The units are defined for phosphorylase stimulation and branching linkage assays as mmol1 min. For the iodine-staining assay, it is defined as a decrease of one absorbance unit at 660 nm/min for amylose (C1) and for amylopectin one absorbance unit at 530 nm (C2).


by chromatography. BEIIa and BEIIb were very similar in their affinity for amylose and the size of chain transferred. When presented with amyloses of different average CL, the BEs had higher activity with the longer chain amylose. But while BEI could still catalyze the branching of an amylose of average CL of 197 with 89% of the activity shown with a CL of 405, the activity of BEII dropped sharply with this change in CL. A study of the in vitro reaction products indicated that the action of BEIIa and BEIIb resulted in the transfer of shorter chains than those transferred by BEI. Thus, BEI catalyzes the transfer of longer branched chains and that BEIIa and BEIIb catalyze the transfer of shorter chains. This may suggest that BEI produces a slightly branched polysaccharide that serves as a substrate for enzyme complexes of SSs and BEII isoforms to synthesize amylopectin. BEII isoforms may also play a predominant role in forming the short chains present in amylopectin. Moreover, BEI may be more involved in producing the more interior B-chains of the amylopectin while BEIIa and BEIIb would be involved in forming the exterior (A) chains. Vos-Scheperkeuter et al. (1989) purified a single form of branching activity of 79 kDa molecular mass from potato tubers. The antibodies prepared against the native potato enzyme was found to react strongly with maize BEI and weakly with maize BEIIb. The antiserum inhibited the activities of both potato tuber BE and maize BEI in neutralization tests. Potato BE thus shows a high degree of similarity to maize BEI and a lesser extent with the maize BEII isozymes. Up to four cDNA clones have been isolated for potato BE, coding for proteins, 91e99 kDa (Khoshnoodi et al., 1993; Kossmann et al., 1991; Poulsen and Kreiberg, 1993). All these allelic clones have sequences similar to the BEI type. The sbeIc allele, codes for a mature enzyme of 830 amino acids and a molecular weight of 95 180. The sbeIc BE protein product, expressed in E. coli, migrates as a 103-kDa protein (Kossmann et al., 1991). It is noteworthy that BE isolated from other plants, bacteria, and mammals have molecular masses ranging from 75 to about 85 kDa. These molecular masses are consistent with the molecular weights obtained from deduced amino acid sequences obtained from isolated genes or cDNA clones. Potato BEII was first characterized and found to be a granule-bound protein in tuber starch (Larsson et al., 1996). In potato, BEII appears to be less abundant than BEI. Both potato tuber BEI and BEII were cloned and expressed in E. coli (Jobling et al., 1999; Rydberg et al., 2001) and the properties of the potato tuber BE isoforms were compared (Rydberg et al., 2001). As seen with the maize BEs, potato BEI was more active on amylose than BEII and BEII was more active on amylopectin than BEI. Mizuno et al. (1992) reported four forms of BE from immature rice seeds. BEI and BE2 (composed of BE2a and BE2b) were the major forms while BE3 and BE4 were minor forms, being less than 10% of the total BE activity. The molecular weight of the BEs were BE1, 82 kDa; BE2a, 85 kDa; BE2b, 82 kDa; BE3, 87 kDa; BE4a, 93 kDa; and BE4b, 83 kDa. However, BE1,


67

BE2a, and BE2b were similar immunologically in their reaction toward maize endosperm BEI antibody. The rice seed BE1, BE2a, and BE2b were very similar in their N-terminal amino acid sequences. The three BE N-terminal sequences were either TMVXVVEEVDHLPIT or VXVVEEVDHLPITDL. The latter sequence is the same as the first but lacking the first two N-terminal amino acids. Although these activities came out in separate fractions from an anion exchange column, most likely they are the same protein on the basis of immunology and N-terminal sequences. BE2a, however, is 3 kDa larger. It is possible that BE2a may be the less proteolyzed form. Antibody raised against BE3 reacted strongly against BE3 but not against, BE1, BE2a, and BE2b. Thus, rice endosperm, as noted for maize endosperm, has essentially two different isoforms of BE. Because of the many isoforms existing for rice seed BEs, Yamanouchi and Nakamura (1992) studied and compared the BEs from rice endosperm, leaf blade, leaf sheath, culm, and root. BE activity could be resolved into two fractions, BE1 and BE2. Both fractions were found in all tissues studied in different ratios of activity. The specific activity of the endosperm enzyme, either on the basis of fresh weight or protein, was 100- to 1000-fold greater than that of the enzyme from other tissues studied. On gel electrophoresis, rice endosperm BE2 could be resolved into two fractions, BE2a and BE2b. Of interest was that electrophoresis of the other tissue BE2 forms revealed only BE2b. BE2a was only detected in the endosperm tissue. In rice, it seems, there are tissue-specific isoforms of BE. Three forms of BE from developing hexaploid wheat (Triticum aestivum) endosperm have been partially purified and characterized (Morell et al., 1997). Two forms are immunologically related to maize BEI and one form with maize BEII. The N-terminal sequences are consistent with these relationships. The wheat BEIB gene is located on chromosome 7B while the wheat BEIAD peptide genes are located on chromosomes 7A and 7D. The BE classes in wheat are differentially expressed during endosperm development in that BEII is constitutively expressed throughout the whole cycle while BEIB and BEIAD are expressed in late endosperm development. McCue et al. (2002) showed that in wheat (Triticum aestivum cv. Cheyenne), cDNAs encoding BE1 and BEII shared extensive identity with BE sequences reported for wheat and other plants. Using the cDNAs they studied the steady state RNA levels of the BEs during development. For BE2 its RNA was detectable 5 days postanthesis (DPA) and reached a maximum 10 days post-DPA. BE1 steady-state levels started rising at 10 DPA peaking at 15 DPA. Levels of all messages declined at 20e25 DPA. In barley, four BE isozymes were identified by separation on FPLC and three were partially purified (Sun et al., 1997). Subsequently, two cDNA clones encoding the barley BEIIa and BEIIb were isolated (Sun et al., 1998). The major structural difference between the two enzymes was the presence of a 94-amino acid N-terminal extension in the BEIIb precursor.


8.10.3 Genetic Studies on Branching Enzyme-Deficient Mutants There are some maize endosperm mutants that appear to increase the amylose contents of starch granules. The normal maize starch granule contains about 25% of the polysaccharide as amylose, with the rest as amylopectin. In contrast, amylose extender (ae) mutants may have as much as 55%e70% of the polysaccharide as amylose and may have an amylopectin fraction with fewer branch points and with the branch chains longer in length compared to those of normal amylopectin. Results with the recessive maize endosperm mutant, ae, originally suggested that ae is the structural gene for either BEIIa or BEIIb as the activity of BEI was not affected by the mutation (Boyer and Preiss, 1978b, 1981; Preiss and Boyer, 1980). In gene dosage experiments, Hedman and Boyer (1982) reported a near-linear relationship between increased dosage of the dominant ae allele and BEIIb activity. Since the separation of form IIa from IIb was not very clear, it is possible that the ae locus was also affecting the level of IIa. Singh and Preiss (1985) concluded that, although some homology exists between the three starch BEs, there are major differences in the structure of BEI when compared with BEIIa and BEIIb as shown by its different reactivity with some monoclonal antibodies and differences in amino acid composition and proteolytic digest maps. Recent studies by Fisher et al. (1993, 1996). Analyzing 16 isogenic lines having independent alleles of the maize ae locus suggest that BEIIa and BEIIb are encoded by separate genes and the BEIIb enzyme is encoded by the ae gene. They isolated a cDNA clone labeled Sbe2b, which had a cDNApredicted amino acid sequence at residues 58e65 exactly the same as the N-terminal sequence of the maize BEIIb that they had purified (Fisher et al., 1993). Moreover, they did not detect in ae endosperm extracts any mRNA with the Sbe2b cDNA clone. There was some BE activity in the ae extracts but on ionexchange chromatography the activity associated with the BEIIa fraction. The finding that the enzyme defect in the ae mutant is BEIIb is consistent with the finding that, in vitro, BEII is involved in transfer of small chains. The ae mutant has not only an increased amount in amylose content but also the amylopectin structure is altered in having fewer and longer chains and a lesser number of total chains. In other words, there are very few short chains. Wrinkled pea has a reduced starch level; 66%e75% of that seen in the normal round seed. The amylose content is about 33% in the WT round form but is 60%e70% in the wrinkled pea seed. Edwards et al. (1988) measured several enzyme activities involved in starch metabolism at four different developmental stages of wrinkled pea and found that BE activity at its highest level was only 14% of that observed for the round seed variety. The other starch biosynthetic enzymes as well as phosphorylase had similar activities in wrinkled and round seeds. Confirmation of these results also showed that the


69

r(rugosus) lesion in the wrinkled pea of genotype rr, was associated with the absence of one isoform of BE (Smith, 1988). Edwards et al. (1988) postulated that the reduction in starch content observed in the BE-deficient mutant seeds is an indirect effect of the reduced BE activity through an effect on SS activity. The authors suggested that, in the absence of BE activity, for the SS isozymes, an a-1,4-glucosyl elongated chain is a poor glucosyl-acceptor (primer) for the SS substrate, ADP-Glc therefore decreasing the rate of a-1,4-glucan synthesis. This had been shown in a similar system, in a study of rabbit muscle glycogen synthase (Carter and Smith, 1978) where it was found that continual elongation of the outer chains of glycogen caused it to become a less efficient primer with a higher Km, thus decreasing the apparent activity of the glycogen synthase. The finding that ADP-Glc in the wrinkled pea accumulated to a higher concentration than in normal pea was considered evidence that the in vivo activity of the SS was restricted. Under in vitro conditions, where a suitable primer such as amylopectin or glycogen is added, the SS activity in the wrinkled pea was equivalent to that found for the WT. Ae mutants have been found in rice (Mizuno et al., 1993). The alteration of the starch structure is very similar to that reported for the maize endosperm ae mutants. The defect is in the BE3 isozyme, BE3 of rice being more similar in amino acid sequence to maize BEII than to BEI (Mizuno et al., 1993; Guan et al., 1994b). Thus, rice BE3 may catalyze the transfer of small chains, rather than long chains. Of interest was the use of gene silencing through RNA interference (RNAi) technology to silence the expression of wheat BEIIa and BEIIb that resulted in a high-amylose (>70%) phenotype (Regina et al., 2006). The critical aspect was to suppress expression of BEIIa. Suppression of BEIIb alone had no effect. Suppression of BEIIa decreased markedly the proportion of glucose CLs of 4e12 with a corresponding increase of CLs greater than 12. When this high-amylose starch was fed to rats in a diet as a whole meal, several indices of large bowel function, including short-chain fatty acids, were improved relative to a standard whole-meal wheat. The results indicated that the high-amylose wheat has a significant potential to improve human health through the high-amylose starch content resistant to digestion (Regina et al., 2006).

8.10.4 Isolation of cDNA Clones Encoding the Branching Enzyme Isozyme Genes The r locus of pea seed was cloned using an antibody toward one of the pea BE isoforms and screening a cDNA library (Bhattacharyya et al., 1990). However, the BE gene in the wrinkled pea contained an 800 bp insertion. Thus expression of the cDNA yielded an inactive BE. The sequence of the 2.7 kB clone had more than 50% homology to the glycogen BE of E. coli (Baecker et al., 1986) and thus it was proposed that the cloned cDNA corresponded to the starch BE gene of pea seed. The glg B gene has been sequenced for a


cyanobacterium (Kiel et al., 1990), and its deduced amino acid sequence is 62% identical extensive to the amino acid sequence in the middle area of the E. coli protein. This is the portion of the sequence that contains the amino acid residues critical for catalysis. Therefore, BEs in nature have extensive homology irrespective of the degree of branching of their products, that is higher (about 10% a-1,6 linkages) in glycogen, the storage polysaccharide in mammals, enteric bacteria, and in cyanobacteria, than in the amylopectin (about 5% a-1,6 linkages) present in higher plants. It would be of interest in determining the difference in catalysis between the BEs that are involved in the synthesis of amylopectin and those involved in the catalysis of bacterial or mammalian glycogen. Can it be due to different interactions of BE with the SSs or glycogen synthases or are the differences inherent in the BE catalysis? The answer to this question could also resolve, in part as to why the starch granules in different plants are unique in their granule structure. cDNA clones of genes representing different isoforms of BE of different plants have been isolated from potato tuber (Khoshnoodi et al., 2004; Kossmann et al., 1991; Poulsen and Kreiberg, 1993), maize kernel (Guan et al., 1994a; Guan and Preiss, 1993; Fisher et al., 1993, 1996), cassava (Salehuzzaman et al., 1992), and rice seeds (BEI (Mizuno et al., 1993; Nakamura and Yamanouchi, 1992), BEIII (Mizuno et al., 1993)). The cDNA clones of maize BEI and BEII have been overexpressed in E. coli and purified (Guan et al., 1994a; Guan and Preiss, 1993). The transgenic enzymes had the same properties as seen with the natural maize endosperm BEs with respect to specific activity and specificity toward amylose and amylopectin (Guan et al., 1994a). It is important to note that in vitro, maize BEI and BEII have different properties. BEI had a higher rate of branching amylose than branching amylopectin and preferentially transferred longer chains. In contrast, maize BEII had a lower rate of branching amylose than branching amylopectin and preferentially transferred shorter chains (Takeda et al., 1993; Guan and Preiss, 1993).

8.10.5 Reserve Tissue Branching Enzyme Is Localized in the Plastid The localization of BE within the plastid has been determined in potato (Kram et al., 1993) using antibodies raised against potato BE and immunogold electron microscopy. The enzyme (i.e., the equivalent of the BEI isoform of maize, as indicated above) was found in the amyloplast, concentrated at the interface between the stroma and the starch granule, rather than throughout the stroma, as it is the case with the ADP-Glc PPase (Kim et al., 1989; Jacquot et al., 1997). This would explain how amylose synthesis is possible when the enzyme responsible for its formation, that is, the Wx protein, GBSS, is capable of elongating both linear and branched glucans. The spatial separation of BE isoforms from the GBSSs, even if only partial,


71

would allow the formation of amylose without it being subsequently branched by the BE. However, even if spatial separation did not exist, starch crystallization may have the same effect, that is, prevent further branching. Unpublished experiments (Morell and Preiss, 1987) found that about 5% of the maize endosperm total BE activity was associated with the starch granule after amylase digestion. Whether this BE was similar to the soluble BEs was not determined. However, other studies (Preiss and Sivak, 1996) showed that a polypeptide of about 80 kD present in maize and pea starch reacted with antibodies raised against maize BE1. However, small amounts of BE are expected to sediment with the starch granules because of its affinity for the polysaccharide. The results are similar to those reported by Mu-Forster et al. (1996).

8.10.6 Branching Enzyme Belongs to the a-Amylase Family Amino acid sequence relationships between that of BE and amylolytic enzymes, such as a-amylase, pullulanase, glucosyl-transferase, and cyclodextrin glucanotransferase, especially at those amino acids believed to be contacts between the substrate and the amylase family enzymes, was first reported by Romeo et al. (1998). Baba et al. (1991) reported that there was a marked conservation in the amino acid sequence of the four catalytic regions of amylolytic enzymes in maize endosperm BEI. As shown in Table 1.10, four regions that putatively constitute the catalytic regions of the amylolytic enzymes are conserved in the starch branching isoenzymes of maize endosperm, rice seed, and potato tuber and the glycogen BE of E. coli. A very extensive analysis of this high conservation in the a-amylase family has been reported by Svensson et al. (Svensson, 1994; Jespersen et al., 1993) with respect to sequence homology and also in the prediction of the (b/a)8-barrel structural domains with a highly symmetrical fold of eight inner, in the family. The (b/a) 8-barrel structural domain is based on crystal structures of some a-amylases and cyclodextrin glucanotransferases. The conservation of the putative catalytic sites of the a-amylase family in the glycogen and SBEs may be anticipated as BE catalyzes two continuous reactions: cleavage of a 1,4-a-glucosidic linkage in a 1,4-a-glucan chain yielding an oligosaccharide chain which is transferred to an O-6 of the same chain, or to another 1,4-a-glucan chain, with synthesis of a new (1e6)a-glucosidic linkage. 8.10.7 Amino Acid Residues That Are Functional in Branching Enzyme Catalysis As indicated in Table 1.10, four regions which constitute the catalytic regions of the amylolytic enzymes are conserved in the starch branching isoenzymes of maize endosperm, rice seed, and potato tuber and the glycogen BEs of E. coli (Svensson, 1994; Jespersen et al., 1993). It would be of interest to know


TABLE 1.10 Comparison of Primary Structures of Various Branching Enzymes With the Four Most Conserved Regions of the a-Amylase Family Region 1

Region 2

Maize endosperm BE I

277

DVVHSH

347

GFRFDGVTS

Maize endosperm BE II

315

DVVHSH

382

GFRFDGVTS

Potato tuber BE

355

DVVHSH

424

GFRFDGITS

Rice seed BE 1

271

DVVHSH

341

GFRFDGVTS

Rice seed BE 3

337

DVVHSH

404

GFRFDGVTS

Escherichia coli glycogen BE

335

DWVPGH

400

ALRVDAVAS

Bacillus subtilis a-amylase

100

DAVINH

171

GFRFDAAKH

Bacillus sphaericus cyclodextrinase

238

DAVFNH

323

GWRLDVANE

Pseudomonas amyloderamosa isoamylase

291

DVVYNH

370

GFRFDLASV

Region 3

Region 4

Maize endosperm BE I

402

TVVAEDVS

470

CIAYAESHD

Maize endosperm BE II

437

VTIGEDVS

501

CVTYAESHD

Potato tuber BE

453

VTMAEEST

545

CVTYAESHD

Rice seed BE 1

396

TIVAEDVS

461

CVTYAESHD

Rice seed BE 3

459

ITIGEDVS

524

CVTYAESHD

E. coli glycogen BE

453

VTMAEEST

517

NVFLPLNHD

B. subtilis a-amylase

204

FQYGEILQ

261

LVTWVESHD

B. sphaericus cyclodextrinase

350

IIVGEVWH

414

SFNLLGSHD

P. amyloderamosa isoamylase

412

RILREFTV

499

SINFIDVHD

The sequences have been derived from references referred to in the text. Three examples of enzymes from the a-amylase family are compared. Svensson’s group (Svensson, 1994; Jespersen et al., 1993) compares over 40 enzymes ranging from amylases, glucosidases, various a-1,6-debranching enzymes as well as branching enzymes. The invariant amino acid residues believed to be involved in catalysis are in bold letters.

whether the seven highly conserved amino acid residues of the a-amylase family listed in bold letters in Table 1.10 are also functional in BE catalysis. Experiments such as chemical modification and analysis of the threedimensional structure of the BEs have been carried out to determine the nature of its catalytic residues and mechanism. The seven highly conserved amino acid residues of the a-amylase family appear to be also functional in BE catalysis. A series of experiments (Kuriki et al., 1996), in which amino acids were replaced by site-directed mutagenesis,


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do suggest that the conserved Asp residues of regions two and four and the Glu residue of region 3 (Table 1.10, in bold letters) are important for BEII catalysis. Arginine residues are also important, as suggested by chemical modification with phenylglyoxal (Cao and Preiss, 1996), as well as histidine residues as suggested by chemical modification studies with diethyl pyrocarbonate (Funane et al., 1998). Also, the regions of the C-terminus and N-terminus that are dissimilar in sequence and size in the various branching isoenzymes, BEI and BEII, of maize endosperm may be of importance in catalysis with respect to substrate preference (amylose or amylopectin) or size of chain transferred during catalysis. Indeed, studies by Kuriki et al. (1997) with respect to the different N- and Ctermini indicate that these amino acid sequence regions are important with respect to BE specificity with respect to substrate preference (amylose or amylopectin) as well as in size of chain transferred and extent of branching. The C-terminal was functional with respect to substrate preference while the N-terminal was functional with respect to the size of chain transferred in the BE catalysis. Furthermore, truncation of 113 amino acids of the N-terminal of the E. coli BE causes it to branch longer branch chains than the WT enzyme (Binderup et al., 2000, 2002; Devillers et al., 2003) further indicating that the N-terminal region was involved in specifying the size of the chain transferred. The crystal structure of a truncated form of the E. coli BE has been ˚ (Abad et al., 2002). The enzyme consists of elucidated at a resolution of 2.3 A three major domains, an NH2-terminal seven-stranded b-sandwich domain, a COOH-terminal domain, and a central a/b-barrel domain having the enzyme active site and the catalytic amino acids shown to be involved in catalytic action. Oligosaccharide binding was modeled for BE using the enzymee oligosaccharide complex structures of various a-amylases and cyclodextrin glucanotransferase. Residues were implicated in oligosaccharide binding. The crystal structure of the Oryza sativa L BE1 has been crystallized (Noguchi ˚ by et al., 2011). Its crystal structure was elucidated at a resolution of 1.9 A molecular replacement using the E. coli glycogen BE as a search model. Asp344 and Glu399, which are amino acids postulated to play an essential role in catalysis as a nucleophile and a general acid/base, respectively, are located at a central cleft in the groove.

8.10.8 Other Enzymes Involved in Starch Synthesis In addition to the three starch biosynthetic enzymes, other enzymes have been shown to have some effect on starch structure and starch synthesis. These enzymes will be briefly reviewed. 8.10.9 Isoamylase As previously indicated, a su1 mutation in maize, which causes a deficiency of isoamylase, an enzyme normally considered to be mainly involved in starch


degradation in plants, maize and rice, or in C. reinhardtii, results in the accumulation of a water-soluble polysaccharide, phytoglycogen instead of starch (Pan and Nelson, 1984; James et al., 1995; Nakamura et al., 1996, 1997; Rahman et al., 1998; Dinges et al., 2001, 2003; Myers et al., 2000; Kubo et al., 1999). In C. reinhardtii (Ball et al., 1996), the mutation results in complete loss of starch but in higher plants the lesser amount of amylopectin seen in the mutant plant may be due to the severity of the enzyme deficiency. It was proposed that “maturation” or trimming of the precursor of amylopectin, “preamylopectin,” was required in order for the amylopectin to aggregate into an insoluble granule structure (Zeeman et al., 1998). An alternative proposal is that during amylopectin synthesis there is a competition between polysaccharide aggregation into granule starch and formation of water-soluble polysaccharide and that the water-soluble polysaccharide, phytoglycogen, is consistently degraded by isoamylase (Zeeman et al., 1998). If isoamylase activity is deficient, then the phytoglycogen accumulates and competes with amylopectin for the starch biosynthetic enzymes. The amount of phytoglycogen accumulation is dependent on the degree of loss of isoamylase activity (Zeeman et al., 1998). This competition concept between amylopectin aggregation and phytoglycogen accumulation can also explain why in certain isoamylase mutations the two polysaccharides, phytoglycogen and amylopectin, may be simultaneously present (Zeeman et al., 1998). Although there is no question that isoamylase deficiency is the cause of phytoglycogen accumulation and lower starch accumulation in the mutant plant, the mechanism for phytoglycogen accumulation is still not completely understood.

8.10.10 Phosphorylase Starch phosphorylase was originally considered an enzyme only involved in the degradation of starch as the Pi concentration was considerably higher than the Glc-1-P concentration and the equilibrium constant is close to one (w2.2) (Hanes, 1940). The reversible reaction phosphorylase catalyzes is seen below. Glucose-1-P þ a-Glucan(n) 4 a-Glucan(n þ 1) þ Pi Phosphorylase in plants exists as two isozymes generally labeled as Pho1 and Pho2. The location of Pho1 is in the plastid while Pho2 is present in the cytoplasm (Okita et al., 1979; Shimomura et al., 1982; Nakano and Fukui, 1986; Mori et al., 1993). Pho1 research results now indicate that the two phosphorylases have different functions in starch metabolism. In Chlamydomonas an isoform of a plastidial phosphorylase, normally considered a degradative enzyme, does play some role in the synthesis of starch (Dauvilleé et al., 2006). Mutants of the plastid enzyme have significant reduced levels of starch, w34% of normal. Moreover, the mutant amylopectin structure is altered with respect to chain size distribution and there is a higher than normal amylose content in the mutant. The starch granule shape was also different.


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A similar result was found in rice (Satoh et al., 2008). In rice, plastidial phosphorylase (Pho1) accounts for 96% of the phosphorylase activity. Mutants of Pho1 resulted in seeds that had variable amounts of starch that varied due to growth at different temperatures. When grown at 30 C starch levels were close to normal while growth at 20 C caused the seeds to be shrunken having 20% of the normal starch levels. Thus at low temperatures Pho1 plays an important role in starch synthesis while at higher temperatures other components can either complement or substitute for the Pho1 function in starch synthesis. In Arabidopsis, mutants having no Pho1 does not cause any significant change in starch accumulation during the day or in its remobilization at night (Zeeman et al., 2004). Starch structure and composition was also unaltered. Thus from different plants the question whether plant starch phosphorylase is involved with another function can be considered. Of interest is a report in rice of an interaction between rice Pho1 and rice disproportionating enzyme (Dpe1) (Hwang et al., 2016). Various binding experiments (coimmunoprecipitation, chromatographic coelution, and electrophoretic comigration) showed that Pho1 and Dpe1 formed a 1:1 complex. Rice Pho1 is capable of synthesizing short-chain maltodextrins (Hwang et al., 2010). In the presence of Dpe1 an accelerated synthesis of larger maltodextrins was observed. Moreover, the Dpe1 increased the Pho1 apparent affinity for Glc-1-P at the lower temperature of 20 C. Further studies may provide information on the possible role of the OsPho1:OsDpe1 complex in initiating starch synthesis. The two Pho isozymes can be distinguished in molecular size, as well as substrate specificity (Albrecht et al., 1998, 2001; Rathore et al., 2009). Plastidial phosphorylases of higher plants have about a 78e80 amino acid insertion located between the N- and the C-terminal domains. This was first shown in potato (Nakano and Fukui, 1986). The plastidial Pho1-form has monomeric size of approximately 105 kDa, and has low affinity to branched glucans, glycogen, and starch. On the other hand, the Pho2-form has a monomeric size of approximately 90 kDa, which has very high affinity to linear and branched glucans. Arabidopsis Pho2, restricted to the cytosol, is involved in maltose metabolism preferring high-molecular weight glycans as glucosyl acceptors (Lu et al., 2006). In leaves starch is hydrolyzed via amylases and maltose is the predominant form transported from the chloroplast to the cytosol. Pho2 acts on soluble heteroglycans in the cytosol along with disproportionating enzyme (Lu et al., 2006; Fettke et al., 2005). Evidence for the role of Pho2 in Arabidopsis was obtained by inactivation of the enzyme with T-DNA insertion. The Pho2 mutant accumulated at night 4-times the night time level of maltose (Lu et al., 2006).

8.10.11 a-1,4-Glucanotransferase (D-Enzyme) Some starch-deficient mutants of Arabidopsis and Chlamydomonas have been shown to be defective in a-1,4-glucanotransferase activity. The enzyme is also known as D-enzyme and the reaction it catalyzes is shown below. Maltotriose þ Maltotriose 4 D-Maltopentaose þ Glucose


Other oligosaccharides can also act as substrates and in the reaction glucose is formed. The transglucosylase of Arabidopsis leaf disproportionates maltotriose and forms higher maltodextrins, much greater in comparison to maltooctaose (Lin and Preiss, 1988). Essentially maltose units are transferred from maltotriose to maltooligosaccharides to increase their size by two glucose units. A C. reinhardtii mutant, STA11, lacking D-enzyme activity has been characterized and has been shown to have significantly lower levels of starch (Colleoni et al., 1999). Other enzymes involved in starch metabolism such as ADP-Glc PPase, granule-bound and soluble SS, BE, phosphorylase, a-glucosidase, amylases, and debranching enzyme activities were not affected (Colleoni et al., 1999). The starch content in the mutant was about 6%e13% of WT and there was an excessive accumulation of maltooligosaccharides up to a polymer size of 16 glucose units. Further studies showed that STA11 is the gene for D-enzyme and its cDNA was cloned (Wattebled et al., 2003). Both the maltooligosaccharide accumulation and starch synthesis were studied in the WT and D-enzyme mutant during day and night cycles. The maltooligosaccharides accumulated during starch synthesis and were degraded during starch degradation in both the mutant and WT. At present it is not clear how D-enzyme deficiency causes a lowering of starch accumulation in C. reinhardtii. Actually an earlier report showed that a 98% reduction in D-enzyme activity in potato tuber and leaves had no effect on starch synthesis (Takaha et al., 1998). The lack of D-enzyme did cause slowing of plant growth but plant development appeared normal. Both starch content or the proportion of amylose in the granule was altered. The amylopectin structure was not altered. Thus in potato the lack of D-enzyme showed no evidence for involvement in synthesis or starch storage (Takaha et al., 1998). Also in Arabidopsis it has been shown that mutants of D-enzyme overproduced leaf starch and the amylose to amylopectin ratio increased (Critchley et al., 2001). The amylopectin structure was unaltered and overproduction of maltodextrins by the mutant occurred only during the process of starch degradation and decreased during starch synthesis (Critchley et al., 2001). The conclusion was that D-enzyme is required for maltodextrin metabolism during starch degradation. The result is different from what was observed for Chlamydomonas where maltodextrin accumulation occurred during starch synthesis and decreased during starch degradation. Whether the D-enzyme plays a role both in starch synthesis as well as in degradation is an intriguing question. In summary, much information in the past 10 years has been obtained on the enzymes involved in starch synthesis. However, a number of problems remain. There is no question that the major allosteric regulation of starch synthesis occurs at the ADP-Glc PPase-catalyzed reaction and is dependent on the ratio of 3PGA to Pi. The detailed process of how that ratio is regulated in storage and other plant nonphotosynthetic tissue still remains to be elucidated.


77

How the multitude of different isoforms of SSs and BEs coordinate their activities with the BE isoforms in synthesizing both amylose and amylopectin is still a formidable problem that requires much more detail. Of recent interest are the findings that enzymes such as isoamylase, D-enzyme, and phosphorylase are important in the synthesis of the starch granule. Their precise roles remain yet to be elucidated.

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Boyer, C.D., Preiss, J., 1978a. Multiple forms of (1 / 4)-a-d-glucan, (1 / 4)-a-d-glucan-6glycosyl transferase from developing Zea mays L. kernels. Carbohydrate Research 61, 321e334. Boyer, C.D., Preiss, J., 1978b. Multiple forms of starch branching enzymes of maize: evidence for independent genetic control. Biochemical and Biophysical Research Communications 80, 169e175. Boyer, C.D., Preiss, J., 1981. Evidence for independent genetic control of the multiple forms of maize endosperm branching enzymes and starch synthases. Plant Physiology 67, 1141e1145. Brown, K., Pompeo, F., Dixon, S., Mengin-Lecreulx, D., Cambillau, C., Bourne, Y., 1999. Crystal structure of the bifunctional N-acetylglucosamine 1-phosphate uridyltransferase from Escherichia coli: a paradigm for the related pyrophosphorylase superfamily. EMBO Journal 18, 4096e4107. Burton, R.A., Johnson, P.E., Beckles, D.M., Fincher, G.B., Jenner, H.I., Naldrett, M.J., Denyer, K., 2002. characterization of the genes encoding the cytosolic and plastidial forms of ADPglucose pyrophosphorylase in wheat endosperm. Plant Physiology 130, 1464e1475. Cakir, B., Shiraishi, S., Tuncel, A., Matsusaka, H., Satoh, R., Singh, S., Crofts, N., Hosaka, Y., Fujita, N., Hwang, S.-K., Satoh, H., Okita, T.W., 2016. Analysis of the rice ADP-glucose transporter (OsBT1) indicates the presence of regulatory processes in the amyloplast stroma that control ADP-glucose flux into starch. Plant Physiology 170, 1271e1283. Cao, H., Preiss, J., 1996. Evidence for essential arginine residues at the active sites of maize branching enzymes. Journal of Protein Chemistry 15, 291e304. Cao, H., Sullivan, T.D., Boyer, C.D., Shannon, J.C., 1995. Btl, a structural gene for the major 3944 kDa amyloplast membrane polypeptides. Physiologia Plantarum 95, 176e186. Cao, H., Imparl-Radosevich, J., Guan, H., Keeling, P.L., James, M.G., Meyers, A.M., 1999. Identification of the soluble starch synthase activities of maize endosperm. Plant Physiology 120, 205e216. Carter, J., Smith, E.E., 1978. Actions of glycogen synthase and phosphorylase of rabbit-skeletal muscle on modified glycogens. Carbohydrate Research 61, 395e406. Caspar, C., Huber, S.C., Somerville, C., 1986. Plant Physiology 79, 1e7. Charng, Y.-Y., Iglesias, A.A., Preiss, J., 1994. Structure-function relationships of cyanobacterial ADP-glucose pyrophosphorylase: site-directed mutagenesis and chemical modification of the activator-binding sites of ADP-glucose pyrophosphorylase from Anabaena PCC 7120. Journal of Biological Chemistry 269, 24107e24113. Chen, B.-Y., Janes, H.W., 1997. Multiple forms of ADP-glucose pyrophosphorylase from tomato fruit. Plant Physiology 113, 235e241. Chen, B.-Y., Janes, H.W., Gianfagna, T., 1998a. PCR cloning and characterization of multiple ADP-glucose pyrophosphorylase cDNAs from tomato. Plant Science 136, 59e67. Chen, B.-Y., Wang, Y., Janes, H.W., 1998b. ADP-glucose pyrophosphorylase is localized to both the cytoplasm and plastids in developing pericarp of tomato fruit. Plant Physiology 116, 101e106. Clarke, B.R., Denyer, K., Jenner, C.F., Smith, A.M., 1999. The relationship between the rate of starch synthesis, the adenosine 50 -diphosphoglucose concentration and the amylose content of starch in developing pea embryos. Planta 209, 324e329. Colleoni, C., Dauvilleé, D., Mouille, G., Buleón, A., Gallant, D., Bouchet, B., Morell, M., Samuel, M., Delrue, B., D’Hulst, C., Bliard, C., Nuzillard, J.-M., Ball, S., 1999. Genetic and biochemical evidence for the involvement of a-1,4 glucanotransferases in amylopectin synthesis. Plant Physiology 120, 993e1004.

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Takaha, T., Critchley, J., Okada, S., Smith, S.M., 1998. Normal starch content and composition in tubers of antisense potato plants lacking D-enzyme (4-a-glucanotransferase). Planta 205, 445e451. Takeda, Y., Guan, H.P., Preiss, J., 1993. Branching of amylose by the branching isoenzymes of maize endosperm. Carbohydrate Research 240, 253e263. Tenorio, G., Orea, A., Romero, J.M., Merida, A., 2003. Oscillation of mRNA level and activity of granule-bound starch synthase I in Arabidopsis leaves during the day/night cycle. Plant Molecular Biology 51, 949e958. Tetlow, I.J., Davies, E.J., Vardy, K.A., Bowsher, C.G., Burrell, M.M., Emes, M.J., 2003. Subcellular localization of ADPglucose pyrophosphorylase in developing wheat endosperm and analysis of the properties of a plastidial isoform. Journal of Experimental Botany 54, 715e725. Thorbjørnsen, T., Villand, P., Kleckowski, L.A., Olsen, O.A., 1996a. A single gene encodes two different transcripts for the ADPglucose pyrophosphorylase small subunit from barley (Hordeum vulgare). Biochemical Journal 313, 149e154. Thorbjørnsen, T., Villand, P., Denyer, K., Olsen, O.-A., Smith, A., 1996b. Distinct forms of ADPglucose pyrophosphorylase occur inside and outside the amyloplasts in barley endosperm. Plant Journal 10, 243e250. Tiessen, A., Hendriks, J.H.M., Stitt, M., Branscheid, A., Gibon, Y., Farre´, E.M., Geigenberger, P., 2002. Starch synthesis in potato tubers is regulated by post-translational redox modification of ADP-glucose pyrophosphorylase: a novel regulatory mechanism linking starch synthesis to the sucrose supply. Plant Cell 14, 2191e2213. Tiessen, A., Prescha, K., Branscheid, A., Palacios, N., McKibbin, R., Halford, N.G., Geigenberger, P., 2003. Evidence that SNF1-related kinase and hexokinase are involved in separate sugar-signaling pathways modulating post-translational redox activation of ADPglucose pyrophosphorylase in potato tubers. Plant Journal 35, 490e500. Toyosawa, Y., Kawagoe, Y., Matsushima, R., Crofts, N., Ogawa, M., Fukuda, M., Kumamaru, T., Okazaki, Y., Kusano, M., Saito, K., Toyooka, K., Sato, M., Ai, Y., Jane, J.-L., Nakamura, Y., Fujita, N., 2016. Deficiency of starch synthase IIIa and IVb alters starch granule morphology from polyhedral to spherical in rice endosperm. Plant Physiology 170, 1255e1270. Tsai, C.Y., 1974. The function of the Waxy locus starch synthesis in maize endosperm. Biochemical Genetics 11, 83e96. Tuncel, A., Cakir, B., Hwang, S.-K., Okita, T., 2014a. The role of the large subunit in redox regulation of the rice endosperm ADP-glucose pyrophosphorylase. FEBS Journal 281, 4951e4963. Tuncel, A., Kawaguchi, J., Ihara, Y., Matsusaka, H., Nishi, A., Nakamura, T., Kuhara, S., Hirakawa, H., Nakamura, Y., Cakir, B., Nagamine, A., Okita, T.W., Hwang, S.-K., Satoh, H., 2014b. The rice endosperm ADP-glucose pyrophosphorylase large subunit is essential for optimal catalysis and allosteric regulation of the heterotetrameric enzyme. Plant and Cell Physiology 55, 1169e1183. Umemoto, T., Yano, M., Satoh, H., Shomura, A., Nakamura, Y., 2002. Mapping of a gene responsible for the difference in amylopectin structure between japonica-type and indica-type rice varieties. Theoretical and Applied Genetics 104, 1e8. Uzoma, D., Arias-Garzon, S., Sayre, L.R., 2006. Genetic modification of cassava for enhanced starch production. Plant Biotechnology Journal 4, 453e465. Van den Koornhuyse, N., Libessart, N., Delrue, B., Zabawinski, C., Decq, A., Iglesias, A., Preiss, J., Ball, S., 1996. Control of starch composition and structure through substrate supply

94 PART j ONE Analyzing and Modifying Starch in the monocellular alga Chlamydomonas reinhardtii. Journal of Biological Chemistry 271, 16281e16287. Ventriglia, T., Ballicora, M.A., Teresa Ruı´z, M., Pedro, M.R., Valverde, F., Preiss, J., Romero, J.M., 2008. Two Arabidopsis ADP-glucose large subunits (APL1 and APL2) are catalytic. Plant Physiology 148, 65e76. Vos-Scheperkeuter, G.H., de Wit, J.G., Ponstein, A.S., Feenstra, W.J., Witholt, B., 1989. Immunological comparison of the starch branching enzymes from potato tubers and maize kernels. Plant Physiology 90, 75e84. Vrinten, P.L., Nakamura, T., 2000. Wheat granule-bound starch synthase I and II are encoded by separate genes that are expressed in different tissues. Plant Physiology 122, 255e263. Van de Wal, M., D’Hulst, C., Vincken, J.-P., Buleón, A., Visser, R., Ball, S.J., 1998. Amylose is synthesized in vitro by extension of and cleavage from amylopectin. Journal of Biological Chemistry 273, 22232e22240. Wang, T.L., Hadavizideh, A., Harwood, A., Welham, T.J., Harwood, W.A., Faulks, R., Hedley, C.L., 1990. An analysis of seed development in Pisum sativum. XIII. The chemical induction of storage product mutants. Plant Breed 105, 311e320. Wang, Y.-J., White, P., Pollak, L., Jane, J.-L., 1993a. Amylopectin and intermediate materials in starches from mutant genotypes of the Oh43 inbred line. Cereal Chemistry 70, 521e529. Wang, Y.-J., White, P., Pollak, L., Jane, J.-L., 1993b. Characterization of starch structures of 17 maize endosperm mutant genotypes with Oh43 inbred line background. Cereal Chemistry 70, 171e179. Wattebled, F., Buleon, A., Bouchet, B., Ral, J.-P., Lienard, L., Delvalle, D., Binderup, K., Dauvillee, D., Ball, S., D’Hulst, C., 2002. Granule-bound starch synthase I. A major enzyme involved in the biogenesis of B-crystallites in starch granules. European Journal of Biochemistry 269, 3810e3820. Wattebled, F., Ral, J.P., Dauvilleé, D., Myers, A.M., James, M.G., Schlichting, R., Giersch, C., Ball, S.G., D’Hulst, C., 2003. STA11, a Chlamydomonas reinhardtii locus required for normal starch granule biogenesis, encodes disproportionating enzyme. Further evidence for a function of a-1,4 glucanotransferases during starch granule biosynthesis in green algae. Plant Physiology 132, 137e145. Wessler, S.R., Baran, G., Varagona, M., Dellaporta, S.L., 1986. Excision of Ds produces waxy proteins with a range on enzymatic activities. EMBO Journal 5, 2427e2432. Wu, M.X., Preiss, J., 1998. The N-terminal region is important for the allosteric activation and inhibition of the Escherichia coli ADP-glucose pyrophosphorylase. Archives of Biochemistry and Biophysics 358, 182e188. Wu, M.X., Preiss, J., 2001. Truncated forms of the recombinant Escherichia coli ADP-glucose pyrophosphorylase: the importance of the N-terminal region for allosteric activation and inhibition. Archives of Biochemistry and Biophysics 389, 159e165. Yamamori, M., Fujita, S., Hayakawa, K., Matsuki, J., Yasui, T., 2000. Genetic elimination of a starch granule protein, SGP-1, of wheat generates an altered starch with apparent high amylose. Theoretical and Applied Genetics 101, 21e29. Yamanouchi, H., Nakamura, Y., 1992. Organ specificity of isoforms of starch branching enzyme (Q-enzyme) in rice plant cell. Plant and Cell Physiology 33, 985e991. Yep, A., Ballicora, M.A., Sivak, M.N., Preiss, J., 2004a. Identification and characterization of a critical region in the glycogen synthase from Escherichia coli. Journal of Biological Chemistry 279, 8359e8367.


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Yep, A., Ballicora, M.A., Preiss, J., 2004b. The active site of the Escherichia coli glycogen synthase similar to the active site of retaining GT-B glycosyltransferases. Biochemical and Biophysical Research Communications 316, 960e966. Yep, A., Ballicora, M.A., Preiss, J., 2006. The ADP-glucose binding site of the Escherichia coli glycogen synthase. Archives of Biochemistry and Biophysics 453, 188e196. Zeeman, S.C., Umemoto, T., Lue, W.L., Auyeung, P., Martin, C., Smith, A.M., Chen, J., 1998. A mutant of Arabidopsis lacking a chloroplastic isoamylase accumulates both starch and phytoglycogen. Plant Cell 10, 1699e1711. Zeeman, S.C., Smith, S.S., Smith, A.M., 2002. The priming of amylose synthesis in Arabidopsis leaves. Plant Physiology 128, 1069e1076. Zeeman, S.C., Thorneycroft, D., Schupp, N., Chapple, A., Weck, M., Dunstan, H., Haldimann, P., Bechtold, N., Smith, A.M., Smith, S.M., 2004. Plastidial alpha-glucan phosphorylase is not required for starch degradation in Arabidopsis leaves but has a role in the tolerance of abiotic stress. Plant Physiology 135, 849e858. Zeeman, S.C., Kossman, J., Smith, A.M., 2010. Starch: its metabolism, evolution and biotechnological modification in plants. Annual Review of Plant Biology 61, 209e234. Zhang, X., Colleoni, C., Ratushna, V., Sirghie-Colleoni, M., James, M.G., Myers, A.M., 2004. Molecular characterization demonstrates that the Zea mays gene sugary2 codes for the starch synthase isoform SSIIa. Plant Molecular Biology 54, 865e879. Zhang, X., Myers, A.M., James, M.G., 2005. Mutations affecting starch synthase III in Arabidopsis alter leaf starch structure and increase the rate of starch synthesis. Plant Physiology 138, 663e674. Zhang, X., Szydlowski, N., Delvalle´, D., D’Hulst, C., James, M.G., Myers, A.M., 2008. Overlapping functions of the starch synthases SSII and SSIII in amylopectin biosynthesis in Arabidopsis. BMC Plant Biology 8, 96e113.

FURTHER READING ´ ., 2014. Starch synthase 4 is located in the Ga´mez-Arjona, F.M., Raynaud, S., Ragel, P., Me´rida, A thylakoid membrane and interacts with plastoglobule-associated proteins in Arabidopsis. Plant Journal 80, 305e316. Mo¨hlmann, T., Tjaden, J., Henrichs, G., Quick, W.P., Haüsler, R., Neuhaus, H.E., 1997. ADPglucose drives starch synthesis in isolated maize endosperm amyloplasts: characterization of starch synthesis and transport properties across the amyloplast envelope. Biochemical Journal 324, 503e509. Nakamura, Y., Imamura, M., 1985. Regulation of ADP-glucose pyrophosphorylase from Chlorella vulgaris. Plant Physiology 78, 601e605.


Chapter 2

Analyzing Starch Molecular Structure Eric Bertoft

Bertoft Solutions, Turku, Finland

1. INTRODUCTION: CHARACTERIZING STRUCTURES OF STARCH COMPONENTS Most naturally occurring starch granules, regardless of the plant source or tissue, contain two principal types of polysaccharides, namely amylose and amylopectin. Both are polymers of solely a-D-glucose connected by (1/4)linkages into shorter or longer chains. Amylopectin, the major component of most starches, consists of a large number of shorter chains that are bound together at their reducing end side by a (1/6)-linkage, which makes this very large polysaccharide extensively branched (Manners, 1989; Pe´rez and Bertoft, 2010). Amylose consists only of either a single or a few long chains, thus making the molecule linear or slightly branched (Takeda et al., 1992b). The amylose content of most starches is 20%e30% (Fredriksson et al., 1998; Jane et al., 1999; Morrison et al., 1984; Bertoft et al., 2008). However, certain mutant plants, commonly called waxy because of the waxy appearance of the seed endosperm, have a much lower content, or even lack the amylose component completely (Jane et al., 1999; Morrison et al., 1984; Banks et al., 1970; Ge´rard et al., 2002; Vasanthan and Bhatty, 1996). Other types possess an increased amylose content (Morrison et al., 1984; Ge´rard et al., 2002; Vasanthan and Bhatty, 1996; Banks et al., 1974; Schwall et al., 2000). The shape of the granules of high-amylose starches are often more or less deformed (Jane et al., 1994), and they may contain an additional polysaccharide component referred to as intermediate material (IM) because of its structure that in many aspects apparently is intermediate to the two main components (Shi et al., 1998). Though the chemical composition of the starch components is very simple, the analysis of their structure is not. For any polymer the sequence of the chemical units, by which it is built up, is of prime importance. Proteins are readily characterized by the sequence of their 20 different types of amino Starch in Food. http://dx.doi.org/10.1016/B978-0-08-100868-3.00002-0 Copyright © 2018 Elsevier Ltd. All rights reserved.

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acids, and nucleic acids are routinely analyzed for their nucleotide sequences. Many polysaccharides are composed of at least two carbohydrate species, but in the starch components, and other polyglucans, it is practically impossible to organize the single type of carbohydrate into a meaningful sequence. Special parameters are therefore in use, by which the starch components are characterized. These parameters are especially useful for analysis of the more complex amylopectin component. Apart from the glucosyl unit, the basic structural unit is the glucosyl chain of which different types and segments have been defined (Fig. 2.1). In the classical nomenclature by Peat et al. (1952), A-chains are defined as

FIGURE 2.1 Definition of types of chains and chain segments in the branched structure of amylopectin. Circles denote glucosyl units, circle with slash is the reducing end, lines denote (1/4), and curved lines with arrows denote (1/6) linkages. The structure of amylopectin is drawn following the cluster model (with two clusters shown) in (a) and (b) and following the backbone model in (c), (d), and (e) with building blocks encircled in (c) and a clusterlike group of building blocks encircled in (d).

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unsubstituted, whereas B-chains are substituted by other chains. The B-chains are further subdivided into Ba-chains, defined (Hizukuri and Maehara, 1990) as those substituted with at least one A-chain (and possibly other A- and/or B-chains), and Bb-chains that are only substituted with one or more B-chains. The macromolecule contains also a single C-chain that carries the sole reducing end group. This basic nomenclature is useful for both amylose and amylopectin. It should be noticed, however, that the C-chain is not distinguished from the B-chains in most experiments. The chains are also characterized as long and short chains. Albeit there is no exact definition of their lengths, long chains in amylopectin generally have a degree of polymerization (DP) > 35 (Bertoft et al., 2008). Note that the definition of long and short chains can be very different in amylose compared to amylopectin, because of the overall much longer chains in amylose. The chains are further divided into characteristic segments. An external chain is the part of a chain that extends from the outermost branch point to the nonreducing end (Manners, 1989). Thus, all A-chains are external by definition, whereas only one part of the B-chain is external. The rest of a B-chain is called the total internal chain and includes all segments between the branches as well as all the glucose residues involved in the branch points (Bertoft, 1991b). An alternative definition is core chain, from which the outermost branched residue is excluded (Yun and Matheson, 1993). Finally, internal chains are defined as the segments of the B-chains between the branches, excluding the branch point residues (Manners, 1989). For practical reasons, the segment at the reducing end side of the molecule is also considered an internal chain. The actual structure of amylopectin depends on the organization of the chains within the macromolecule. Two structural models exist that aims to explain the arrangement of the chains, but consensus as of which model is more likely valid has not been achieved so far. In the older, cluster model, the short unit chains are clustered together (Nikuni, 1969, 1978; French, 1972; Robin et al., 1974; Manners and Matheson, 1981), and the units of clusters are interconnected by longer chains (Fig. 2.1; Hizukuri, 1986). In this model, A-chains (generally considered as short chains) together with short B-chains, named B1-chains, form the cluster, whereas B2, B3, etc., are long chains that span over two, three, or more clusters, thereby interconnecting them. In a more recent model, known as the building block backbone model (Fig. 2.1), the macromolecule has a backbone consisting of mostly interconnected B2- and B3-chains (i.e., long chains), but the presence of shorter B1-chains is not excluded (Bertoft, 2013). Short A- and B1-chains extends from the backbone and form very small, tightly branched units known as building blocks, which are the basic structural units in this model as opposed to clusters in the cluster model. The building blocks are separated by interblock segments with approximate lengths of 5e8 glucose residues. Groups of building blocks are eventually separated by longer segments and these groups have been considered as clusters, albeit structurally different from the traditional concept


(Bertoft, 2013). It should be noted that in the backbone model, the nomenclature of the B-chains remains similar as in the cluster model, but it refers only to either short (B1) or long (B2 and B3) chains, not to a function as intraor intercluster chains, respectively. The parameters above are all conveniently obtained by specific hydrolysis of the starch components with enzymes. There are three major classes of enzymes useful for analytical purposes (The Amylase Research Society of Japan, 1988). Most used are the debranching enzymes that specifically hydrolyze the a-D-(1/6)-linkages, thereby releasing the units of chains from the macromolecule. The second group are the exo-acting enzymes that attack the a-D-(1/4)-linkages near the nonreducing ends. Because the enzymes cannot bypass a branch point, most of the external chains are removed, leaving a limit dextrin in which all branches are preserved. Glucoamylases are, however, an exception and can, under certain circumstances, also attack the branch and thereby eventually degrade the starch components completely into glucose. The third group is the endo-acting enzymes, for which internal chain segments are the principle substrate. External chains are also attacked, however. When enzymes are used in structural studies, it is of greatest importance that the enzyme preparation is free from any disturbing activity that could influence on the final results. Generally, only enzyme preparations intended for analytical purposes should be considered, whereas preparations used for industrial scale production of starch-derived products may contain considerable levels of minor activities. In a few situations, the disturbing influence of a side activity can be reduced, or eventually obliterated, by changing the analytical method for measuring the products. The general principles for structural analysis of the starch components, focused on enzymic methods and amylopectin as the major subject, are highlighted in this chapter. It should be noticed that almost every starch sample possesses unique properties and therefore may require certain modifications of the general methods. This is probably one of the reasons to the great diversity of method modifications found between different laboratories. Often it starts already with the choice of method for starch granule solubilization. The reader is therefore strongly advised to consult the original sources referred to in the text before planning any experiments in detail.

2. FRACTIONATION OF STARCH When a structural analysis is to be performed, the starch sample generally has to be fractionated into its components amylose, amylopectin and, in some cases, IM. It is necessary to remove the fatty material from the starch granules by Soxhlet extraction in 85% methanol (Schoch, 1942a) and then completely dissolve the sample before the fractionation. Granules are dissolved in 90% dimethyl sulfoxide (DMSO) by stirring at room temperature or on a boiling water bath (Wang et al., 1993a; Klucinec and Thompson, 1998): 6e10 M urea


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solution (Patil et al., 1974) or a mixture of 90% DMSO and 10% 6 M urea has also been used (Morrison and Laignelet, 1983). As an alternative, 0.5 M KOH or NaOH act as an optimal solvent for starch (Lehtonen, 1988). In many applications the starch granules are dispersed in 1e2 M hydroxide solution and then diluted (Bruun and Henriksna¨s, 1977; Chen et al., 1997; Blennow et al., 2001). When extremely high pH is used, heat should be used very carefully, or preferentially avoided, to prevent any alkaline degradation of the carbohydrates. Molecular aggregates of amylopectin tend to remain in the solution, however (Chen et al., 1997; Blennow et al., 2001; Millard et al., 1997). To obtain a more completely destructured sample, the dissolved starch is precipitated in ethanol and then redissolved (Wang et al., 1993a; Klucinec and Thompson, 1998; Chen et al., 1997). The classical method for starch fractionation is that of Schoch (1942b), and modifications thereof (Adkins & Greenwood, 1969; Jane & Chen, 1992), in which an unsoluble complex between amylose and n-butanol is allowed to settle. Amylopectin remains in the supernatant. Lansky et al. (1949) used a commercial mixture of amyl alcohols (Pentasol) to precipitate amylose. In a slightly modified procedure a mixture of aqueous n-butanol and 3-methyl-nbutanol was used (Hizukuri et al., 1981; Takeda et al., 1986). A very highmolecular weight amylopectin component was obtained from some maize starches as a middle, loose layer on the amylose fraction in a similar butanol mixture (Takeda and Preiss, 1993). Several reprecipitations increase the purity of the fractions (Takeda et al., 1984, 1986). Banks and Greenwood (1967) described a procedure suitable for fractionation of cereal starches, in which the amylose was precipitated as a thymol complex. Another approach for fractionating starch was described by Matheson and Welsh (1988). In this method the amylopectin component, rather than the amylose, is precipitated as a complex with the lectin concanavalin A. The lectin (a protein) is then destroyed by hydrolysis with a protease (Matheson, 1990). Though the method gives very pure amylopectin preparations, they will also contain branched IM if that exists in the original starch sample (Matheson and Welsh, 1988; Ge´rard et al., 2001). The binding of concanavalin A to amylopectin is dependent on the concentration of the lectin and the structure of the polysaccharide (Colonna et al., 1985). As the lectin is comparatively expensive to purchase on the market, this method is restricted to small-scale separation. Gel-permeation chromatography (GPC) has also been used for small-scale fractionation (Koch et al., 1998; Lloyd et al., 1996). Amylopectin is eluted at the void volume of the column and is easy to collect (Koch et al., 1998), whereas amylose is partly included into the gel particles. In some cases, IMs were reported to elute with volumes intermediate to that of amylopectin and amylose (Wang et al., 1993a; Ge´rard et al., 2001). A common media is Sepharose CL 2B eluted with an alkaline solution (Bertoft et al., 2008; Rollings et al., 1982; Colonna et al., 1988; Boyer and Liu, 1985).


3. ANALYSIS OF AMYLOSE Though amylose is the minor component in most granules, it has a large influence on the properties of starch. The traditional definition of amylose is a molecule composed of a long, linear chain of (1/4)-linked a-D-glucosyl units. Most starch preparations contain, however, also slightly branched amylose molecules. There is no specified smaller size for the definition of amylose. In some cases even comparatively short linear dextrins, obtained by hydrolysis of amylopectin, were called amylose. This is unfortunate, because it tends to be a source of confusion. Chains of lengths comparable to the short chains of amylopectin should preferentially be called linear oligosaccharides.

3.1 Amylose Content of Starch Amylose was probably the first biopolymer for which a helical structure was proposed (Hanes, 1937). The well-known deep-blue complex formed with iodine was later proved to involve amylose in a helical conformation (Rundle and French, 1943), details of which have been a matter of dispute until present time (Nimz et al., 2003; Calabrese and Khan, 1999; Minick et al., 1991; Ziegast and Pfannemu¨ller, 1982). The color and intensity of the complex depends on the chain length (CL) of the amylose (Bailey and Whelan, 1961; Banks et al., 1971; Manners and Stark, 1974; John et al., 1983). At CL > 80 the wavelength maximum (lmax) of the absorption of light is >610 nm and is typical for amylose. lmax shifts to lower wavelengths and the color shifts to red for shorter chains (Bailey and Whelan, 1961). Thus, the short chains of amylopectin possess a lmax at 530e575 nm (Archibald et al., 1961; Shibanuma et al., 1994; Takeda et al., 1999; Hung and Morita, 2007; Annor et al., 2014b; Waduge et al., 2014; Gayin et al., 2016b). The blue value (Gilbert and Spragg, 1964) (BV) is defined as the absorbance at 680 nm (though sometimes measured at 640 nm) of 1 mg starch in 100 mL of a mixture containing 2 mg I2 and 20 mg KI. For a “true” BV, the absorbance should be multiplied with the factor 4 (because old colorimeters used 4-cm cuvettes rather than 1-cm cuvettes in modern instruments). BV for amylose (Takeda et al., 1984, 1986, 1999; Takeda and Preiss, 1993; Shibanuma et al., 1994; Nilsson et al., 1996; Schulman et al., 1995) is 1.01e1.63, whereas that for amylopectin (Takeda and Preiss, 1993; Shibanuma et al., 1994; Takeda et al., 1999; Nilsson et al., 1996; Schulman et al., 1995) is 0.08e0.38. Though BV is easy to measure, it should be considered mainly as a qualitative test for amylose. The most frequently used quantitative test for amylose is to measure the iodine affinity (IA) potentiometrically (Schoch, 1964). An automated amperometric titration method was also described (Takeda et al., 1987b). In most cases approximately 20 g of iodine is bound per 100 g amylose, in contrast to only 0.5e1.1 g iodine/100 g amylopectin (Takeda et al., 1986, 1987b, 1999; Takeda and Preiss, 1993; Schulman et al., 1995). Assuming the IA value of


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amylose is 20, the amylose content is calculated from IA of a defatted starch sample as (Takeda et al., 1987b): % Apparent amylose ¼ IAstarch/20 100

(2.1)

It should be noted that the amylopectin component in some starches, notably high-amylose starches, possesses unusually long chains (Klucinec and Thompson, 1998; Baba and Arai, 1984; Montgomery et al., 1964; Takeda et al., 1993c; Bradbury and Bello, 1993), which increases IA for the starch and leads to overestimation of the amylose content. Also, the amylopectin of indica varieties of rice contain unusually high levels of long chains (Takeda et al., 1987b). It is, however, possible to correct the obtained apparent amylose values to true values if the IA of the purified amylopectin is taken into account (Takeda et al., 1987b): % Amylose ¼ (IAstarch IAamylopectin)/(IAamylose IAamylopectin) 100 (2.2) A considerable part of the amylose in many starches, especially cereal starches, is complexed with lipids, mainly lysophospholipids (Fredriksson et al., 1998; Morrison et al., 1984, 1993; Andersson et al., 1999). These lipideamylose complexes (LAM) have no affinity for iodine. A colorimetric determination of LAM and the total (true) amylose content was described by Morrison and Laignelet (1983). In this, starch granules are dissolved in hot 90% DMSOe10% 6 M urea, treated with a mixture of I2 and KI, and the absorbance at 635 nm is measured from which the fraction of apparent amylose (corresponding to free amylose, FAM) is obtained. A part of the dissolved sample is defatted in ethanol before the measurement is repeated to obtain the total amylose. In both cases the content is calculated from the general equation: % Amylose ¼ (28.414 absorbance) 6.218

(2.3)

LAM is obtained as the difference between the total amylose and FAM (Morrison and Laignelet, 1983). Chrastil (1987) developed an iodine-binding method suitable for both defatted starch and flours. A stable absorbance at 620 nm was obtained by developing the amyloseeiodine complex in acidic conditions using trichloroacetic acid. A rapid method for the estimation of amylose in maize starches, including high-amylose starch, was developed by Knutson and Grove (1994). The starch granules are gelatinized without heat in 3 M CaCl2 and then further sonicated at 60e70 C in an iodineeDMSO mixture. After dilution in water, the absorbance is read at 600 nm and the amylose content is determined from a standard curve. A microscale method was described by Mohammadkhani et al. (1998), in which only 2e3 cereal seeds are needed for the amylose determination. Zhu et al. (2008) recommended a method in which the absorbance of the amyloseeiodine complex is measured at both


620 and 510 nm. More recently, Kaufman et al. (2015) developed a 96-well plate IA assay suitable for screening large sets of samples. Campbell et al. (2002) suggested that near-infrared transmittance spectroscopy could partly replace the iodine-staining methods and enable rapid screening of apparent amylose contents in breeding programs. Amylose can also be quantified using the lectin concanavalin A. As mentioned above, the lectin forms an insoluble complex with amylopectin and possible intermediate, branched molecules. The amylose content in starch is obtained as the difference between the carbohydrate content in the solution before and after precipitation of the amylopectineconcanavalin A complex (Gibson et al., 1997). This method is also applicable on flour samples if the total starch and amylose concentrations are measured enzymatically using a-amylase and amyloglucosidase (Gibson et al., 1997). The released glucose is then usually measured with a glucose oxidaseeperoxidase method (commonly known as GOPOD) (Gibson et al., 1997; Karkalas and Tester, 1992). Amylose contents were also estimated by size-fractionation of starches using GPC (Wang et al., 1993a; Blennow et al., 2001; Boyer et al., 1980) or high-performance size-exclusion chromatography (HPSEC) (Bradbury and Bello, 1993; Kobayashi et al., 1985; Mua and Jackson, 1995; Simsek et al., 2013). There is a risk, however, that solubilization problems related to the starch components affect the result (Chen et al., 1997; Gidley et al., 2010). Other problems may arise from shear scission of amylopectin in the column (Gidley et al., 2010; Cave et al., 2009). Though no baseline separation of amylose and amylopectin normally is obtained, GPC on a column packed with TSK HW75 S was found to be the most accurate among several different amylose determination methods (Ge´rard et al., 2001). The fraction of amylose in GPC is identified by measuring the BV (Krisman, 1962) of the collected fractions (Wang et al., 1993a; Gayin et al., 2016b; Manelius and Bertoft, 1996; Vamadevan et al., 2014), or online by postcolumn iodine injection in the case of HPSEC (Suortti and Pessa, 1991; Pessa et al., 1992; Autio et al., 1992). An alternative method is to enzymatically debranch the starch sample before GPC (Fredriksson et al., 1998; Bertoft et al., 2008; Wang et al., 1993a; Sargeant, 1982) or HPSEC (Bradbury and Bello, 1993; Wang and White, 1994a). The long amylose chains are eluted from the column before the short chains of the amylopectin (Karkalas and Tester, 1992). Also in this case, however, a baseline separation between the two fractions is difficult to achieve.

3.2 Structural Analysis of Amylose The size of amylose molecules is more frequently given as the DP than as molecular weight and is obtained by light scattering (Hizukuri and Takagi, 1984), from the limiting viscosity number (Cowie and Greenwood, 1957), or by reducing end analysis with the ParkeJohnson reagent (Park and Johnson, 1949) as modified by Hizukuri et al. (Hizukuri et al., 1981, 1983). The


105

number-average DP (DPn) from different plants varies between 0.51 and 6.34 103 (Hizukuri et al., 1981; Takeda et al., 1984, 1986, 1988; Shibanuma et al., 1994; Schulman et al., 1995; Hizukuri and Takagi, 1984; Biliaderis et al., 1981; Lii and Chang, 1991). Weight-average values are higher (Table 2.1; Takeda et al., 1986, 1999; Hizukuri and Takagi, 1984). The size distribution was analyzed by HPSEC on Toyosoda TSK-GEL PW columns, coupled to simultaneous refractive index (RI) and low-angle laser light scattering (LALLS) detection, for a range of samples (Hizukuri and Takagi, 1984; Takagi and Hizukuri, 1984). Broad distributions were obtained for amylose from potato (DPw 0.84e21.8 103) and tapioca (0.58e22.4 103), whereas amylose from kudzu possessed a more narrow distribution (0.48e12.3 103). More recently, multi-angle light scattering (MALS) detectors have largely replaced LALLS for molecular weight estimations (Ong et al., 1994; You and Lim, 2000; You and Izydorczyk, 2002; Zhong et al., 2006; Chen and Bergman, 2007). A considerable portion of the amylose fraction, between 10% and 70% depending on the sample, contains slightly branched macromolecules (Hizukuri et al., 1981; Manners and Bathgate, 1969; Takeda et al., 1987a). These are generally larger than the linear amyloses (Takeda et al., 1992b, 1993a) and possess between 5 and 20 chains per molecule (Table 2.1). The Cchain of branched amylose from maize was tritium labeled at the reducing end by treatment with sodium [3H]-borohydride and CL ranged from 200 to 710 (Takeda et al., 1992a). Takeda et al. (1987a, 1989a, 1992b) studied branched amyloses by preparing the b-amylolysis limit dextrins (b-LDs) of the amylose fraction. All linear amylose, together with the external chains of the branched amylose, is hydrolyzed (see Section 4 for a more detailed description of the use of enzymes.). The molar fraction of branched amylose (MFbranched) is calculated from (Shibanuma et al., 1994; Takeda et al., 1987a): MFbranched ¼ NCwhole

amylose sample/(NCb-LD

1)

(2.4)

in which NC is the average number of chains per molecule. Takeda et al. (1990) subfractionated maize amylose in aqueous n-butanol. A minor, branched component with high-molecular weight remained in the supernatant. It was shown that the component contained a fraction of very short chains that were suggested to form small “immature” clusters. A somewhat similar conclusion was made by Takeda et al. (1993a) for rice amylose. After debranching the rice amylose (with the enzyme isoamylase), these authors labeled the unit chains with tritium and found that the amylose not only contained long chains typical of amylose but also short chains possessing a peak at DP 21 when analyzed by GPC. In fact, these short chains had lengths comparable to chains typically found in amylopectin. Moreover, on a number basis the short chains predominated: the molar ratio of very long (DP > 200), long (DP 30e200), and short chains (DP 10e30) was 0.8:0.6:4.0. Hanahiro et al. (2013) confirmed the existence of short chains in rice amylose

Branched Fraction as b-limit Dextrin

Whole Fraction Source

DPw

DPn

DPw

DPn

CLn

NC

MF

Rice (japonica)

2820e2950

1100e1110

3080e3500

890e1030

105e115

8.5e9.0

0.31e0.43

Rice (indica)

2290e2720

920e1040

2480e3460

790e890

90e105

5.7e9.7

0.32e0.49

Maize

2270e2500

930e990

2700e3000

790e850

140e160

5.3e5.4

0.44e0.48

Wheat

2360e5450

830e1570

1670e5880

700e1430

50e71

12.9e20.7

0.26e0.44

Barley

5580

1570

5930

1440

120

12.0

0.35

Water chestnut

4210

800

4150

1200

108

11.1

0.11

Tapioca

e

2660

e

1970

115

17.1

0.42

Sweet potato

e

3280

e

1770

130

13.6

0.70

CL, chain length; DP, degree of polymerization; MF, molar fraction of branched amylose; NC, average number of chains per branched amylose molecule. a Data are based on values given in a collection of references: Takeda et al. (1986, 1987a, 1988, 1989a, 1999), Shibanuma et al. (1994), Hizukuri et al. (1988).


TABLE 2.1 Properties of Amylosesa


107

using fluorescent labeling with 2-aminopyridine. Based on several details in the size-distribution pattern of the chains, Hanahiro et al. (2013) concluded that the branching pattern of these short chains in amylose was distinct from the branching pattern of amylopectin.

4. ANALYSIS OF AMYLOPECTIN STRUCTURE The molecular weight of amylopectin is considerably larger than that of amylose and no suitable media are available for a size-distribution analysis by GPC or SEC. Because of the tendency to form molecular aggregates (Blennow et al., 2001; Millard et al., 1997; Gidley et al., 2010; Callaghan and Lelievre, 1985), as well as a risk for fragmentation (Cave et al., 2009; Yokoyama et al., 1998; Vilaplana and Gilbert, 2010); it is also difficult to obtain accurate average estimations of the true size of this gigantic macromolecule. Mw-values range between 2 and 700 106, depending on the source of plant, method of determination, and solvent for the amylopectin sample (Banks et al., 1970; Millard et al., 1997; Simsek et al., 2013; Callaghan and Lelievre, 1985; Yokoyama et al., 1998; Aberle et al., 1994; Thurn and Burchard, 1985; Lelievre et al., 1986; Franco et al., 2002; Matalanis et al., 2009). Yoo and Jane (2002) found that waxy starches tend to have larger amylopectin molecules than their normal counterparts; waxy rice having the largest Mw of 5680 106 among a series of 23 different starches. The last decades flow field-flow fractionation (FFFF, more commonly abbreviated F4), or more precisely asymmetrical F4 (AF4), has become an alternative method to GPC and SEC for separation of starch components (Perez-Rea et al., 2015, 2016). The advantage of this technique is the absence of a stationary medium through which the macromolecules are forced, thus minimizing the risk of sample fragmentation. Another advantage is the practically unlimited sizefractionation range (Litzeń, 1993; Wahlund, 2000; Wahlund and Nilsson, 2012). With this technique, Wahlund et al. (2011) reported Mw values for amylopectin samples from maize, wheat, rice, potato, tapioca, and peas to range between 0.45 and 4.5 108 g/mol. Rolland-Sabate´ et al. (2011) also analyzed samples from maize, wheat, rice, and potato with varying amylose content and reported Mw values for the amylopectin component between 1.0 and 4.8 108 g/mol, i.e., similar to that of Wahlund et al. (2011). Mn values for amylopectin are much lower than Mw values (Stacy and Foster, 1957; Potter et al., 1953). When the reducing power was measured with the modified Park-Johnson method (Hizukuri et al., 1981, 1983), DPn values were 4.8e15.0 103 in different samples (Takeda and Preiss, 1993; Shibanuma et al., 1994; Takeda et al., 1988, 1989b; Hizukuri et al., 1989, 1988), which is only slightly higher than those of amyloses and corresponds to Mn 0.8e2.5 106. Overall, the Mw/Mn ratio is of the magnitude 102 suggesting a very broad range of molecular sizes. By fluorescent labeling of the reducing end followed by GPC, the amylopectin component from several plants was


found to consist of three molecular species (Takeda et al., 2003). Large molecules had DPn values between 13.4 and 26.5 103, medium molecules 4.4 and 8.4 103, and small molecules 0.7 and 2.1 103. The unit chain distribution appeared to be fairly similar between the size groups, however, suggesting a similar basic arrangement of the chains (Takeda et al., 2003). The use of different enzymes to investigate the amylopectin structure is outlined in Fig. 2.2. Different amylases are active in a concentration of 2.5 M DMSO or less (Lineback and Sayeed, 1971), which should be taken into account if the sample is dissolved in DMSO before the enzymatic degradation. Note that several of the methods described in this section also are applicable to amylose and IMs.

FIGURE 2.2 Principles of the use of enzymes in structural studies of amylopectin and other branched starch components. Amylopectin is drawn following the cluster model concept.


109

4.1 Unit Chain Length and Distribution The most common, though not necessarily the most informative, structural analysis is to measure the length and the size distribution of the unit chains in amylopectin. The average CL is obtained from: CL ¼ Gtot/NC (2.5) in which Gtot is the total number of glucose residues (or total carbohydrate content) and NC is the number of chains in the sample. NC equals the number of nonreducing ends and is obtained by a modified (Hizukuri et al., 1981; Takeda et al., 1984) rapid Smith degradation method (Hizukuri and Osaki, 1978). The glycerol liberated from the terminal nonreducing ends is then measured enzymatically (Hizukuri et al., 1981). Alternatively, the sample is first debranched enzymatically, as described below, and then the reducing end of each chain is measured with a modified (Hizukuri et al., 1981, 1983; Takeda et al., 1993b) Park-Johnson reagent (Park and Johnson, 1949). A sensitive alternative is the 2,2ʹ-bincinchoninate reagent (Waffenschmidt and Jaenicke, 1987; McFeeters, 1980), which also was adopted to microscale applications (Fox and Robyt, 1991). CL can also be measured from the signals obtained by 1H- and 13C-NMR spectroscopy (Gidley, 1985). CL equals approximately the ratio of a-(1/4) to a-(1/6) linkages, because amylopectin is a very large molecule. The H-1 proton at the nonreducing terminal was assigned a chemical shift separate from the other H-1 protons, which increases the precision (Nilsson et al., 1996). With NMR there is no need to debranch the sample before the analysis, and the agreement with the enzymatic methods is good (Nilsson et al., 1996; Gidley, 1985). Table 2.2 shows typical CL values of selected amylopectin samples. Considerably more information than given by the CL value is obtained from the unit chain distribution, often called the unit chain profile. Before the analysis, the sample is debranched with isoamylase or pullulanase (Manners, 1989; Fig. 2.2). The enzymes have different action patterns against branched oligosaccharides but also against intact amylopectin (Kainuma et al., 1978). Isoamylase attacks the branches by an endo-attack pattern, whereas pullulanase was characterized to perform an exo-attack (Harada et al., 1972). The Km value for isoamylase with amylopectin as the substrate is lower than for pullulanase (Yokobayashi et al., 1973), and it is easier to obtain a more complete debranching with the former (Harada et al., 1972; Yokobayashi et al., 1970). Whether all the branches have been hydrolyzed is conveniently controlled by the addition of b-amylase after the debranching experiment. This enzyme degrades the sample completely into maltose (and small amounts of maltotriose) if no branches remain (Yokobayashi et al., 1970). The unit chain profile have been analyzed by GPC (Jane and Chen, 1992; Takeda and Preiss, 1993; Boyer and Liu, 1985; Akai et al., 1971; Bertoft,


TABLE 2.2 Chain Lengths and Molar Ratios of Selected Amylopectinsa Source

AP Type

CL

ECL

ICL

S:L

A:B

Barley

1

18

11

5

19.4

1.0

Wheat

1e2

18

12

4

16.2

1.4

Waxy maize

2

18

12

5

13.2

1.0

Waxy rice

2

18

12

5

10.5

0.8

Sweet potato

2e3

20

23

6

10.1

1.1

Cassava

3

19

12

5

11.0

1.3

b

Pea

n.k.

26

16

9

11.5

1.4

Potato

4

21

14

6

6.6

1.5

A:B, ratio of A- to B-chains; AP type, structural type of amylopectin; CL, chain length; ECL, external chain length; ICL, internal chain length; S:L, ratio of short to long chains. a Data are based on values given in a collection of references: Bertoft et al. (1993b, 1999, 2008), Yun and Matheson (1993), Matheson (1990), Bertoft (2004), Enevoldsen and Juliano (1988), Zhu and Bertoft (1996), Bertoft and Koch (2000), Zhu et al. (2011a), Kalinga et al. (2013). b Not known.

1991a; Yusuph et al., 2003) or HPSEC (Fredriksson et al., 1998; Hizukuri and Maehara, 1990; Klucinec and Thompson, 2002a; Wong and Jane, 1997; Hizukuri, 1985) for a broad range of amylopectins. With HPSEC the postcolumn detection is frequently performed with an RI detector. Debranched samples were also labeled with the fluorescent dye 2-aminopyridine and analyzed by HPSEC using a fluorescence detector, which gives the molar size distribution of the chains (Hanashiro et al., 2002, 2011; Charoenkul et al., 2006; Thitipraphunkul et al., 2003). High-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) (Koizumi et al., 1991; Wong and Jane, 1995; Lu et al., 1997) has largely increased the resolution of individual chains up to the order of DP 50e70 (Jane et al., 1999; Blennow et al., 2001; Takeda et al., 1999; Silverio et al., 2000; Shi and Seib, 1995; Hanashiro et al., 1996; Bertoft, 2004). Unfortunately, the amperometric detection is not directly proportional to the carbohydrate content (Koizumi et al., 1991; Ammeraal et al., 1991), but this is possible to adjust by calibration of the response signal with fractions of known CL and carbohydrate content (Koch et al., 1998). The problem was also avoided by coupling a short column with immobilized glucoamylase after the main column. The glucoamylase hydrolyses the chains completely into glucose before entering the PAD detector thus giving a proportional response regardless of the CL (Wong and Jane, 1997). An additional benefit is that the sensitivity of PAD for longer chains increases. The unit chain profile can also be analyzed by fluorophore-assisted carbohydrate electrophoresis (FACE)


111

(Morell et al., 1998, 2003; Srichuwong et al., 2005; Butardo et al., 2011; Fujita et al., 2006, 2007; Nakamura et al., 2002; Regina et al., 2012; Yao et al., 2005). In this method, the chains of the debranched sample are labeled at their reducing ends with the negatively charged fluorophore 8-amino1,3,6-pyrenetrisulphonic acid, which is registered by a fluorescence detector (Morell et al., 1998; O’Shea and Morell, 1996; O’Shea et al., 1998). The resolution is at least as high as in HPAEC, and the result represents directly the molar distribution of the unit chains. All amylopectin samples, regardless the source, possess typically a major group of short chains and a minor group of long chains, the division between the groups being generally approximately at DP 36 (Bertoft et al., 2008; Hanashiro et al., 1996). Two unit chain profiles, obtained by HPAEC and presented as bar graphs, are shown in Fig. 2.3. Waxy maize is a typical A-crystalline starch sample (Hizukuri, 1985) with a comparatively high molar ratio of short:long chains (S:L ratio, Table 2.2). A typical B-crystalline starch is represented by the amylopectin from an amylose-free potato, which has a lower S:L ratio. Note that the shortest chain in both samples is DP 6, which is a general feature of all amylopectins described so far (though trace amounts of shorter chains have been reported). The distribution pattern of the shortest chains with DP 6e8 is characteristic for amylopectins from certain plants, thus giving a fingerprint of the source (Koizumi et al., 1991). These short chains are (most probably) A-chains and were consequently named “fingerprint A-chains” (Afp-chains) (Bertoft, 2004). High-amylose maize amylopectin samples of mutant plants, which have B-crystalline granules (Hizukuri, 1985), possess a fingerprint pattern similar to normal maize amylopectin (Jane et al., 1999). C-crystalline starches, being mixtures of A- and B-polymorphs (Cairns et al., 1997; Buleón et al., 1998), possess intermediate patterns (Koizumi et al., 1991; McPherson and Jane, 1999). The peak of short chains possesses in some samples a shoulder at approximately DP 12 or 14 and was therefore subdivided (Hizukuri, 1986; Shibanuma et al., 1994; Takeda et al., 1999; Hanashiro et al., 2002). The shortest chains were suggested to represent A-chains, whereas the other subgroup was short B-chains (B1-chains) (Hizukuri, 1986; Hanashiro et al., 1996). It was later shown, however, that there is a considerable overlap of Aand B1-chains leading to a serious underestimation of the actual A-chain number and too low ratio of A:B chains (Bertoft et al., 2008). Instead, an accurate estimation of the relative number of A-chains has to be done with limit dextrins (see Section 4.2). The unit chain profile was also transformed into gaussian distributions, by which two groups of short A-chains were described, in addition to several groups of B-chains (Ong et al., 1994). Most amylopectin samples have a polymodal size distribution of their chains, which is best detected by HPSEC (Hizukuri, 1986; Klucinec and Thompson, 1998; Shibanuma et al., 1994; Takeda et al., 1993c, 1999; Ong et al., 1994). The long chains are subdivided into B2 chains and B3 chains at

BSmajor

Long chains

Afp 6-8

Waxy maize φ,β -limit dextrin

A Bfp

B2

B2

B3 B3

Amylose-free potato

Short chains

A Bfp

Afp 6-8

BSmajor

Amylose-free potato φ,β -limit dextrin

Long chains B2

B2 B3

B3

FIGURE 2.3 Bar graphs redrawn from HPAEC-PAD chromatograms showing the unit chain distribution of two amylopectin samples and their limit dextrins. Bars for DP > 60 are approximate. Groups of different chain categories are shown.


Waxy maize

Short chains


113

approximately DP 60 (Fig. 2.3; Hizukuri, 1986). Extremely long chains with DP up to the order of 103 were also described for several samples (Hizukuri and Maehara, 1990; Shibanuma et al., 1994; Takeda et al., 1999, 1989b; Hanashiro et al., 2005; Inouchi et al., 2006). The length of these chains, which are named “extra-long” or “super-long” chains, are comparable to amylose chains and they are synthesized by the same enzyme as amylose, namely granule-bound starch synthase I (GBSS I) and found exclusively in nonwaxy starches (Aoki et al., 2006; Hanashiro et al., 2008). Most extra-long chains appears to be sparsely branched B-chains (Takeda et al., 1989b; Hanashiro et al., 2005; Laohaphatanaleart et al., 2009) and are common especially in Indica varieties of rice (Takeda et al., 1987b), but they were also reported in starch from several other plants (Shibanuma et al., 1994; Takeda et al., 1988; Charoenkul et al., 2006; Hanashiro et al., 2005; Laohaphatanaleart et al., 2009; Noda et al., 2005; Zhu et al., 2013a). The distribution of the C-chain of amylopectin was described by Hanashiro et al. (2002). Samples were labeled at the reducing end with 2-aminopyridine before the debranching and then fractionated by SEC. C-chains are distributed over the DP range 10e130, with a peak around DP 40 (Takeda et al., 2003; Hanashiro et al., 2002; Thitipraphunkul et al., 2003). The C-chain appears therefore to be of similar kind as most of the B-chains and is not a type of extra-long chain.

4.2 External Chain Length and Internal Chain Distribution Though the unit CL distribution provides knowledge about the overall composition of chains, a more detailed understanding of the fine structure of amylopectin needs information about the positions of the chains within the macromolecule (Thompson, 2000). When exo-acting enzymes remove the external chains, limit dextrins containing the internal parts together with all original branches remain and are used for the study of the internal structure (cf. Fig. 2.2). In addition, information about different types of chains is better achieved than when analyzing the unit chains of the intact amylopectin. The enzyme b-amylase hydrolyses every second a-D-glucosidic (1/4)linkage from the nonreducing ends, thereby producing maltose (Manners, 1989). The enzyme cannot bypass the branches, thus leaving a resistant b-limit dextrin (b-LD) with remaining short external chain stubs of characteristic lengths (Summer and French, 1956; Lee, 1971; Fig. 2.4). By performing the hydrolysis inside a dialysis membrane, the maltose is directly removed from the b-LD (Lii and Lineback, 1977; Qi et al., 2003). Alternatively, the dialysis is made after the reaction (Peat et al., 1952; Yun and Matheson, 1993; Biliaderis et al., 1981; Lee, 1971; Manners and Marshall, 1972; Hood and Mercier, 1978), or the b-LD is precipitated in methanol (Bender et al., 1982; Bertoft, 1989a; Lundqvist et al., 2002) or ethanol (Klucinec and Thompson,


FIGURE 2.4 Action pattern of sweet potato b-amylase and rabbit muscle phosphorylase a on a branched substrate. The residual external chain stubs of the b-limit dextrin, and the 4- and 4,b-limit dextrins, are shown as filled circles. (a) Adapted from Summer, R., French, D., 1956. Action of b-amylase on branched oligosaccharides. Journal of Biological Chemistry 222, 469e477. (b) Adapted from Bertoft, E., 1989b. Partial characterisation of amylopectin alphadextrins. Carbohydrate Research 189, 181e193.

2002b). Maltose was also removed by tangential flow filtration (TFF) (Bertoft et al., 2008) or by GPC on a short desalting column (Ge´rard et al., 2000). The b-limit value is defined as the relative amount of maltose formed in the reaction. Typically, amylopectins possess b-limit values in the order of 50% e60% (Yun and Matheson, 1993; Colonna et al., 1985; Takeda et al., 1987b, 1999; Hizukuri et al., 1989; Bertoft, 2004; Banks and Greenwood, 1973; Biliaderis et al., 1979; Enevoldsen and Juliano, 1988), but the amylopectin component of amylose-extender mutants (ae-starches) have somewhat higher values (Klucinec and Thompson, 2002b). The b-limit value is used to calculate the average external chain length (ECL) as (Manners, 1989): ECL ¼ CL (%b-limit/100) þ 2 (2.6) in which it is assumed that the sample possesses an equal number of A- and Bchains, as well as of chains with odd and even number of residues, and thus the average length of the external stubs in the b-LD is two glucosyl residues. Phosphorylase a, an enzyme isolated from rabbit muscle, produces glucose 1-phosphate from the nonreducing ends (Manners, 1989) and leaves at average 3.5 residues next to a branch point (Bertoft, 1989b, 2004; Fig. 2.4). The cleavage of the glucosidic linkage proceeds by phosphorolysis, rather than hydrolysis, and the phosphate is provided as a phosphate buffer. The reaction is readily reversible (Hestrin, 1949), and to obtain a true f-LD, the concentration ratio of phosphate:glucose 1-phosphate should exceed 10 at the end of the reaction (Bertoft, 1989b). If the reaction is made inside a dialysis bag (Lee, 1971), the concentration of glucose 1-phosphate is continuously kept at a low level. Because the residual external chain stubs are longer than in the b-LD, the f-limit value is typically somewhat lower than the b-limit value (Colonna


115

et al., 1985; Bertoft, 1989b, 2004; Hestrin, 1949; Liddle and Manners, 1957; Walker and Whelan, 1960) and the ECL is obtained from (Bertoft, 1989b): ECL ¼ CL (%f-limit/100) þ 3.5.

(2.7)

If the f-LD is further treated with b-amylase (Yokobayashi et al., 1970; Bertoft, 1989b, 2004; Lii and Lineback, 1977; Bertoft et al., 1999; Zhu and Bertoft, 1996), each external chain stub will be attacked once and the molar amount of maltose formed equals the number of unit chains (Bertoft, 1989b). The average length of the external stubs of the resulting limit dextrin (f,b-LD) is 1.5 and the structure (Bertoft, 1989b, 2004) equals the smallest possible b-LD (Fig. 2.4). Because the number of internal chains equals the number of branches, which are one less than the NC, the internal chain length (ICL) is obtained from (Bertoft, 1989b): ICL ¼ [(CL ECL) NC]/(NC 1) 1.

(2.8)

When the NC is large or unknown (the typical case for amylopectin), the following approximation is used (Manners, 1989): ICL ¼ CL ECL 1.

(2.9)

Typical ECL and ICL for some amylopectins are shown in Table 2.2. Pullulanase attacks short maltosyl chain stubs more efficiently than isoamylase (Hizukuri and Maehara, 1990) and is used to completely debranch b- or f,b-LDs. The chain distribution can be analyzed chromatographically and the maltose obtained from the f,b-LD equals the number of A-chains, whereas all longer chains represent the B-chains (see Fig. 2.4; Bertoft et al., 2008, 2011a; Laohaphatanaleart et al., 2009; Zhu et al., 2011a, 2013a; Bertoft and Koch, 2000; Annor et al., 2014a; Gayin et al., 2016a; Kalinga et al., 2014a; Kong et al., 2008). From a b-LD, half of the A-chains are obtained as maltose and half as maltotriose (Biliaderis et al., 1981; Akai et al., 1971; Shi and Seib, 1995; Morell et al., 1998; Lii and Lineback, 1977; Hood and Mercier, 1978; Klucinec and Thompson, 2002b; Atwell et al., 1980; Asaoka et al., 1985; Inouchi et al., 1987; Mercier, 1973; Reddy et al., 1993; Robin, 1981; Yuan et al., 1993). A small amount of the shortest B-chains are also found as maltotriose, however, and might interfere slightly with the estimation of A-chains. The ratio of A:B-chains of some amylopectins estimated from limit dextrins are shown in Table 2.2. The distribution of the B-chains of f,b-LDs represents their total internal lengths plus a single external residue (Fig. 2.4). The subdivision of long internal B-chains corresponds to the division of the long unit chains of the native samples, but the internal chains are shorter because the external segment is lacking. The short internal B1-chains are also found as subgroups, which are not readily distinguished from the chain profile of the intact amylopectin (Fig. 2.3). The subgroups of short chains were designated “fingerprint B-chains” (Bfp-chains) with DP 3e7 and a major group of short


B-chains (BSmajor) with DP 8e23, the upper range being dependent on the starch sample (Bertoft, 2004). Just as with Afp-chains, the profile of Bfpchains appears to be a fingerprint in HPAEC chromatograms, hence the name (Bertoft et al., 2008). Based on the profiles of the internal chains of a range of amylopectin samples, the structure of amylopectin was divided into four different types (Bertoft et al., 2008). Type 1 amylopectins possess typically a broad size distribution of BSmajor-chains up to DP 26 or 27. As a result, the groove, which normally is found between short and long internal chains, is practically abolished. The relative molar amount of short chains is high in these starches, giving a high ratio of short B- to long B-chains (BS:BL) in the order of 7.0e9.3. The ratio of short to long chains (S:L) in the amylopectin, which includes the A-chains, is also high: approximately 14e22. Type 1 includes e.g., amylopectins from barley, oats, and rye (Bertoft et al., 2008). Type 2 amylopectins have a narrower size distribution of BSmajor-chains and more B2-chains, which results in a clear groove between short and long chains in the chromatograms (Bertoft et al., 2008). In addition, the relative content of Bfp-chains is high. The ratios of BS:BL (4e7) and S:L (12e14) are lower compared to type 1 amylopectins. Among cereals, type 2 was found for maize (cf. Fig. 2.3) and rice starches (Bertoft et al., 2008), whereas wheat appeared to possess a structure intermediate of type 1 and 2 (Kalinga et al., 2013). Also, amylopectin from the pseudocereal amaranth possessed type 2 structure (Kong et al., 2009). In addition, type 2 was characteristic of amylopectin from sago palm (trunk) and kudzu (root) starches, which both possess C-crystalline granules (Bertoft et al., 2008). The structure of type 3 amylopectin tend to have somewhat more of the long B3-chains, whereas Bfp-chains are found in smaller number as in type 2. Therefore, the ratios of BS:BL and S:L are somewhat lower than for type 2. Type 4 amylopectins have characteristically considerably more B2- and B3chains than any of the other groups and possess the lowest ratio of short to long chains. Type 3 starches includes, e.g., mung bean and tapioca, whereas Bcrystalline starches appear to have predominantly type 4 amylopectin, and include, e.g., edible canna, lesser yam and potato (the latter shown in Fig. 2.3; Bertoft et al., 2008).

4.3 Units of Clusters According to the cluster model, the unit chains of amylopectin are packed into clusters with densely grouped branches (Nikuni, 1978; French, 1972; Robin et al., 1974; Manners and Matheson, 1981; Imberty and Pe´rez, 1989). The cluster model largely relies on indirect evidences, because comparatively few attempts to study the clusters in more detail have been published. Thurn and Burchard (1985) presented a model in which one cluster contains on average only 4.22 chains, whereas Hizukuri (1986) suggested that some 22e25 chains


117

are involved. Gallant et al. (1997) concluded that a cluster is built up by 18e34 chains in the form of double helices. Endo-acting enzymes have been used to cleave longer internal chain segments to release smaller dextrins from the amylopectin macromolecule. Such long segments are potentially found between the units of clusters, enabling a method to isolate the clusters for further structural investigations (cf. Fig. 2.2). Because different enzymes have different action patterns and affinity to the chain segments between branches, the results are partly dependent on the properties of the enzyme, besides the branching pattern of the substrate molecule. Besides two attempts to isolate clusters with the maltotetraoseforming amylase from Pseudomonas stutzeri (Finch and Sebesta, 1992) and cyclodextrin glycosyl transferase from Klebsiella pneumoniae (Bender et al., 1982), clusters have exclusively been isolated by using the a-amylase of Bacillus amyloliquefaciens (earlier called liquefying amylase of B. subtilis) by Bertoft et al. (Bertoft, 1991b, 2007a; Zhu and Bertoft, 1996; Bertoft et al., 2011a, 2012a; Kong et al., 2009; Kalinga et al., 2014b; Laohaphatanaleart et al., 2010; Wikman et al., 2011; Zhu et al., 2011b, 2013b, 2015a,b). This enzyme contains nine subsites unevenly distributed around the catalytically active site (Robyt and French, 1963; Thoma et al., 1970), and attack at the external chains preferentially results in the formation of maltohexaose and other linear, small oligosaccharides (Bertoft, 1989a; Laohaphatanaleart et al., 2010; Robyt and French, 1963). Simultaneously, attack at the internal chains occurs (Goesaert et al., 2010), preferential at the comparatively long intercluster segments (Bertoft, 1989b). Because of the constitution of subsites in the enzyme, these intercluster segments have nine or more residues (Kong et al., 2009; Bertoft, 2007a; Laohaphatanaleart et al., 2010; Zhu et al., 2011b; Bertoft et al., 2011b), whereas segments inside the clusters are shorter. This enabled the only definition of a cluster suggested so far, namely a group of chains in which the distances between branches are shorter than nine glucosyl units (Bertoft, 2007b). For the preparation of clusters, a comparatively dilute solution of the B. amyloliquefaciens a-amylase is used. The reaction is initially fast when all subsites are filled with D-glucosyl residues, but it slows down considerably when this criterion is not fulfilled (Kong et al., 2009; Kalinga et al., 2014b; Zhu et al., 2011b). At this stage, units of clusters predominate in the reaction mixture and can be recovered and size-fractionated in methanol (Bertoft and Spoof, 1989). The size distribution of clusters in preparations from diverse plants have been analyzed by GPC and is generally broad (Bertoft et al., 2012a). The estimated average DP of clusters in the form of limit dextrins from a range of amylopectin sources is shown in Table 2.3. Generally, clusters in type 1 and 2 amylopectins tend to be large, whereas type 3 and 4 have smaller clusters (Bertoft, 2007a; Bertoft et al., 2012a). The chain distribution of the cluster preparations is obtained by debranching, using the same techniques as for amylopectin and its limit

Distribution of Groups of Building Blocks (mol%) Sample

AP Type

DPcltr

NCcltr

DPBbl

NBbl

IB-CL

2

3

1

76.9

12.9

11.5

5.8

5.8

54.0

26.5

1

70.1

11.8

11.0

5.7

5.7

54.6

Wheat

1e2

82.4

14.2

10.7

6.3

6.4

Rice

2

65.8

11.1

13.0

4.2

2

75.2

12.7

13.3

4.8

b

Barley Oat

c

Sago b

4

5

6

9.2

7.8

2.4

27.2

9.4

7.1

1.7

57.0

24.5

9.6

7.6

1.3

6.5

48.5

26.4

10.1

10.4

4.5

6.4

46.2

27.8

10.7

10.1

Sweet potato

2e3

73.8

11.5

12.1

6.6

7.3

43.1

32.5

13.6

Mung bean

3

61.3

9.7

11.0

4.6

6.4

53.4

28.4

9.2

b

Potato

4

47.4

5.9

11.8

3.0

8.1

49.7

29.7

14.0

Lesser yam

4

59.7

8.5

11.9

3.9

7.4

51.3

26.4

11.4

5.2 d

10.8 7.3

1.6 d

6.7 8.2

2.7

AP type, structural type of amylopectin from which the clusters were isolated; DPBbl, average DP of building blocks isolated from the clusters; DPcltr, average DP of clusters; IB-CL, interblock chain length; the groups of building blocks are described in the text; NBbl, average number of building blocks in the clusters; NCcltr, average number of chains in the clusters. a Data are based on values given in a collection of references: Bertoft et al. (2011a,b, 2012a), Bertoft (2007a,b), Kalinga et al. (2014b), Zhu et al. (2011b,c). b Data are estimated average values based on data given in the reference. c Data for the population of large granules in wheat. d Group 5 þ 6.


TABLE 2.3 Building Block Structure of Clusters From Selected Amylopectin Samplesa


119

dextrins. The average NC of a single cluster varies between approximately 5 and 16 (Table 2.3; Ge´rard et al., 2000; Kong et al., 2009; Bertoft, 2007a; Bertoft et al., 2012a; Kalinga et al., 2014b; Zhu et al., 2011b). Because the a-amylase cleaves longer chains in the amylopectin into shorter fragments, many of the chain categories in the resulting cluster preparations are not directly comparable to those found in amylopectin. It was therefore suggested that the chains in the a-amylase products are designated by lowercase letters as opposed to capital letters for chains in amylopectin (Bertoft et al., 2012a). The chains in clusters are further numbered according to the proposed number of interblock chain segments that they are involved in. Thus, in f,b-limit dextrins of clusters, b0-chains (DP 4e6) lack the interblock segment because they are completely embedded in the building blocks, b1-chains (DP 7e18) have one interblock segment, b2-chains (DP 19e27) have two segments, and b3-chains (DP 28) have three or more segments (Bertoft et al., 2012a). B2- and B3chains of amylopectin are preferentially attacked by the a-amylase and this is detected as a decreased number of b3-chains in the clusters. Instead, the a-amylase gives rise to increased number of the shorter chains, especially b0chains and chains with DP 3, which represents a mixture of a- and b-chains (Bertoft and Koch, 2000; Bertoft et al., 2012a), but b1- and b2-chains are produced as well (Zhu et al., 2011b, 2013a,b; Kong et al., 2009; Bertoft et al., 2012a). With type 4 amylopectins (B-crystalline starches), b2-chains are produced in larger quantities than from the other types of amylopectin structures (Bertoft, 2007a; Bertoft et al., 2012a). In some cases, the ratio of a:b chains in the cluster preparation is different from the A:B ratio of the original amylopectin (Zhu and Bertoft, 1996; Bertoft, 2007a), which suggests that the cleavage of intercluster chain segments gives rise to proportionally more of either new a-chain stubs or new b-chain stubs. In other cases the ratio appears indifferent (Bertoft, 1991b; Bertoft and Koch, 2000; Zhu et al., 2011b), in which case the a-amylolysis results in equal numbers of new a- and b-chains. Such differences between samples were suggested to reflect certain preferential modes of cluster interconnection (Zhu and Bertoft, 1996) and may contribute with details to the structural analysis of the macromolecule. In all cases analyzed so far, the cluster preparations contained some b2and b3-chains (Bertoft and Koch, 2000; Bertoft, 2007a; Kalinga et al., 2014b; Laohaphatanaleart et al., 2010; Zhu et al., 2011b, 2013b, 2015a,b), which are comparable to the long chains in amylopectin. In fact, the average number of long chains in isolated clusters appears generally to correspond to between 0.5 and 1 chain (i.e., every second or every cluster carries the chain, respectively) (Bertoft et al., 2010, 2011a; Kalinga et al., 2014b; Zhu et al., 2011c, 2013b). This is not expected based on the traditional cluster model, in which long chains must be cleaved to release clusters from the macromolecule, and therefore clusters should contain only short chains (Fig. 2.5a). The building block backbone model is, however, compatible with this result. With this


FIGURE 2.5 The principles of isolation of clusters and building blocks from amylopectin with the a-amylase of B. amyloliquefaciens. In (a) the theoretical events are shown based on the cluster model and in (b) based on the backbone model. Only the internal part of the molecules are shown to highlight the endo-attack by the enzyme. IB-S, interblock segment; IC-S, intercluster segment. Circles are glucosyl units in the enlargements of clusters and building blocks.

model, the long chain in clusters represents a part of the backbone structure and contains two or more interblock chain segments (Fig. 2.5b). The apparent cluster is actually a group of building blocks and this group is separated from other similar groups of building blocks by (apparent) interluster chain segments.


121

4.4 Units of Building Blocks Whereas a diluted a-amylase preparation is used for the isolation of clusters, a 100 or 200 times more concentrated solution is used to produce a-LD from the clusters (Fig. 2.5b; Bertoft et al., 1999; Bertoft, 2007a). The composition of building blocks have been analyzed by GPC using Superdex 30 or by HPAEC with CarboPac PA-100 as the column (Bertoft et al., 2012a,b). Different groups of building blocks are distinguished. The smallest building blocks belong to group 2 and have only two chains (and thus one branch). These blocks have DP between 5 and 9. Building blocks with DP 5 correspond to the pentasaccharide 62-a-maltosylmaltotriose (French et al., 1972, Fig. 2.5b). Blocks with DP 6, albeit giving a single peak by HPAEC, consist actually of three individual molecular species, namely 62-a-maltosylmaltotetraose, 63a-maltosylmaltotetraose, and 62-a-maltotriosylmaltotriose (Umeki and Yamamoto, 1972; Jodelet et al., 1998). Blocks with DP 7 consist of six individual molecular species (Umeki and Yamamoto, 1975) and the composition becomes increasingly complex with larger sizes. Building blocks of group 3 have DP 10e14 and consist of three chains (Bertoft et al., 2010, 2011b, 2012b; Zhu et al., 2011c). Two possible conformations of the chains within the dextrins exist (Bertoft et al., 2012b). In the so-called Haworth conformation (named after the Nobel laureate Walter N. Haworth), the chain with the reducing end (the c-chain) carries a b-chain, which in turn carries an a-chain. Thus, the ratio of a:(b þ c) is 0.5. In the other alternative, known as Staudinger conformation (named after the Nobel laureate Hermann Staudinger), the c-chain carries two a-chains and the ratio is 2.0. Umeki and Yamamoto (1975b) showed that ICL is at least 1 with both conformations and, thus, two branches cannot exist immediately adjacent to each other. Building blocks of group 4 with four chains possess increasingly diverse molecular species between DP 15 and 19. In HPAEC at least two peaks are obtained for each DP and the pattern is slightly different in clusters isolated from different starch sources (Bertoft et al., 2012b). The possible arrangements of the chains include also mixed conformations as shown by the fact that the ratio of a:(b þ c) chains is intermediate between 0.5 and 2.0 (Bertoft et al., 2012b). Building blocks of group 5 are not distinguished readily by HPAEC but can be detected by GPC and by which they also were isolated as a group with DP between approximately 20 and 35 (Bertoft et al., 2011b, 2012b; Zhu et al., 2011c). These building blocks have between five and seven chains with an average around six chains. The largest building blocks are included in group 6 and possess an average NC up to 10e11 (Bertoft et al., 2012b). The general structure of building blocks in clusters from different starches is remarkably similar. It was found, however, that clusters from cereal samples (amylopectin structure of type 1 or 2) tend to possess somewhat lower ratios of a:(b þ c) chains than clusters from amylopectins with type 3 or 4 structure


(Bertoft et al., 2012b). This suggests that cereal amylopectins have building blocks with a more preferred Haworth conformation, whereas other amylopectins tend to have more preferred Staudinger conformation. It is possible that such differences at least partly contributes to the functional properties of starch. It should be noted that the structure of building blocks described above are a-LDs typically obtained when using the a-amylase of B. amyloliquefaciens. Other enzymes give rise to different a-LDs. For example, Umeki and Yamamoto (1975b) made a detailed investigation of the a-LDs produced from the b-LD of waxy rice amylopectin by the so-called saccharifying a-amylase from Bacillus subtilis. These authors found one single-branched dextrin with DP 4, three double-branched dextrins with DP 6 and 7, and eight triplebranched dextrins with DP 9 and 10, of which the single-branched dextrin was most common and represented about 73% of the a-LDs by number. With the a-amylase of B. amyloliquefaciens, the single-branched dextrins (i.e., group 2 building blocks) predominate in clusters regardless of the plant source and represent about 43%e60% of the building blocks (Table 2.3; Kong et al., 2009; Bertoft et al., 2010, 2011b, 2012a; Bertoft, 2007b; Zhu et al., 2011c). Group 3 building blocks is the second-most common type (typically about 25%e33%), whereas the larger blocks are found in decreasing amounts. Because the size distribution of building blocks is quite similar in clusters from different sources, the number of building blocks inside a cluster largely depends on the size of the cluster (Table 2.3). The average number of building blocks (NBbl) in a cluster can be estimated as (Bertoft et al., 2010): NBbl ¼ (wt%)Bbl/100 DPcltr/DPBbl

(2.10)

in which (wt%)Bbl is the proportion by weight of all branched building blocks in the cluster, and DPcltr and DPBbl is the average DP of the clusters and building blocks, respectively. The data are obtained by analysis of the size distributions of the clusters and the building blocks. The unit chains in building blocks are very short. CL of group 2 blocks is around 3.3e3.7, whereas larger blocks possess somewhat longer chains (Bertoft et al., 2012b). The size distribution of the chains is narrow: the broadest range is found in group 6, which have chains of DP 2e15. Most blocks possess a peak of b-chains at DP 5e7 (Zhu et al., 2011c; Bertoft et al., 2012b). Average ICL is between 1 and 3 in all groups of building blocks. The density of branches (DB) is therefore high, typically between 14% and 21% (Bertoft et al., 2010, 2011b, 2012a,b; Bertoft, 2007b; Zhu et al., 2011c). This can be compared with the DB of clusters which mostly range from 10% to 17% (Bertoft et al., 2011a, 2012a; Kong et al., 2009; Bertoft, 2007a; Kalinga et al., 2014b; Zhu et al., 2011b, 2013b). Besides the branched building blocks, small carbohydrates representing glucose up to maltohexaose are also produced from the clusters by B. amyloliquefaciens a-amylase. These dextrins have no branches and can be


123

hydrolyzed into glucose, maltose, and maltotriose by b-amylase, whereby they are clearly separated from the branched building blocks of DP 5, when analyzed by GPC or HPAEC (Bertoft et al., 2012a). This group of small carbohydrates (named group 1 as they consist of only a single chain) derives from the internal chain segments between the building blocks and can be used for the estimation of the interblock chain length (IB-CL). The a-amylase leaves at average approximately two glucose residues adjacent to any side of a branch point (Umeki and Yamamoto, 1975a). Thus, at each side of the interblock segment a similar stub remains and the (approximate) total length of the segment equals (Bertoft et al., 2010): IB-CL ¼ (mol%)Group 1 DPGroup 1/(mol%)Bbl þ 4

(2.11)

in which (mol%)Group 1 and (mol%)Bbl is the proportion by mole of the carbohydrates in group 1 and the branched building blocks, respectively, and DPGroup 1 is the average DP of group 1. The average IB-CL varies between 5.7 and 8.1 glucosyl units and depends on the type of amylopectin molecular structure (Table 2.3). Typically, type 1 amylopectins possess the shortest IBCL and type 4 the longest IB-CL, whereas types 2 and 3 have intermediate lengths (Bertoft et al., 2012a). Interestingly, IB-CL was shown to correlate with the thermal properties of starch granules, so that starches with short IB-CL gelatinize at lower temperatures than starches with long IB-CL (Vamadevan et al., 2013a). It was suggested that longer IB-CL stabilize the crystalline structure in the granules by enabling a more perfect packing of the crystallites, whereas short IB-CL might introduce disorder in the structure (Vamadevan et al., 2013b).

4.5 Starch Phosphate Esters Some starches, in particular from potato but also from other tubers or roots (Blennow et al., 1998a, 2001; McPherson and Jane, 1999), possess phosphate esters substituted mostly at C-6 and C-3 positions of the D-glucosyl units of the amylopectin component (Hizukuri et al., 1970), whereas the amylose is essentially free from phosphate (Schoch, 1942b). The enzyme a-glucan, water dikinase (GWD) is primarily responsible for the phosphorylation at the C-6 position (Lorberth et al., 1998; Ritte et al., 2002), whereas phosphoglucan, water dikinase (PWD) catalyzes phosphorylation at C-3 (Baunsgaard et al., 2005; Ko¨tting et al., 2005). Though the total phosphorus content is low, in potato starch corresponding to approximately 1 phosphorylated residue in 200e500 (Blennow et al., 1998a, 2001), it greatly differs between cultivars (Bay-Smidt et al., 1994; Muhrbeck and Tellier, 1991) and is of importance for the functionality of starch. Surprisingly, amylose-free potato starch was reported to contain similar levels (Visser et al., 1997) or less phosphorus than normal starch (McPherson and Jane, 1999), whereas a high-amylose-containing potato starch (from antisense starch branching enzyme mutation) contained


higher levels (Schwall et al., 2000). The reason is the presence of long chains in the amylopectin component (Wikman et al., 2011), for which GWD possesses high affinity (Ritte et al., 2002). Cereal starches contain also phosphorus, but this is only to a minor part covalently bound to amylopectin (Hizukuri and Maehara, 1990; Shibanuma et al., 1994; Takeda et al., 1987b, 1988, 1989b). Instead, most of this phosphorus is found as lysophospholipids that are complexed with the amylose fraction of the granules and not covalently linked to amylopectin (Morrison, 1995; Shamekh et al., 1999). The esterified phosphorus content and positions on the D-glucosyl residues have been analyzed by 31P-NMR (Blennow et al., 1998a, 2000a; Bay-Smidt et al., 1994; Muhrbeck and Tellier, 1991; Lim and Seib, 1993; Kasemsuwan and Jane, 1994). Total starch-bound phosphorus was also analyzed chemically (Bay-Smidt et al., 1994; Lanzetta et al., 1979; Bergthaller, 1971). After controlled acid hydrolysis of all glycosidic linkages in hot 0.7 M HCl for 4 h (Hizukuri et al., 1970), the amount of glucose 6-phosphate residues was analyzed enzymatically by the glucose 6-phosphate dehydrogenase catalyzed reduction of NADPþ (Hizukuri et al., 1970; Bay-Smidt et al., 1994; Michal, 1984). Glucose 6-phosphate and glucose 3-phosphate were also separated by HPAEC-PAD (Blennow et al., 1998b). However, the quantitative estimation of the more labile component glucose 3-phosphate is difficult because of partial degradation. Generally, about 70% of the phosphorus is found at the C-6 position and 30% at C-3 (Hizukuri et al., 1970; Muhrbeck and Tellier, 1991; Blennow et al., 2000a). The location of phosphate esters in the amylopectin macromolecule was analyzed using the enzyme glucoamylase of Rhizopus delemar (Hizukuri et al., 1970; Takeda and Hizukuri, 1986) or Aspergillus niger (Zhu and Bertoft, 1996; Lim and Seib, 1993; Takeda and Hizukuri, 1986; Abe et al., 1982). This is an exo-acting enzyme that produces glucose by hydrolysis of the a-D-(1/4)linkages. If the entire external part of the B-chain is removed together with all a-D-(1/4)-linkages of the A-chain, the residual single glucosyl stub is also removed by attack at the a-D-(1/6)-linkage, and the amylopectin is completely degraded (Fig. 2.6; The Amylase Research Society of Japan, 1988). If, however, the requirements for attack at the (1/6)-linkage are not fulfilled, a g-limit dextrin is produced. This is the case if a substituent, like a phosphate group, is found in either the A- or the B-chain. Such groups act as barriers and cannot be by-passed by the enzyme (Fig. 2.6; Takeda and Hizukuri, 1986; Abe et al., 1982). Only a minor part of the macromolecule remains resistant to glucoamylase attack; reported g-limit values of amylopectin are 81%e97% (Zhu and Bertoft, 1996; Abe et al., 1982). Phosphorylated amylopectin has also been debranched enzymatically and the phosphorylated chains were isolated by anion-exchange chromatography (Takeda and Hizukuri, 1981, 1986). By further treatment with b-amylase, Takeda and Hizukuri (1982) showed that the phosphate is largely associated with the long B-chains of potato amylopectin, and the degree of


125

FIGURE 2.6 Action pattern of fungal glucoamylase on branched and phosphorylated branched and linear substrates. [, phosphate ester at C-6; Y, phosphate ester at C-3. The structures of limit dextrins are adapted from Takeda, Y., Hizukuri, S., 1986. Actions of Aspergillus oryzae alphaamylase, potato phosphorylase, and rabbit muscle phosphorylase a and b on phosphorylated (1/4)-a-D-glucan. Carbohydrate Research 153, 295e307 and The Amylase Research Society of Japan, 1988. Handbook of Amylases and Related Enzymes, first ed. Pergamon Press plc, Oxford.

phosphorylation seems to increase with the length of the chains (Blennow et al., 1998a). Moreover, no phosphate is found closer than nine D-glucosyl residues from the nonreducing ends or in the vicinity of the branches (Takeda and Hizukuri, 1982). Wikman et al. (2011) debranched the b-LD of potato amylopectin and found that 8.2% of the chains by number were phosphorylated. The phosphate was apparently only associated with the B-chains. Wikman et al. (2011) also isolated phosphorylated clusters by anion-exchange chromatography and found that in normal potato starch, these clusters represented 24% of all clusters. The phosphorylated clusters were larger than their nonphosphorylated counterparts, possessed longer CL and ICL but were less densely branched. Partial acid hydrolysis of potato starch granules, after which mainly the crystalline lamellae remains, showed that most of the phosphate is associated with the amorphous parts of the granule, but a substantial amount is also included in the crystalline lamellae (Blennow et al., 2000a; Wikman et al., 2013). C-6 phosphate was assumed to disturb the crystallization of amylopectin (Muhrbeck et al., 1991) and, accordingly, the major part of the C-6substituted phosphate is found in the amorphous parts, whereas the phosphate at C-3 position is more equally distributed between the amorphous and crystalline areas (Blennow et al., 2000a; Wikman et al., 2014). In addition, Blennow et al. (Blennow et al., 1998a, 2000b) analyzed a series of phosphorylated starches and concluded that the degree of phosphorylation is strongly correlated to the distribution of the nonphosphorylated chains in the amylopectin component. Jane and Shen (1993) used a chemical solubilization technique, in which the granules are gradually solubilized in 4 M CaCl2 (Jane, 1993). These


authors showed that the inner region of potato starch granules is more phosphorylated than the peripheral parts, which later was confirmed by particle induced X-ray emission data (Blennow et al., 2005). It also correlated with a longer length of the long B-chains of the amylopectin component toward the interior of the granules (Jane, 1993). In contrast, Buleón et al. (2014) used synchrotron X-ray microfluorescence mapping of potato granules and found more phosphate in their peripheral parts than in their interior. Jane and Shen (1993) showed that small potato starch granules, which possess lower amylose content, contain a higher degree of phosphorylation than large granules. In addition, Bay-Smith et al. (1994) found that individual potato tubers are more phosphorylated at their interior parts. Thus, the phosphate esters are unevenly distributed on both the molecular and the granular levels, as well as on the tuber level.

5. ANALYSIS OF INTERMEDIATE MATERIALS Some starches, notably the high-amylose types, contain IM. This material has gained interest because it appears to contribute to the fraction of resistant starch (Li et al., 2008), which is considered beneficial for human health. Besides mutant maize varieties (Ge´rard et al., 2001, 2002; Shi et al., 1998; Wang et al., 1993a; Boyer and Liu, 1985; Baba and Arai, 1984; Vamadevan et al., 2014; Li et al., 2008; Ikawa et al., 1978; Inouchi et al., 1983; Perera et al., 2001; Yun and Matheson, 1992) and wrinkled peas (Banks et al., 1974; Matheson and Welsh, 1988; Lloyd et al., 1996; Colonna and Mercier, 1984; Bertoft et al., 1993a), IM was also described in high-amylose varieties of barley (Banks et al., 1974) and potato (Schwall et al., 2000). Moreover, IM was found in nonmutant (normal) starches from potato (Yoon and Lim, 2003), maize (Klucinec and Thompson, 1998), oats (Banks and Greenwood, 1967; Yoon and Lim, 2003; Paton, 1979; Wang and White, 1994b), wheat (Banks and Greenwood, 1967; Dais and Perlin, 1982), rye, and barley (Banks and Greenwood, 1967). The nature of this material is somewhat confusing and might be unique for each type of starch (Banks et al., 1974). The method of fractionation of the starch could also influence on the result, especially when IM is isolated together with either the amylose or the amylopectin fraction. In such cases IM might even remain unknown for the investigator and influence on the structural analysis of the other components. In several cases, the measured amylose content of high-amylose starches by iodine binding is largely overestimated due to IM with a high-IA or amylopectin of longer CL than normal (Ge´rard et al., 2001; Boyer et al., 1980; Perera et al., 2001; Wang et al., 1993b), or both (Shi et al., 1998; Wang et al., 1993a,b; Baba and Arai, 1984; Wang and White, 1994b). Because IM is structurally related to either amylose or amylopectin, it is analyzed by the same methods. Banks and Greenwood (1967) described a method adopted to cereal starches, in which an amyloseethymol complex was separated from the


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amylopectin component. The complex was reprecipitated in butanol into amylose and a soluble fraction containing anomalous amylose and/or amylopectin, the latter with unusually long CL. Adkins and Greenwood (1969), working with maize starches, precipitated amylose in 1-butanol and from the supernatant (the amylopectin fraction) they precipitated a glucan in complex with iodine. The amount of the complex increased with increasing amylose content of the maize and was characterized as short-chain amylose material. Wang et al. (1993b) also found IM included in the supernatant fraction together with amylopectin that they isolated from a range of mutant maize samples. IM was fractionated by GPC on Sepharose CL 2B and found to be a branched component smaller than the amylopectin (Li et al., 2008). It possessed similar types of chains as in amylopectin, but in different proportions depending on the type of mutation (Wang et al., 1993b). Klucinec and Thompson (1998) precipitated amylose together with an intermediate fraction from the amylopectin in an aqueous mixture of 6% 1-butanol and 6% isoamyl alcohol. The amylose was then reprecipitated in 1-butanol, whereas IM remained in the supernatant. They found that IM in normal maize had rather similar types of chains as amylopectin, but in high-amylose starches the composition changed, and IM possessed a large group of long chains. The apparently true amylopectin component also contained increased amounts of the long chains (Klucinec and Thompson, 1998). More, and/or longer, long chains is a typical feature associated with amylopectin in maize starches containing the amylose-extender mutation (Boyer and Liu, 1985; Baba and Arai, 1984; Vamadevan et al., 2014; Ikawa et al., 1978; Inouchi et al., 1983; Wang et al., 1993b; Liu et al., 2012). Similar results have been found for highamylose-containing rice starches (Asaoka et al., 1986; Nishi et al., 2001; Kubo et al., 2010). Klucinec and Thompson (1998) concluded that IM, like the amylopectin, is branched, though its structural features results in altered physical behavior, as shown by its precipitation in the 1-butanoleisoamyl alcohol mixture. In a double (dull:waxy) and triple mutant (amyloseextender:dull:waxy) maize starch, an IM component was found in high concentration (40% and 80%, respectively; Bertoft et al., 2000). The material, which contained only slightly altered chain distribution, was apparently more resistant to a-amylase attack than the normal amylopectin and the behavior of the a-dextrins in methanol was different. Bertoft et al. (2000) suggested that this IM possessed a more regularly branched structure that prevented the action of the a-amylase and rendered the molecule the altered behavior. Colonna and Mercier (1984) isolated a very low-molecular weight component from wrinkled pea starch (also a high-amylose type starch). This IM was branched and possessed similar types of chains as the amylopectin component, but the IA was high and the S:L chain ratio low. Bertoft et al. (1993a) size-fractionated the IM and found that the proportion of the long chains slightly increased with decreasing molecular weight. In fact, the molecular weight of the small IM resembled that of the clusters of the


amylopectin component and was suggested to be composed of small, clusterlike structures interconnected by the long chains, thus increasing the proportion of these chains (Bertoft et al., 1993a). The IM of wrinkled pea has also been described as a mixture of very short, linear amylose chains and branched, either normal (Banks et al., 1974) or long-chained amylopectin (Boyer et al., 1980). Biliaderis et al. (1981) also reported long-chained materials and Matheson (1990) found that both ECL and ICL were larger than in the normal amylopectin of smooth pea starch. The different opinions regarding the nature of IM might reflect differences among varieties of wrinkled peas. In the developing endosperm of wheat kernel starch, material with sizes corresponding to amylose was considered as IM due to its intermediate iodinestaining properties (Waduge et al., 2014). At maturity the material was not present anymore, which suggested that it was a precursor molecule to either amylose or amylopectin. In fact, it was suggested that the linear structure of amylose and the backbone structure of amylopectin are related (Bertoft, 2013). The linear amylose molecule is an extreme example of a backbone with comparatively low DP and high-IA. Branched amylose has shorter chains that combine into a backbone and few side branches to the backbone, together having higher DP and lower IA than linear amylose. Amylopectin is the other extreme example with a backbone consisting of a large number of comparatively short chains and extensive side branches, which render the molecule a high-DP and low-IA. In this scenario, IM possess a structure in between the extremes with an intermediate backbone, DP, and iodine-staining properties.

6. FUTURE TRENDS Research on starch has lasted for two centuries, and it is amazing to find that a final consensus about its molecular structure has not been achieved yet. The same is true with regards to the structure of the starch granules. (For an overview of the history of research on starch, the reader is referred to a series of essays by Seetharaman and Bertoft, 2012a,b,c,d, 2013a,b.) The challenge to understand starch structure is not simply to record data and search for correlations, e.g., between unit CL profiles and starch properties. Rather, it is to understand the arrangement of the unit chains inside the macromolecules into a final three-dimensional structure. The unit chains can, in theory, be arranged into virtually any kind of branched structure (Bertoft, 2004), among which there is only one structure that fully will explain the properties of the starch granules and the functionality of starch in food systems. There are a range of techniques, which are outside the scope of this chapter but discussed in other parts of this book, that are frequently in use to study starch structure, especially to analyze the granular structure. To these belong specific microscopic imaging techniques (Gallant et al., 1997), such as atomic force microscopy (Baker et al., 2001; Baldwin et al., 1998; Chauhan and Seetharaman, 2013; Dang et al., 2006; Liu et al., 2001; Park et al., 2011; Ridout


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et al., 2006; Szymonska et al., 2003; Waduge et al., 2010) and confocal microscopy, the latter combined with fluorescence labeling (Ambigaipalan et al., 2011; Apinan et al., 2007; Glaring et al., 2006; Han and Hamaker, 2002; Manca et al., 2015; Naguleswaran et al., 2011; Varatharajan et al., 2011). Synchrotron microfocus mapping of single starch granules was shown to be a powerful tool to probe the architecture and constituents of the granules (Buleón et al., 1997, 1998, 2014; Popov et al., 2009). Other techniques, used almost on a routine basis include differential scanning calorimetry (DSC) (Srichuwong et al., 2005; Qi et al., 2003; Alvani et al., 2012; Ambigaipalan et al., 2013; Biliaderis, 2009; Gomand et al., 2010; Jayakody and Hoover, 2008; Lan et al., 2008; Liu et al., 2009; Ratnayake and Jackson, 2007; Xia et al., 2010), wide-angle X-ray scattering (WAXS) (Varatharajan et al., 2011; Lan et al., 2008; Ratnayake and Jackson, 2007; Xia et al., 2010; Jiang et al., 2010; Tziotis et al., 2005; Vermeylen et al., 2005; Wang et al., 2012; Saibene and Seetharaman, 2006; Kubo et al., 2008), small-angle X-ray scattering (SAXS) (Regina et al., 2012; Vermeylen et al., 2004, 2005; Wang et al., 2012; Sanderson et al., 2006; Daniels and Donald, 2003; Donald et al., 2001), or NMR (Gidley, 1985; Falk et al., 1996; McIntyre et al., 1990; Tamaki et al., 1997, 1998). Combinations of two or more of these techniques give novel insights into how the starch macromolecules are arranged inside the granules (Cooke and Gidley, 1992; Waigh et al., 1999; Jenkins and Donald, 1998; Kiseleva et al., 2005; Genkina et al., 2004, 2007; Koroteeva et al., 2007a,b; Kozlov et al., 2006, 2007a,b), details of which are found in other chapters of this book. Genetic modifications of plants by biotechnology have made it possible to manipulate the biosynthesis of starch (Keeling and Myers, 2010; Robyt and Mukerjea, 2013; D’Hulst & Me´rida, 2010; Tetlow, 2011; Zeeman et al., 2010; Kossmann and Lloyd, 2000). Tailor-made starches for different industrial applications are predicted to become increasingly important in the future. The development of these starches and the understanding of the interaction of the enzymatic activities involved in their biosynthesis demands a range of suitable methods for the analysis of the structure of the altered macromolecules. As starches from mutant plants often are available in very small quantities, microscale methods for their structural analyses are needed and experiments toward that direction have been made (Zhu et al., 2013c, 2014). Future technology should therefore to a significant extent contribute with more sensitive detection methods. It is, however, somewhat difficult today to foresee that analyses regarding the fine structure of starch and its components within a near future could be done on a routine basis for screening of a large number of samples. Certain structural details may, however, correlate with fairly easily analyzed parameters and explain important functional properties, thereby enable faster screening. The enzymatic techniques to analyze starch structure belong to the most powerful tools available to date. The underlying methodology in any new technique will, therefore, to a large extent remain dependent on the methods described in this chapter.


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146 PART j ONE Analyzing and Modifying Starch Takeda, Y., Shibahara, S., Hanashiro, I., 2003. Examination of the structure of amylopectin molecules by fluorescent labeling. Carbohydrate Research 338, 471e475. Tamaki, S., Hisamatsu, M., Teranishi, K., Yamada, T., 1997. Structural change of potato starch granules by ball-mill treatment. Starch/Sta¨rke 49, 431e438. Tamaki, S., Hisamatsu, M., Teranishi, K., Adachi, T., Yamada, T., 1998. Structural change of maize starch granules by ball-mill treatment. Starch/Sta¨rke 50, 342e348. Tetlow, I.J., 2011. Starch biosynthesis in developing seeds. Seed Science Research 21, 5e32. The Amylase Research Society of Japan, 1988. Handbook of Amylases and Related Enzymes, first ed. Pergamon Press plc, Oxford. Thitipraphunkul, K., Uttapap, D., Piyachomkwan, K., Takeda, Y., 2003. A comparative study of edible canna (Canna edulis) starch from different cultivars. Part II. Molecular structure of amylose and amylopectin. Carbohydrate Polymers 54, 489e498. Thoma, J.A., Brothers, C., Spradlin, J., 1970. Subsite mapping of enzymes. Studies on Bacillus subtilis amylase. Biochemistry 9, 1768e1775. Thompson, D.B., 2000. On the non-random nature of amylopectin branching. Carbohydrate Polymers 43, 223e239. Thurn, A., Burchard, W., 1985. Heterogeneity in branching of amylopectin. Carbohydrate Polymers 5, 441e460. Tziotis, A., Seetharaman, K., Klucinec, J.D., Keeling, P., White, P.J., 2005. Functional properties of starch from normal and mutant corn genotypes. Carbohydrate Polymers 61, 238e247. Umeki, K., Yamamoto, T., 1972. Enzymatic determination of structure of singly branched hexaose dextrins formed by liquefying a-amylase of Bacillus subtilis. Journal of Biochemistry 72, 101e109. Umeki, K., Yamamoto, T., 1975a. Structures of singly branched heptaoses produced by bacterial liquefying a-amylase. Journal of Biochemistry 78, 889e896. Umeki, K., Yamamoto, T., 1975b. Structures of multi-branched dextrins produced by saccharifying a-amylase from starch. Journal of Biochemistry 78, 897e903. Vamadevan, V., Bertoft, E., Seetharaman, K., 2013a. On the importance of organization of glucan chains on thermal properties of starch. Carbohydrate Polymers 92, 1653e1659. Vamadevan, V., Bertoft, E., Soldatov, D.V., Seetharaman, K., 2013b. Impact on molecular organization of amylopectin in starch granules upon annealing. Carbohydrate Polymers 98, 1045e1055. Vamadevan, V., Hoover, R., Bertoft, E., Seetharaman, K., 2014. Hydrothermal treatment and iodine binding provide insights into the organization of glucan chains within the semicrystalline lamellae of corn starch granules. Biopolymers 101, 871e885. Varatharajan, V., Hoover, R., Li, J., Vasanthan, T., Nantanga, K.K.M., Seetharaman, K., Liu, Q., Donner, E., Jaiswal, S., Chibbar, R.N., 2011. Impact of structural changes due to heat-moisture treatment at different temperatures on the susceptibility of normal and waxy potato starches towards hydrolysis by porcine pancreatic alpha amylase. Food Research International 44, 2594e2606. Vasanthan, T., Bhatty, R.S., 1996. Physicochemical properties of small- and large-granule starches of waxy, regular, and high-amylose barleys. Cereal Chemistry 73, 199e207. Vermeylen, R., Goderis, B., Reynaers, H., Delcour, J.A., 2004. Amylopectin molecular structure reflected in macromolecular organization of granular starch. Biomacromolecules 5, 1775e1786. Vermeylen, R., Goderis, B., Reynaers, H., Delcour, J.A., 2005. Gelatinisation related structural aspects of small and large wheat starch granules. Carbohydrate Polymers 62, 170e181.


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148 PART j ONE Analyzing and Modifying Starch Wong, K.S., Jane, J., 1997. Quantitative analysis of debranched amylopectin with a postcolumn enzyme reactor. Journal of Liquid Chromatography 20, 297e310. Xia, L., Wenyuan, G., Juan, W., Qianqian, J., Luqi, H., 2010. Comparison of the morphological, crystalline, and thermal properties of different crystalline types of starches after acid hydrolysis. Starch/Sta¨rke 62, 686e696. Yao, Y., Guiltinan, M.J., Thompson, D.B., 2005. High-performance size-exclusion chromatography (HPSEC) and fluorophore-assisted carbohydrate electrophoresis (FACE) to describe the chain-length distribution of debranched starch. Carbohydrate Research 340, 701e710. Yokobayashi, K., Misaki, A., Harada, T., 1970. Purification and properties of Pseudomonas isoamylase. Biochimica et Biophysica Acta 212, 458e469. Yokobayashi, K., Akai, H., Sugimoto, T., Hirao, M., Sugimoto, K., Harada, T., 1973. Comparison of the kinetic parameters of Pseudomonas isoamylase and Aerobacter pullulanase. Biochimica et Biophysica Acta 293, 197e202. Yokoyama, W., Renner-Nantz, J.J., Shoemaker, C.F., 1998. Starch molecular mass and size by size-exclusion chromatography in DMSO-LiBr coupled with multiple angle laser light scattering. Cereal Chemistry 75, 530e535. Yoo, S.H., Jane, J.L., 2002. Molecular weights and gyration radii of amylopectins determined by high-performance size-exclusion chromatography equipped with multi-angle laser-light scattering and refractive index detectors. Carbohydrate Polymers 49, 307e314. Yoon, J.-W., Lim, S.-T., 2003. Molecular fractionation of starch by density-gradient ultracentrifugation. Carbohydrate Research 338, 611e617. You, S., Izydorczyk, M.S., 2002. Molecular characteristics of barley starches with variable amylose content. Carbohydrate Polymers 49, 33e42. You, S.G., Lim, S.T., 2000. Molecular characterization of corn starch using an aqueous HPSECMALLS-RI system under various dissolution and analytical conditions. Cereal Chemistry 77, 303e308. Yuan, R.C., Thompson, D.B., Boyer, C.D., 1993. Fine structure of amylopectin in relation to gelatinization and retrogradation behavior of maize starches from three wx-containing genotypes in two inbred lines. Cereal Chemistry 70, 81e89. Yun, S.-H., Matheson, N.K., 1992. Structural changes during development in the amylose and amylopectin fractions (separated by precipitation with concanavalin A) of starches from maize genotypes. Carbohydrate Research 227, 85e101. Yun, S.-H., Matheson, N.K., 1993. Structures of the amylopectins of waxy, normal, amyloseextender, and wx:ae genotypes and of the phytoglycogen of maize. Carbohydrate Research 243, 307e321. Yusuph, M., Tester, R.F., Ansell, R., Snape, C.E., 2003. Composition and properties of starches extracted from tubers of different potato varieties grown under the same environmental conditions. Food Chemistry 82, 283e289. Zeeman, S.C., Kossman, J., Smith, A.M., 2010. Starch: its metabolism, evolution, and biotechnological modification in plants. Annual Review Plant Biology 61, 209e234. Zhong, F., Yokoyama, W., Wang, Q., Shoemaker, C.F., 2006. Rice starch, amylopectin, and amylose: molecular weight and solubility in dimethyl sulfoxide-based solvents. Journal of Agricultural and Food Chemistry 54, 2320e2326. Zhu, Q., Bertoft, E., 1996. Composition and structural analysis of alpha-dextrins from potato amylopectin. Carbohydrate Research 288, 155e174. Zhu, T., Jackson, D.S., Wehling, R.L., Geera, B., 2008. Comparison of amylose determination methods and the development of a dual wavelength iodine binding technique. Cereal Chemistry 85, 51e58.


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Chapter 3

Understanding Starch Structure and Functionality Yongfeng Ai1, Jay-lin Jane2

1 University of Saskatchewan, Saskatoon, SK, Canada; 2Iowa State University, Ames, IA, United States

1. INTRODUCTION Starch, the major energy reserve in green plants, is commonly found in seeds (e.g., cereal grains and pulses), tubers (e.g., potato), roots (e.g., cassava and sweet potato), fruits (e.g., banana and squash), stems (e.g., sago), and leaves (e.g., tobacco). Starch is the predominant component of cereal grains, pulses, and tuber and root crops. For example, milled rice kernels contain up to 90% starch (dry basis, db) (Zhou et al., 2002), maize kernels up to 80% starch (db) (Orman and Schumann, 1991), and pulse grains up to 53% starch (db) (Wang, 2008). In higher plant tissues, starch is synthesized using glucose-1-phosphate through a series of complex enzymatic reactions as discussed in Chapter 1. Biosynthesis of starch granules is initiated at the hilum, the organic center of the granule, and the starch chains are elongated radially toward the periphery by apposition. The radial arrangement of starch chains is evidenced by the Maltese cross observed for starch granules under a polarized light microscope. Starch molecules are effectively organized in semicrystalline granules, which has a density of around 1.5 g/cm3 (Imberty et al., 1991). The greater density of starch granules than that of water allows easy isolation and purification of starch by gravity sedimentation. The semicrystalline structure of starch granules maintains the granular integrity and prevents the dispersion of the granules in water at ambient temperature. Heating starch in the presence of water (as a plasticizer) can gelatinize and disperse native starch granules. Starches from different botanical origins display characteristic gelatinization properties, reflecting distinct structures of starch molecules, and the organization of double-helical crystalline structures inside starch granules (Jane et al., 1999). Without the presence of water or other plasticizers, starch does not gelatinize at high temperature. Instead it will decompose when the temperature reaches 250 C and beyond (Jang and Pyun, 1996). Starch in Food. http://dx.doi.org/10.1016/B978-0-08-100868-3.00003-2 Copyright © 2018 Elsevier Ltd. All rights reserved.

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Most starch is composed of two major polysaccharides: amylose and amylopectin. Amylose is a primarily linear polysaccharide of a-1,4-linked D-glucopyranose with a few branches of a-1,6 linkages (Takeda et al., 1990). Amylopectin, however, is a highly branched polysaccharide with a-1,4linked linear chains of different lengths connected by approximately 5% a-1,6 branch linkages (Hizukuri, 1986). The two main components of starch have distinctly different properties. In an aqueous dispersion, amylose has a greater tendency to recrystallize (known as retrogradation), forms strong gels and films, and develops a dark-blue color after complexing with iodine. In contrast, amylopectin retrogrades more slowly, forms weak gels and brittle films, and displays a purple to red color after complexing with iodine. Amylose content and branch chain length distribution of amylopectin directly determine many functional properties of starch, including gelatinization, pasting, gelling, and retrogradation/syneresis (Jane et al., 1999, 2003). Other components in starch, including endogenous lipids in normal cereal starches and phosphate monoesters in potato starch, also have significant impacts on the functional properties of starch (Jane et al., 1999; Debet and Gidley, 2006). Depending on the botanical source, starch displays characteristic gelatinization temperatures, paste viscosity and clarity, gelling ability, and retrogradation rate (relating to syneresis). Understanding of relationships between structures and functional properties of starch is essential for the development of new starch varieties with desirable functionality for intended applications in the food and other industries. In this chapter, structures of various components (e.g., amylose, amylopectin, intermediate components, lipids, phosphate monoesters, and proteins) and granular structures (e.g., size, shape, and internal organization) of starches important for food and other applications will be reviewed and discussed. Functional properties of the starches will also be examined and related to their structural features.

2. COMPONENTS OF STARCH GRANULES 2.1 Structures and Properties of Amylose Amylose is an essentially linear polysaccharide of a-1,4-linked D-glucopyranose with a degree of polymerization (DP) between hundreds to tens of thousands. Amylose was once considered to be a linear molecule, but b-amylolysis of the molecule could only give a degree of hydrolysis at 72%e95% (Takeda et al., 1986; Hizukuri et al., 1988). Complete hydrolysis of amylose into maltose can be achieved when a mixture of b-amylase and pullulanase is used (Takeda et al., 1987). Experimental data indicate that amylose molecules have branched glycosidic bonds. Using a combination of debranching treatment, chemical analyses, and gel permeation chromatography, Takeda et al. (1984, 1990) characterized the structure of amylose isolated from different starches and reported multiple branches of amylose.

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The authors have further illustrated that amylose of a small molecular weight tends to have a linear molecule, whereas that of a large molecular weight (e.g., potato and cassava amylose) consists of multiple extra-long chains (DP > 2730 in maize amylose), long chains (DP > 230), as well as immature clusters of short branch chains (DP w 18) as shown in Fig. 3.1(a). When amylose is dispersed freshly in an aqueous solution, the molecule is in a random-coil conformation. The random-coil conformation, however, is meta-stable because of the coexistence of an array of hydrophobic hydrocarbon groups and hydrophilic hydroxyl groups in the chain of a-1,4-linked D-glucopyranose units. In the presence of a compound (i.e., complexing agent) that carries a hydrophobic moiety, such as polyiodide ion and n-butyl alcohol, amylose in the aqueous dispersion can instantly form an inclusion helical complex with the complexing agent, where the hydrophobic side of the amylose chain interacts with the hydrophobic moiety of the compound. The helical complex has the complexing agent included in the central cavity of the helix, resembling the structure of an inclusion complex of a cyclodextrin

FIGURE 3.1 (a) Structure of branched amylose molecule consisting of extra-long chain (EL), long chain (L), short chain (S), and reducing end (Ø). (b) A cluster model of amylopectin with A (—), B1 (—), B2 (—), and B3 (—) chains. The chain carrying the reducing end (Ø) is the C chain. d: a-1,4 glucan chain; /: a-1,6 linkage. C. L., chain length. (a) Reprinted from Takeda, Y., Shitaozono, T., Hizukuri, S., 1990. Structures of subfractions of corn amylose. Carbohydrate Research 199, 207e214; (b) Reprinted from Hizukuri, S., 1986. Polymodal distribution of the chain lengths of amylopectins, and its significance. Carbohydrate Research 147, 342e347.


and a guest molecule. The helices will further align and fold into lamellar crystallites and display a V-type crystalline pattern (Kong and Ziegler, 2014). Amylose-polyiodide helical complex shows a characteristic dark blue color with the absorption lmax at 640 nm. The lmax of the complex decreases to a shorter wavelength with the decrease in the chain length of amylose, and the color of the complex changes to purple, red, and eventually orange when the DP of amylose chain decreases to 19e25 (Mould and Synge, 1954). The color differences reflect the numbers of iodine molecules that can line up to develop a polyiodide ion in the cavity of the inclusion helix. The longer the polyiodide ion forms in the cavity, which provides a longer electron relay, the darker the color displays. The inclusion-complex formation between amylose and polyiodide ions can be utilized to determine the amylose content of starch (Duan et al., 2012; Chrastil, 1987). The inclusion complex between amylose and n-butyl alcohol can readily crystallize and precipitate out from the starch dispersion, which can be employed to fractionate amylose from amylopectin in starch (Schoch, 1942). Studies have further demonstrated that chemicals in the families of alcohols, carboxylic acids, ketones, and aldehydes have a tendency to complex with amylose, whereas those of amines and amino acids have little tendency for the complex formation and subsequent precipitation. Free fatty acids, mono- and diglycerides, and phospholipids are known to form helical complex with amylose. Triglycerides, however, were once considered not being able to complex with amylose because the mixture does not show the characteristic V-type X-ray diffraction pattern nor does it display a thermal transition peak of amyloseelipid dissociation at around 95 C in a differential scanning calorimetry (DSC) thermogram. A study using 13C-nuclear magnetic resonance (NMR) spectroscopy, however, demonstrates that triglycerides in corn oil indeed form a helical complex with amylose (Ai et al., 2013). Depending on the size of the cross section of the complexing agent, the size of the amylose inclusion helix varies: for linear-chain complexing agents, such as n-butyl alcohol and free fatty acids, amylose forms a helical complex of six glucose units per turn (Rundle and Edwards, 1943; Takeo et al., 1973); for branchedchain compounds possessing larger cross sections, such as iso-butyl alcohol, tert-butyl alcohol, and tetra-chloroethane, amylose forms a helical complex of seven glucose units per turn (Zaslow, 1963; Yamashita and Hirai, 1966); and for compounds possessing even larger cross section, such as a-naphthol, amylose forms a helical complex of eight glucose units per turn (Yamashita and Monobe, 1971). Without the presence of a complexing agent in the dispersion, the hydrophobic array of an amylose chain will interact with that of an adjacent amylose chain to form a double helix. The double-helical conformation of amylose has hydrophobic arrays of the amylose chains folded inside the double helix, away from the aqueous medium, to reach a lower-energy and more stable state. Consequently, free amylose chains in the aqueous dispersion have a natural


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tendency to form double helices and then develop a gel or precipitate out from the dispersion, a phenomenon known as retrogradation. Once the double helices are formed between amylose molecules, it requires heating up to 170 C to dissociate the double-helical structure (Sievert and Wursch, 1993). The rate of the double-helix formation is determined by several factors, including the chain length of amylose, the concentration of amylose in the dispersion, and the temperature. The optimal chain length of amylose for the retrogradation is around DP 100 (90e110) (Gidley and Bulpin, 1989). A minimum chain length of DP 10 is required for the formation of double helices in a pure oligosaccharide solution (Pfannemuller, 1987). Short amylose chains of DP 6 can cocrystallize with longer chains into the double-helical form with merely one single turn (Pfannemuller, 1987). When amylose chains reach an average DP of 1100, the amylose molecules develop into a gel network instead of precipitate. At a low concentration (e.g., 3.5 mg/mL) and low temperature (e.g., 5 C), amylose retrogrades into a shape of interconnected nodules, whereas at a higher temperature amylose tends to retrograde into a gel form (Lu et al., 1997). At a greater concentration (e.g., 15 mg/mL), amylose only forms a gel at a wide range of incubation temperature (Leloup et al., 1992). When the incubation temperature increases from 5 to 45 C, the doublehelix content of the retrograded amylose decreases from 58.8% to 7.1%, but the average chain length of the crystalline lamellae increases from DP 34 to DP 40 (Lu et al., 1997). Retrograded amylose prepared at a refrigeration or an ambient temperature displays a B-type polymorph, which has an average chain length of DP 31 for the helix. Short-chain amylose (amylodextrin), however, can retrograde into A-, B-, or C-type polymorph, depending on the chain length and concentration of the amylodextrin, temperature, and the presence of salts or other chemicals. Pfannemuller (1987) uses synthesized amylodextrins with uniform chain lengths for retrogradation studies and reports that chains of DP 10e12 form the A-type polymorph at the room temperature, whereas those of DP > 13 give the B-type polymorph. At a higher temperature (e.g., 50 C) and a lower moisture content (e.g., 25% dry solids content), amylodextrin tends to crystallize into the A-type polymorph (Cai and Shi, 2014). With the presence of other compounds in the aqueous solution, such as sodium chloride and acetone, amylodextrin retrogrades into the A-type polymorph rather than the B-type (Montesanti et al., 2010).

2.2 Structures and Properties of Amylopectin Amylopectin is a highly branched molecule, consisting of branch chains of DP 6 to DP w 100 (Fig. 3.1(b)). The branch chain length of amylopectin shows a bimodal distribution, different from the single-modal distribution of the branch chain length of glycogen or phytoglycogen (Yoo et al., 2002; Ratnayake et al., 2001). Starches of different botanical origins have characteristic branch chain


length distributions of the amylopectins. Amylopectin of native starch with the A-type X-ray diffraction pattern is composed of a larger percentage of short branch chains (i.e., DP 6e12) that exist within one cluster, and a smaller percentage of long branch chains that extend through more than one cluster (Fig. 3.1(b)). In contrast, amylopectin of the native starch with the B-type polymorph consists of a larger percentage of long branch chains and a smaller percentage of short branch chains (Jane et al., 1999). C-type starch, consisting of a mixture of A- and B-type polymorph (Bogracheva et al., 1998), has large proportions of both very long and very short branch chain in the amylopectin. Average branch chain length distributions of amylopectins of collected A- (a total of 13 varieties), B- (5 varieties), and C-type (3 varieties) starches are shown in Fig. 3.2 and summarized in Table 3.1. Amylopectins of high-amylose maize starch (amylose-extender, ae mutant) and potato starch, both displaying the B-type polymorph, are known to consist of a larger proportion of long branch chains. And the long branch chains can also bind with iodine to develop a blue color. Consequently, the amylopectin dispersion has the absorption lmax between 573 and 575 nm (Takeda et al., 1993), which can inflate the value of amylose content when an iodine colorimetric or an iodine affinity method is used to determine the amylose content. In contrast, amylopectins of japonica rice starch and maize sugary-2 (su2) mutant starch are known to consist of large proportions of short branch chains, resulting from the deficiency of starch synthase IIa (Liu et al., 2012; Umemoto et al., 2002; Perera et al., 2001). Because amylopectin branch chains are responsible for the formation of crystalline structure in starch granules, the branch chain length of amylopectin molecules significantly affects the gelatinization and retrogradation properties of starch (Section 4.1 and 4.4).

FIGURE 3.2 Average branch chain length distributions of amylopectins of A-, B-, and C-type starch. Data from Jane, J., Chen, Y.Y., Lee, L.F., Mcpherson, A.E., Wong, K.S., Radosavljevic, M., Kasemsuwan, T., 1999. Effects of amylopectin branch chain length and amylose content on the gelatinization and pasting properties of starch. Cereal Chemistry 76, 629e637.

TABLE 3.1 Gelatinization Properties of Starches With A-, B-, and C-Type Polymorphic Structure Determined Using Differential Scanning Calorimetry, and Average Branch Chain Length (BCL) of Amylopectins of the Starches Analyzed Using a HighPerformance Anion-Exchange Chromatography System Equipped With an Enzyme Column Reactor and a Pulsed Amperometric Detector Tp ( C)

Tc ( C)

Range ( C)

DH (J/g)

Average BCL of Amylopectin

Normal maize

64.1 0.2

69.4 0.1

74.9 0.6

10.8

12.3 0.0

24.4

Waxy maize

64.2 0.2

69.2 0.0

74.6 0.4

10.4

15.4 0.0

23.5

du Waxy maize

66.1 0.5

74.2 0.4

80.5 0.2

14.4

15.6 0.2

23.1

Normal rice

70.3 0.2

76.2 0.0

80.2 0.0

9.9

13.2 0.6

22.7

Waxy rice

56.9 0.3

63.2 0.3

70.3 0.7

13.4

15.4 0.2

18.8

Sweet rice

58.6 0.2

64.7 0.0

71.4 0.5

12.8

13.4 0.6

21.6

Wheat

57.1 0.3

61.6 0.2

66.2 0.3

9.1

10.7 0.2

22.7

Barley

56.3 0.0

59.5 0.0

62.9 0.1

6.6

10.0 0.3

22.1

Waxy amaranth

66.7 0.2

70.2 0.2

75.2 0.4

8.5

16.3 0.2

21.8

Cattail millet

67.1 0.0

71.7 0.0

75.6 0.0

8.5

14.4 0.3

21.5

Mung bean

60.0 0.4

65.3 0.4

71.5 0.4

11.5

11.4 0.5

24.8

Chinese taro

67.3 0.1

72.9 0.1

79.8 0.2

12.5

15.0 0.5

23.4

Tapioca

64.3 0.1

68.3 0.2

74.4 0.1

10.1

14.7 0.7

27.6

A-Type Starch


To ( C)a

Starch

157 Continued

To ( C)a

Tp ( C)

Tc ( C)

Range ( C)

DH (J/g)

Average BCL of Amylopectin

ae Waxy maize

71.5 0.2

81.0 1.7

97.2 0.8

25.7

22.0 0.3

29.5

Amylomaize V

71.0 0.4

81.3 0.4

112.6 1.2

41.6

19.5 1.5

28.9

Amylomaize VII

70.6 0.3

N.D.

129.4 2.0

58.8

16.2 0.8

30.7

Potato

58.2 0.1

62.6 0.1

67.7 0.1

9.5

15.8 1.2

29.4

Green leaf canna

59.3 0.3

65.4 0.4

80.3 0.3

21.0

15.5 0.4

28.9

Lotus root

60.6 0.0

66.2 0.0

71.1 0.2

10.5

13.5 0.1

25.4

Green banana

68.6 0.2

72.0 0.2

76.1 0.4

7.5

17.2 0.1

26.7

Water chestnut

58.7 0.5

70.1 0.1

82.8 0.2

24.1

13.6 0.5

26.4

Starch B-Type Starch

b

C-Type Starch

To, onset temperature; Tp, peak temperature; Tc, conclusion temperature; TcTo, range of gelatinization; DH, enthalpy change. Not detectable. Adapted from Jane, J., Chen, Y.Y., Lee, L.F., Mcpherson, A.E., Wong, K.S., Radosavljevic, M., Kasemsuwan, T., 1999. Effects of amylopectin branch chain length and amylose content on the gelatinization and pasting properties of starch. Cereal Chemistry 76, 629e637. a

b


TABLE 3.1 Gelatinization Properties of Starches With A-, B-, and C-Type Polymorphic Structure Determined Using Differential Scanning Calorimetry, and Average Branch Chain Length (BCL) of Amylopectins of the Starches Analyzed Using a HighPerformance Anion-Exchange Chromatography System Equipped With an Enzyme Column Reactor and a Pulsed Amperometric Detectordcont’d


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Amylopectin, with a molecular weight ranging from 7.0 107 to 5.7 109 g/mol (Yoo and Jane, 2002a), is also the primary component responsible for the swelling power and viscosity development of the starch paste after the starch granules are gelatinized and swell (Tester and Morrison, 1990) (Section 4.2). Amylopectins of some normal and high-amylose cereal starches contain extra-long branch chains, which can have a DP of 700e1000 (Shibanuma et al., 1994; Yoo and Jane, 2002b). The extra-long branch chains, however, are absent in waxy starch (Yoo and Jane, 2002b; Inouchi et al., 2005). The content of extra-long chains is related to the dosage of Waxy gene that encodes the granular-bound starch synthase I (GBSSI), and it has been demonstrated that GBSSI is responsible for the biosynthesis of extra-long branch chains (Hanashiro et al., 2008; Yangcheng et al., 2016). The presence of extra-long branch chains in amylopectin significantly influences the pasting properties of starch (Section 4.2).

2.3 Structures and Properties of Intermediate Components Intermediate components are present in some mutants of maize, including ae and su2 maize mutant (Kasemsuwan et al., 1995; Perera et al., 2001). Intermediate components of starch possess branched structures and molecular weights similar to amylose but smaller than amylopectin. Branch chain lengths of intermediate components (DP 52 and 21 for the long and short branch chains, respectively) are larger than that of the amylopectin counterpart (DP 45 and 19, respectively) (Kasemsuwan et al., 1995). After the fractionation of starch using n-butyl alcohol, intermediate components remain in the supernatant with amylopectin, whereas amylose is precipitated by forming inclusion helical-complex crystallites with n-butyl alcohol (Kasemsuwan et al., 1995).

2.4 Structures and Properties of Phytoglycogen Phytoglycogen, a water-soluble glucan present in nature, is commonly found in sugary-1 (su1) mutant of crops, such as rice and maize (Inouchi et al., 1987; Wong et al., 2003). Phytoglycogen has a substantially shorter average branch chain length (DP 10.3) than amylopectin (DP 18.5 for waxy maize) (Inouchi et al., 1987). Phytoglycogen displays a single-modal branch chain length distribution instead of the bimodal distribution of amylopectin. Because of the much greater density of branch linkages (w10%) and the shorter chain length, phytoglycogen fails to develop a semicrystalline structure as amylopectin does and it is present in an amorphous structure in the grain endosperm. Biosynthesis of phytoglycogen in the endosperm is a result of the deficiency of debranching enzymes encoded by the Su1 gene (Rahman et al., 1998) (Chapter 1).


2.5 Lipids and Phospholipids Lipids are present in normal cereal starches as a minor component ( 5 > 9 (with solubility at pH 7 being the same as the control). They also hypothesized that molecular degradation took place at the acidic pH values, but that has not been established and is questionable, especially at pH 5. Both native and chemically modified starches can be pregelatinized. Pregelatinized products are used when heat cannot be applied to the product because of thermal lability of an ingredient, no processing step requires sufficient heat to cook the starch, no heat is available, or no heating equates to use convenience (especially in mixes to be used in the home). They are established articles of commerce and are used for product thickening, moisture control, and texture provision and control. Applications of pregelatinized starches include dry-mix products designed for home use, cake mixes, puddings, cream fillings, snack foods, frostings, and toppings. Pregelatinized modified starches retain much of the attributes contributed by the modification. Because they hydrate rapidly, powders of pregelatinized starches of small mesh size need to be handled rather like hydrocolloids when dispersing them in water, but products of larger mesh sizes, which are designed to impart some graininess or pulpiness desired in some applications, disperse easily.

2.2 Granular Cold-Water-Swelling Starch Another group of “instant” starches consists of products which contain gelatinized, intact granules, i.e., amorphous granules, that swell extensively (without cooking) when placed in an aqueous system at room temperature (BeMiller and Huber, 2015). They are a special kind of pregelatinized starch and are often called cold-water-soluble starches; but the classic pregelatinized starches are usually more room-temperature water soluble (without application of shear) than are these products, so the author prefers the term GCWS or simply cold-water-swelling (CWS) starches to describe them. GCWS starches produce viscosities and gel characteristics more like those of cook-up starches

Physical Modification of Starch Chapter j 5

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when added to room-temperature aqueous systems than do classic pregelatinized starches. They can be made by four general methods: (1) heating an amylose-containing starch in an aqueous solution of an alcohol (Eastman and Moore, 1984; Rajagopalan and Seib, 1991, 1992a,b; Sun et al., 2009), (2) rapidly heating a starch dispersion in a special spray-drying nozzle and drying the droplets in a spray drier (Pitchon et al., 1981), (3) treating the starch with an alkaline, aqueous solution of an alcohol at room temperature (Jane and Seib, 1991; Chen and Jane, 1994), (4) instantaneous controlled pressure drop (DIC) (BeMiller and Huber, 2015). In addition, Zhang et al. (2012) developed a new aqueous ethanol procedure for producing GCWS starches from both Aand B-type starches. Conversion of high-amylose starch into a GCWS product useful for the manufacture of confectionery, convenience, and other food products using a modified spray-drying system has been claimed (Berckmans et al., 2013). Products made from a single starch using different methods, by varying the method conditions using a single starch, and by using a single method with different starches vary in characteristics, but they have the common characteristic that their granules rapidly hydrate, swell, and lose their crystalline order without granule disintegration (even when little or no shear is applied) when placed in an aqueous system at room temperature. In the end, they produce the functionalities of the cook-up starches from which they are made without application of heat. A drawback is that, when GCWS starch granules of an amylose-containing starch are added to unheated water without shear, ensuing retrogradation can result in poor-quality dispersions. However, when those granules are dispersed in a sucrose or glucose syrup with rapid stirring, the resulting dispersion sets to a rigid gel that can be sliced to make gum candies, and the ability to swell in unheated aqueous systems can be useful for making desserts in the home and in muffin batters containing fruit or nut pieces that would settle to the bottom when the batter thins as a result of heating before the gelatinization temperature of the starch is reached and the batter thickens. CGWS starch can be used in instant pudding mixes (Jane, 1992) and as a fat mimetic in spoonable salad dressings (Bortnowska et al., 2014). When waxy maize starch alone is heated in aqueous ethanol, it loses its granular structure, but a product can be made in this manner from a mixture of waxy and normal maize starch. Method 3 is almost always used in the laboratory because special equipment is not required and at least six process variables can be studied. Both native and chemically modified amylosecontaining starches can be converted into GCWS products. Kaur et al. (2011) confirmed that, during conversion of sago starch into a GCWS starch using an alkaline aqueous ethanol treatment, the C-type crystallites were changed into V-type crystallites as a result of starch polysaccharide molecules complexing with ethanol molecules, and Dries et al. (2014) confirmed that, during conversion of maize starch into a GCWS starch


using hot, aqueous ethanol, the A-type crystallites were also changed into V-type crystallites. However, Jivan et al. (2014) found that the alkaline, aqueous alcohol method reduced the crystallinity of a B-type starch without changing the crystalline polymorph. Both Lu et al. (2013) and Jivan et al. (2014) reported significant increases in solubility after conversion into GCWS products. Majzoobi et al. (2015a) found that the solubility of GCWS maize starch in a dilute acetic acid solution increased (as it does for the classic pregelatinized starch), and the dispersion viscosity decreased with increasing concentration of acetic acid. They attributed the increase in solubility to acid-catalyzed depolymerization, which for reasons presented earlier, this reviewer is skeptical of. They also reported that acetic acid increased the turbidity of GCWS gels and that its gels were not as soft and had reduced viscosity, consistency, and cohesiveness as compared to gels of the classic pregelatinized starch. Hedayati et al. (2016) reported that the physical properties of room-temperature water dispersions of GCWS maize starch did not change as much with changes in pH as did the classic pregelatinized starch.

2.3 Heat-Moisture Treatment In 1944, Sair and Fetzer reported that, as a result of heat-moisture treatment (HMT), the water vapor sorptive capacity and X-ray pattern of potato starch (a starch with a B-type X-ray diffraction pattern) became more like that of cereal starches (starches with an A-type diffraction pattern). Because at the time, it was not considered that such changes improved the economic value of the starch, the phenomenon was not further investigated, except by Sair (1967), who much later published a complete description of his results and first used the term HMT. In the 1967 paper, he reported that little or no chemical change in HMT starches occurred at or below 100 C, and that above 100 C, degradation was appreciable; the X-ray patterns of those starches with A-type patterns were unchanged, while those with B-type patterns were changed to (A þ B)-, i.e., C-type, patterns. Compared to the native starches, the capacities of the starches for sorption of water vapor and swelling decreased, gelatinization temperatures increased, and pastes of the starches had changed viscosities, were more opaque, and formed gels with changed properties. He also noted that “The moisture content of the starch is an important factor in effecting this physical change” and that “Water within the granule apparently permits starch molecules, or parts of them to rotate,” allowing molecular rearrangements within granules. Today, HMT is known as a potentially valuable hydrothermal process that consists of heating starch granules at a temperature above the starch’s glass transition temperature (at the moisture content employed) in a closed and sealed vessel. Moisture contents employed generally have been adjusted to from 10% to 40%. Processing temperatures generally range from 84 to 140 C,


229

with most investigations using temperatures above 94 C. Treatment times vary from 1 to >24 h. This chapter focuses on changes effected by this traditional method. HMT is by far the most studied method of physical modification of starch. The large literature on HMT has been reviewed by Jacobs and Delcour (1998), Hoover (2010), Zavareze and Dias (2011), and BeMiller and Huber (2015), each of which have provided details on specific changes in specific starches under specific conditions and which, along with original papers, should be consulted for those details. Because of the several variables (type of starch, moisture content, temperature, time of heating) using the conventional method alone, there is for all practical purposes, no limit to the conditions of HMT. For that reason, as pointed out in the previous reviews, when a specific attribute of a treated starch is compared to that of the native starch, increases, decreases, and no change are likely to have been reported. Rather than re-report the specific observed changes, this section focuses on general changes that have been proposed to take place in granule structures and why. Before that, however, it can be generally stated that structural changes occur in both crystalline and amorphous regions of granules and that the contradictory results occur because effects are a function primarily of the type of starch, its moisture content, and the temperature to which it is heated, which can vary considerably. Changes that are generally agreed upon are that starches with A-type crystallinity undergo no change in the type of crystallinity upon HMT (although there may be an increase in X-ray diffraction intensities), that starches with B-type crystallinity have their crystallinity changed to a C-type (partial conversion) or completely to A-type crystallinity with an accompanying decrease in X-ray diffraction intensities (although not by all moistureetemperature combinations), and that starches with C-type crystallinity have their crystallinity changed (partially or completely) to a greater proportion of the A-type crystallinity, with the B- to A-type crystallinity conversion being favored by higher temperatures and moisture contents. There is also general agreement that, even at the relatively low-moisture contents of HMT processes, the relatively high temperatures allow increased mobility of both starch polysaccharide chain segments and helical structures in both amorphous and crystalline regions of granules. To explain the decrease in the degree of crystallinity often observed in B-type starches, Hoover and Vasanthan (1994) proposed that adjacent helices move apart owing to the rupture of water bridges, resulting in less perfect ordering. The proposed mechanism of Vermeylen et al. (2006) involves disturbances in lamellar structures wherein double helices move laterally within high-density lamellae and also along their helix axes during the B to A transition. Vermeylen et al. (2006) and others (Chung et al., 2009; Li et al., 2011; Jiranuntakul et al., 2012; Kim and Huber, 2013) observed thermal degradation of certain starches at temperatures of 120e130 C, while others


(Varatharajan et al., 2011; Ambigaipalan et al., 2014) did not. Noting that the thermal cleavage of glycosidic linkages coincided with an increase in granule crystallinity, Vermeylen et al. (2006) suggested that such cleavage resulted in greater mobility of released segments, allowing them to align with other segments or structures to form larger and/or more perfect crystallites. To explain the increase in the degree of crystallinity often observed in A-type starches, it has been logically proposed that the thermal energy and the plasticizing effect of water molecules allows double helices to move within crystallites so as to form more ordered and closely packed structures; or to put it in another way, HMT disrupts the least-stable structures and allows the growth and/or more perfect alignment of the more stable native crystallites (Hoover and Vasanthan, 1994; Jacobs and Delcour, 1998). Possibly related to the increases in crystallinity are increases in the onset (To), peak, and conclusion (Tc) gelatinization temperatures and a broadened phase-transition temperature range (Tc To) (owing to a greater increase in Tc than in To) often, but not always, observed. However, these effects have been attributed to changes in amorphous regions of granules, viz., increased amyloseeamylose and amyloseeamylopectin associations and amyloseelipid complexes, the latter in cereal starches (Hoover, 2010; Zavareze and Dias, 2011). Often found after HMT are decreases in swelling power and leaching of amylose from the swollen granules, with the reductions increasing with increasing moisture content and temperature during treatment. Reductions in granule swelling and amylose leaching have been attributed to increased disruption of crystallites, increases in crystallinity, polymorphic B / A transitions, formation of amyloseelipid complexes, and/or changes in amylosee amylose and/or amylopectineamylopectin chain interactions (Hoover, 2010). Varatharajan et al. (2011) concluded that structural reorganizations (changes in chain interactions) predominated at lower temperatures (80e100 C), while A / B polymorphic transitions occurred at higher temperatures. Almost universally, HMT starches have increased RVA pasting temperatures, decreased peak viscosities and breakdown, and increased hot-paste viscosities, giving the starch pasting characteristics similar to those of a lightly cross-linked starch (Hoover, 2010; BeMiller and Huber, 2015). Watcharatewinkul et al. (2010) pointed out that while HMT starches generally possess the improved heat and shear stabilities characteristic of cross-linked starches, the starches also exhibit reduced granule swelling and viscosity development, which may require greater use level; but the greater use level may result in increased body. Contents of slowly digestible (SDS) and RS starch and the effects of physical treatments on them are of considerable interest. HMT results in both disruption of some native structures and formation of new, more-ordered structures with relative amounts of these processes depending on the specific starch, its moisture content, and the temperature and duration of


231

treatment. Both increases and decreases in SDS and RS have been reported. Assays to determine the relative digestibilities of HMT starches have used the method developed by Englyst et al. (1992) or a modification of it, but when the Englyst et al. method is applied to starch, it measures relative contents of SDS and RS in raw starch. Because the preparation of most food products for commercial sale and consumption involves at least one cooking step, heating under similar conditions should be used in the assay so that thermostable SDS and RS fractions are measured. In fact, Qi and Tester (2016) state that “when starches are annealed or heat-moisture treated, gelatinization should be avoided” because gelatinization “accelerates amylase driven hydrolysis.” They were referring to the processes themselves, but the same great increase in digestibility is true of gelatinization after a hydrothermal or other physical treatment. The majority of those who have studied this aspect reported that HMT starches contain slightly to moderately more thermostable SDS and/or RS contents (Kweon et al., 2000; Chung et al., 2009, 2010; Gu¨zel and Sayar, 2010; Sui et al., 2011; Lee et al., 2012). Kim and Huber (2013) found that uncooked, native potato starch had total (i.e., thermostable and nonthermostable) SDS and RS contents of 2.0% and 93%, respectively, and that the same starch heated 3 h at 120 C increased in SDS content as the moisture content of the starch increased, reaching a maximum content of 22%e23% at 20% and 25% moisture, while the maximum RS content (83%) was in the HMT starch treated at 15% moisture. However, when the same native and HMT potato starches were cooked, the control had SDS and RS contents of only 1.6% and 5.9%, respectively. The thermostable SDS content increased as the moisture content of the starch being heated increased, but reached a value of only 6.4% at 25% moisture, while the RS content was not significantly different when the starch had moisture contents of 15%, 20%, or 25% when treated, being in the 15.5%e16.9% range. These values for thermostable SDS and RS in the cooked HMT starch (about 27% and 20%, respectively, of those for uncooked HMT potato starch) are more realistic approximations of the values expected for a consumed food product. Using optimized conditions of heating time and moisture content, Hoyos-Leyva et al. (2015) found that HMT Morado banana starch achieved thermostable SDS and RS contents of 12% and 31%, respectively, while the native starch had heat-stable contents of 7.5% and 13%, respectively. Hung et al. (2016) changed the thermostable SDS and RS contents of high-amylose rice starch from 3.7% to 4.5% and from 6.3% to 22%, respectively, of normal rice starch from 14% to 11% and from 6.5% to 24%, respectively, and of waxy rice starch from 13% to 24% and from 10% to 19%, respectively, via HMT. Wang et al. (2016) determined that HMT normal maize starch reached a maximum thermostable SDS content of 19% when the starch was treated at 30% moisture and a maximum thermostable RS content of 14% when the starch was treated at 20% moisture (from respective contents of 6.2% and 2.1% in the native starch), while amylomaize V starch treated at 30% moisture reached maximum contents of


thermostable SDS of 19% and thermostable RS of 30% (from respective contents of 7.4% and 6.6% in the native starch). They concluded that the amylomaize starch was more susceptible to HMT (because of its B-type crystallinity) than was the normal maize starch (A-type crystallinity). Not considered in the thermostable RS analysis is that the cooked starch in some products may undergo retrogradation rather rapidly, producing RS; but the assays used (employing different heating conditions (mostly different times)) are indications of relative increases in thermostable SDS and RS. Addition of lauric acid to maize starch before HMT resulted in the formation of amyloseelauric acid V-type complexes. The HMT product contained a maximum of 10% thermostable SDS and 18% thermostable RS when the moisture content of the treated starch was 50% (as compared to 5.0% and 6.6%, respectively, in the native starch) (Chang et al., 2014). When the moisture content of the treated starch increased from 10% to 50% the RVA pasting temperature increased. When the moisture content of the treated starch increased from 10% to 30%, the peak viscosities, breakdown, and setback decreaseddthe increase in pasting temperature and the decrease in peak viscosity being attributed to reduced granule swelling owing to the presence of amyloseelauric acid complexes. However, when the moisture content of the treated starch increased from 30% to 50%, peak viscosities, breakdown, setback, and final viscosities increased, with setback increasing dramatically. It was concluded that the dramatic increase in setback resulted from formation of new amyloseelauric acid complexes from noncomplexed lauric acid molecules, many of which arose from dissociation of the original complexes during the heating process. Based on earlier findings of Brumovsky and Thompson (2001) and Lin et al. (2011) that prehydrolysis increased the thermostable RS content, Kim and Huber (2013) and Hung et al. (2014, 2016) reported that when HMT was conducted under slightly acidic conditions, thermostable RS contents increased to 24% for potato starch, to 30%e39% for rice starch, to 40% for sweet potato starch, and to 46% for yam starch. The conclusions were that very mild acidic conditions during HMT promoted limited hydrolysis of amylopectin molecules (primarily at branch points) and facilitated realignment of starch chains to more thermostable arrangements. Sun et al. (2015) conducted HMT of maize starch at pH 4 (different moisture contents and temperatures) and determined the properties of the products but did not determine digestibilities. They reported decreases in swelling power, peak and hot-paste viscosities, and the phase transition temperature range and increases in solubility, gelatinization and pasting temperatures, and gel hardness. The large number of recent papers that reported changes in the properties of various starches effected by HMT offers no new insights as to granular transformations that might occur in the process and are not presented here, although some papers report results of investigations on the effects of different


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experimental conditions (e.g., Sui et al., 2015). Use of alditol solutions in place of water alone as plasticizers was investigated by Sun et al. (2014a,b) and Juansang et al. (2015). The traditional method for HMT, which has been used for most laboratory investigations, is a procedure that would be expensive and difficult to do on a large scale; so alternative methods have been investigated. These alternative methods include microwave heating (Niu et al., 2013) (see Section 3.5 for a discussion of), infrared heating (Ismailoglu and Basman, 2015, 2016), direct steam injection, heating in an aqueous alcohol, direct-vapor HMT, and reduced pressure HMT (BeMiller and Huber, 2015) (see also Section 2.6). HMT has been practiced before, after, and simultaneously with chemical or other modification of starches (BeMiller and Huber, 2015). Dual treatments have been carried out (Klein et al., 2013). Hoover (2010) concluded that HMT starches have good potential to be used as “unmodified” thickeners in processed food products owing to their having temperature, acid, and shear stabilities during cooking similar to those of lightly cross-linked starches. It was found that addition of HMT rice starch to a poor quality rice flour enabled the production of noodles of acceptable quality (Hormdok and Noomhorm, 2007), that a HMT of a poor quality rice flour enabled use of that flour to produce dried and semi-dried noodles with appropriate tensile strength and hardness (Cham and Suwannaporn, 2010), and that noodles could be made from HMT sweet potato starch (Pranoto et al., 2014). However, other results obtained from trial applications in products such as noodles, doughs, bakery products, and pie fillings were both positive and negative. HMT starches have been investigated as ingredients in other food products (BeMiller and Huber, 2015), and HMT has been applied to flours and whole grains.

2.4 Annealing Annealing, another hydrothermal process, consists of holding starch granules in an excess of water (generally >39% w/w) at a temperature that is above the starch’s glass transition temperature and below its gelatinization temperature. The annealing process has been conducted for periods of time ranging from minutes to days. As with HMT, there is a large literature on annealing that has been reviewed previously by Jacobs and Delcour (1998), Tester and Debon (2000), Jayakody and Hoover (2008), Zavareze and Dias (2011), and BeMiller and Huber (2015). Like HMT, the variables are the specific starch used, the temperature, and the duration of heating. (Moisture content is not a variable because the process is conducted in excess water.) Also like HMT, when a specific attribute of a treated starch is compared to that of the native starch, increases, decreases, and no change are likely to have been reported because of the large numbers of starches and conditions that may be employed. Several of the observed property changes are in the same direction as produced by HMT,


while there are differences in others. The proposed mechanisms have some similar features. There seems to be less consensus (than with HMT) on the changes in starch properties imparted by annealing. One thing there seems to be general agreement about is that annealing increases the onset and peak gelatinization temperatures and decreases the phase-transition temperature range (Liu and Du, 2013; Wang et al., 2014; Wu and Du, 2014; BeMiller and Huber, 2015; Zhang et al., 2015a; Zeng et al., 2015; Liu et al., 2015a, 2016b; Bhattacharjya et al., 2015). The preferred explanation for this change (and others resulting from annealing) is that, because of the high degree of plasticization of the molecules in starch granules by water, the increased temperature increases the mobility of double-helical chain segments, allowing them to improve their alignment, the extent of which is determined by the temperature employed. In other words, the least stable structures within both amorphous and crystalline regions are disrupted, and crystallization, perfection of existing crystallites, and/or increased molecular ordering forms more stable and more homogeneous structures, without increasing the number of double helices (in normal or waxy starch granules) (BeMiller and Huber, 2015). However, annealing of high-amylose starches has been reported to produce new double helices and amyloseeamylose, amyloseeamylopectin, and amylopectineamylopectin associations (Tester and Debon, 2000; Lin et al., 2009; Gomand et al., 2012). Gomand et al. (2012) concluded that for normal and waxy potato starches, annealing resulted in increased crystallite surface stability as a result of relaxation of conformationally strained chain segments at crystallite borders and their migration into amorphous regions. They also concluded that for a high-amylose potato starch, annealing produced additional cocrystallization of amylose and amylopectin chains and crystal thickening (Kiseleva et al., 2005). In addition to annealing affecting the thermal properties of starches via changes in crystalline regions of granules, annealing has also been hypothesized to effect rearrangements of structures within amorphous regions, as indicated by increases in glass transition temperatures (Seow and Teo, 1993; Tester and Debon, 2000). Liu et al. (2009) concluded that annealing primarily increased the helical length of short amylopectin helices, particularly in the amylopectin of nonwaxy starches. Starches are often subjected to annealing conditions during isolation and/or chemical modification. For example, the wet-milling process for isolation of maize starch involves steeping of maize kernels (usually for at least 24 h) at a temperature of 50 2 C, and chemical modifications are usually accomplished in starch slurries of elevated pH values at a similar temperature in solutions of a swelling inhibiting starch to prevent gelatinization under the alkaline conditions. In fact, Krueger et al. (1987) presented evidence indicating that maize starch is indeed annealed during its isolation by the commercial wet-milling process, and Sui et al. (2011) found that the conditions used for hydroxypropylation and cross-linking with phosphoryl chloride


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significantly changed the pasting and paste properties of maize starch in the direction that annealing at neutral pH would. There is generally no change in gelatinization enthalpy (DH), although small increases and decreases have been reported (BeMiller and Huber, 2015), indicating little or no changes in the total amounts of glassy and crystalline structures. Likewise, annealed starches usually retain their crystalline packing arrangements, although a partial C / A transition (which means a partial B / A transition) and a partial C / B transition (which means a partial A / B transition) have been reported (BeMiller and Huber, 2015; Zhang et al., 2015a). Together, these findings suggest that molecular rearrangements occur within both amorphous and crystalline arrangements in granules and that the changes within the crystallites primarily result in their perfection (rather than in the amount of crystalline material or in the crystal type), although increases in relative crystallinities (Yu et al., 2015; Liu et al., 2015a, 2016b; Zeng et al., 2015) and in the size of crystallites have been proposed (Gomand et al., 2012; Vamadevan et al., 2013). Alvani et al. (2014) and Gomand et al. (2012) obtained evidence that in both single- and multistep annealing processes, the extent of change in starch properties is the greatest in the starches with the greatest amounts of structural organization disorder and the least in starches with the least organizational defects. Based on results such as the finding that annealing did not change the swelling power of waxy maize starch, but reduced the swelling powers of normal maize and amylomaize starches, Wang et al. (2014) concluded that “amylose molecules play an important role in the structural reorganization of starch granules during annealing” and proposed that annealing “enhances the long-range interaction of amylopectin clusters by the rearrangement of amylose molecules.” Vamadevan et al. (2013) hypothesized that changes in gelatinization behavior effected by annealing were related to the average distances between branch points within amylopectin clusters interblock chain lengths (IB-CL) (Bertoft et al., 2012) and found that they could classify the 16 starches examined into four categories: (1) a group of starches with unfavorably short IB-CL and presumably restricted movement and alignment of double helices that were least responsive to annealing; (2) a group of starches suggested to have almost ideal IB-CL and external chain lengths that give them crystalline lamellae with few defects so that the crystallites can undergo little improvement upon annealing; (3) a group of starches with low phosphate content and long IB-CL, which gives them greater flexibility and movement of double-helical segments, enabling them to improve their alignment during annealing; (4) a group of starches that also have long IB-CL, but with relatively high-phosphate ester contents, which disrupt the usual crystalline order, giving them the greatest change upon annealing (Muhrbeck and Svensson, 1996; Muhrbeck and Wischmann, 1998). (Groups 1 and 2 seem to contain mostly A-type starches; group 3 seems to contain A- and C-type starches, and group 4 seems to contain B- and C-type starches.)


As a consequence of the increased alignment of double helices and perfection of crystallites, annealed amylose-containing starches usually have reduced swelling powers, solubilities, viscosities, and pasting temperatures (Liu and Du, 2013; Wang et al., 2014; Wu and Du, 2014; Falade and Ayetigbo, 2015; Zhang et al., 2015a; Liu et al., 2015a,b, 2016b; BeMiller and Huber, 2015), i.e., characteristics of lightly cross-linked starches. Increased water and decreased oil sorption capacities have also been reported (Liu et al., 2015b). The presence of swollen granules in a gel gives the gel increased hardness/ firmness (Chung et al., 2010; Jyothi et al., 2011; Yadav et al., 2013). As with HMT starches and any native or modified starch, the only important data on digestibility are the thermostable RS and SDS values. Only slight to no increases in thermostable SDS and RS contents following annealing, then cooking, have been found (Chung et al., 2009, 2010; Alvani et al., 2014; Liu et al., 2015a, 2016b). Annealing has been conducted as single- or multistep processes, before or following HMT, with starches before isolation (in situ), and before and after modification (BeMiller and Huber, 2015; Zeng et al., 2015). Use of annealed starches and flours in food products has been less studied than has use of HMT starches and flours, with again the main application being their use in preparation of various noodles (Bhattacharjya et al., 2015). It has been reported that annealing of a poor-quality rice flour gives fresh noodles the required soft texture (Cham and Suwannaporn, 2010).

2.5 Heating Dry Starch In a patented process, Chiu et al. (1998) heated starch with 30 in the acetonitrileewater eluent is a limiting factor (Henshall, 1996). There was clearly a need for a better detection method for carbohydrates (and starch in particular) that is more specific and sensitive than RI and is compatible with gradient elution. This led to the development and use of highperformance anion-exchange chromatography (HPAE-PAD) (also referred to as high-pH anion-exchange chromatography or HPAEC-PAD), which is


proving to be a powerful tool for the analysis of starch and complex carbohydrates. The technique is direct (no derivatization required), highly sensitive and specific, and compatible with gradient elution procedures (Hauffe, 1997). On the other hand, resolution of polysaccharides up to DP > 60 can also be achieved routinely in w1 h. Nowadays this technique is currently being applied in research and quality control laboratories in the characterization and determination of starch in many food products (Levine et al., 2005). Several interesting approaches have been reported in the nineties concerning the determination of starch in foodstuffs by liquid chromatography. A reversed-phase HPLC method using a 5 mm Spherisorb NH2 column, a RI detector, and 85:15 v/v acetonitrile/water as mobile phase was developed and applied to green beans and other starchy vegetables (Lo´pez-Hernańdez et al., 1994). Starch is extracted from the samples and hydrolyzed with amyloglucosidase solution. After the elution process, the starch-derived glucose concentration, C, is calculated by comparison with the chromatogram peak of a glucose standard run before the sample: C ¼ Cs A=As where Cs is the concentration of the glucose standard, A is the area of glucose peak from the sample, and As is the area of glucose peak from the standard. Starch concentration as a weight percentage of the original sample, is determined by Starch ð%Þ ¼ 0.90 100 C ðmg=mLÞ=WðmgÞ ¼ 90 C=W where W is the mass of sample and the factor 0.90 corrected for incorporation of a molecule of water in each glucose unit during hydrolysis (Latimer, 2012). Another interesting proposal is a separation scheme for the determination of starch in processed food (plain cereals, sugar-coated cereals, canned fruits, canned vegetables, crackers, cookies, and so on). It is based on AOAC Method 985.29, some modifications having been added (Casterline et al., 1999). Samples are milled to a fine powder and, if necessary, defatted with petroleum ether. pH 6.0 phosphate buffer is added and samples are stored at 4 C overnight to ensure hydration of the matrix. After centrifugation, the two fractions obtained (soluble and insoluble materials) are separated and the following procedure is applied to each one of them: They are treated with heat-stable a-amylase and amyloglucosidase to hydrolyze starch. Acetonitrile is then added to precipitate substances such as soluble proteins and fibers, which create backflow and thus interfere with the chromatographic resolution of peaks; residues are removed by centrifugation and filtration through a nylon filter, and the filtrate is passed through a solid-phase extraction cartridge to remove HPLC-interfering substances, the eluate being analyzed for starch (soluble or insoluble, depending on the fraction analyzed) by liquid chromatography. Starch content is calculated from the increase in the amount of glucose.

Measuring Starch in Food Chapter j 6

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3.2.7 Gas Chromatography A general scheme was developed (Li, 1996) to determine starch in half-gram freeze-dried samples of selected high-consumption foods such as bread, rice, spaghetti, potatoes, and beans. Samples are extracted for free sugars with 80% methanol, and the residues are incubated with a solution containing amyloglucosidase in acetate buffer. Starch hydrolyzates are then centrifuged and aliquots are removed for glucose determination by GC, after the corresponding derivatization to trimethylsilylated oximes or ethers. Samples are thereafter ready for GC analysis or may be kept in a refrigerator for several weeks. As for the chromatographic determination, a 25-m capillary column containing crosslinked methyl silicone is used. The usual operating conditions are as follows: injection volume, 1 or 2 mL; oven temperature programmed from 160 C (with 1 min hold) to 270 C at 6 C/min; injection port temperature, 250 C; detector temperature, 310 C; carrier gas (helium) constant flow pressure, 20 psi; split ratio, 70:1. Starch is then calculated according to the following formula: Starch content ¼ glucose ðg=100 gÞ 0:9 GC has also been used for the determination of resistant starch (the sum of starch and products of starch degradation not absorbed in the small intestine of healthy individuals) in cooked dried beans. As an alternative to the abovementioned procedure, the pretreatment before enzyme hydrolysis consists of autoclaving the samples at 121 C in the presence of dimethyl sulfoxide (Li and Zhao, 1997). This treatment leads to extensive if not complete solubilization of the starches in the analyzed samples.

3.2.8 Near-Infrared Spectroscopy Near-infrared spectroscopy analysis is an instrumental method for rapidly and reproducibly measuring the chemical compositions of samples with little (grinding, for instance in case of beans) or no sample preparations (Hermida et al., 2006). Since the near-IR spectroscopic analysis offers four principal advantages: speed, simplicity of sample preparation, multiplicity of analyses from a single spectrum, and intrinsic nonconsumption of the sample, it has been widely used to measure constituents of many agricultural commodities and food products as well as online analysis at food-processing sites. Especially, it makes the analyses of starch in foodstuffs an easy, rapid, and nondestructive routine analysis. This is very important, for instance, when the viscosity of gravy in gravy-containing food products is to be evaluated (viscosity is a major factor affecting the final quality of the products). The viscosity of gravy depends on the type and content of the starch used in preparation, the former being directly proportional to the amount of starch and to its chemical structure (the more branched the starch prepared for the gravy is, the higher viscosity the gravy has). Therefore, direct determination of the starch content in gravy is important for further monitoring the quality of


canned products. In this sense, near-IR spectroscopy has proved to be very useful when compared to labor-intensive, time-consuming, and destructive conventional wet chemistry methods (Zeng et al., 1996). The procedure is based on the use of a near-IR spectrum composition analyzer equipped with a built-in computer that integrates with the instrument and can independently carry out the analysis according to the calibration constants. Modified food starch, pure corn starch, and beef flavoring may act as gravy models. This method can be used at a varied range of temperatures, and permits the online analysis of starch content during gravy-containing food processing.

3.2.9 Capillary Electrophoresis Capillary electrophoresis (CE) has been playing an increasingly important role in liquid-phase chemical analysis. Several features including speed of analysis, high resolution, and efficiency account for the present acceleration in the acceptance of this technique. High electric fields are used in CE to force ionic solutes to migrate through a buffer- or gel-filled capillary. The species, injected in minute amounts, are separated along the tube on the basis of charge, size, or both and are subsequently detected near the capillary end (Gu¨nzler and Williams, 2001). The various forms of this technique have been successfully demonstrated in the analysis of carbohydrates, starch being no exception. In this sense, a comprehensive monograph (Volpi, 2007) offers a detailed look at the latest breakthroughs and improvements in CE and CE techniques applied to monosaccharides up to complex oligosaccharides and polysaccharides (including starch). In the case of starch, the absence of charge does not offer any problem by resorting to the use of iodine complexation to impart charge and permit detection of starch components in CE, for example the soluble amylose content and the amyloseeamylopectin ratio (Herrero-Martıńez et al., 2004). Amylopectin and amylose in corn, rice, potato, and wheat starch are resolved in less than 7 min using iodine-containing buffers (over the pH range 4.0e7.5) in an uncoated fused-silica “bubble cell” capillary. The primary basis for separation is shown to be iodine-binding affinity, which can be manipulated through control of temperature and iodine concentration. Iodine concentrations between 0.1 and 0.5 mg/mL have yielded the best results. At lower iodine concentrations amylopectin is not resolved, whereas Joule heating due to the high conductivity of KI solutions prevents separations in 50 mm capillaries at higher concentrations. Furthermore, in contrast to other approaches for CE of carbohydrates, lengthy (4e24 h) derivatization steps are avoided, and separations can be carried out using commonly available detectors. 3.2.10 Flow Injection Analysis Flow Injection Analysis (FIA) is a technique which has gained increasing popularity in laboratories all around the world (Trojanowicz, 2008). Many of


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the time-consuming batch procedures can be replaced by fully automated flow injection systems which can dramatically decrease the time and cost for analysis. In this sense, the use of immobilized enzyme reactors combines the selectivity of enzyme reactions with the speed and simplicity of FIA (Karkalas, 1991). Starch-hydrolyzing enzymes are commonly used to convert this polysaccharide into glucose, which may then be determined by different flow injection procedures, such as the one proposed by Peris-Tortajada et al., whose scheme is shown in Fig. 6.2. In this case, the sample (glucose) solution is injected into a CuII-neocuproine (2,9-dimethyl-1,10-phenanthroline) stream and later merged with a basic (NaOH) stream, the reaction product being monitored at 460 nm. Moreover, since the reduction of glucose requires drastic conditions for sufficiently fast development, the reactor in which the chemical reaction takes place is immersed in a water thermostatic bath at a suitably high temperature (70 C). Starch has also been determined using an FIA system composed of an immobilized glucoamylase reactor followed by pulsed amperometric detection of the glucose produced. Amyloglucosidase, immobilized onto porous silica and packed into a short stainless steel column, is capable of nearly quantitative (98%) conversion of the starch to glucose. The sensitivity of pulsed amperometric detection for soluble starch is increased 26-fold by first passing the starch through the immobilized glucoamylase reactor. The proposed method turned out to be simple, rapid, and sensitive for starch (Larew et al., 1988). Additionally, the half-life of immobilized glucoamylase was estimated to be over 500 days, which means that the reactor can be used during reasonably long periods of time. Another interesting FIA approach also based on the use of immobilized enzymes has been developed for the determination of starch from different origins (wheat, corn, rice). However, in this case the glucose obtained by hydrolyzing starch reacts with NADþ (nicotinamide adenine dinucleotide)

FIGURE 6.2 Flow injection configuration for the analysis of hydrolysis products (sugars) of starch. D, UV-V spectrophotometric detector; IV, injection valve; P, peristaltic pump; R, reactor; TB, water thermostatic bath; W, waste.


conveniently immobilized, and the resulting NADH is monitored spectrophotometrically (Emneus et al., 1986; Emneus et al., 1993). The authors of this approach immobilize a heat-stable a-amylase on controlled pore glass and amyloglucosidase on a ceramic silica support. These enzyme supports are packed into two separate immobilized enzyme reactors which together with a third reactor containing coimmobilized glucose dehydrogenase/mutarotase are incorporated into a flow injection system. a-amylase and amyloglucosidase convert (hydrolyze) the starch into glucose which, by entering the glucose dehydrogenase/mutarotase reactor, is oxidized by glucose dehydrogenase to þ D-gluconate in the presence of NAD which at the same time is reduced to NADH (measured at 340 nm); this product is then proportional to the amount of glucose produced from the hydrolyzed starch samples. In this case, mutarotase plays an important role, since glucose dehydrogenase is only active on the b-anomeric form of glucose, and therefore the produced a-D-glucose also formed in the enzymatic hydrolysis of starch (and through spontaneous mutarotation) needs to be converted to the b-form by mutarotase. An alternative to the UV spectrophotometric measurement is the electrochemical oxidation of the hydrogen peroxide formed after the complete oxidation of the produced glucose in a third reactor containing coimmobilized mutarotase and glucose oxidase (instead of glucose dehydrogenase) (Emneus and Gorton, 1990). Finally, a fully automated method for the determination of starch in foodstuff was also developed (Velasco-Arjona and Luque de Castro, 1996). The method is based on the standard procedure involving leaching of sugars from the sample, hydrolysis of the analyte, and colorimetric determination of the hydrolysis products (sugars) by the neocuproine method (Peris-Tortajada et al., 1992). A focused microwave digester is used as peripheral for the robotic station to expedite analyses by accelerating the hydrolysis step. The station is connected to a flow injection manifold, which minimizes the dilution and derivatization times. In this way, duplicate analyses of three solid samples take 5 h rather than 15 h required when the whole analytical sequence is performed by the robotic station and 5 h/ sample taken by the manual (conventionally applied) method. The use of microwaves as auxiliary energy for accelerating slow steps such as the removal of sugars and starch hydrolysis and automating the overall method as much as possible was further abridged by other researchers (Caballo-Lo´pez and Luque de Castro, 2003). With this aim, a flow injection (FI) manifolddalso based on the neocuproine methoddwas designed, parts of which were located in the irradiation zone of a focused microwave digestor. This enhanced procedure was successfully applied to the determination of starch in flour and bread. It should be remarked that, although this method is only partially automated, the total time of analysis is shorter than that of the fully automated robotic approach proposed by Velasco-Arjona and Luque de Castro (1996).


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3.2.11 Potentiometry (Nonenzymatic Electrochemical Detection) In the present decade, starch has often been indirectly determined in foodstuffs through its previous hydrolysis and subsequent nonenzymatic detection of the resulting glucose. For this purpose, a great number of glucose electrodes have been proposed and developed, them being based on the use of the metals platinum, gold, nickel, copper, of alloys and bimetals, of carbon materials (including graphene and graphene-based composites), and of metalemetal oxides and layered double hydroxides. This has led to a lot of interesting examples of electrochemical detection of glucose (from starch) in foodstuffs, which have recently been reviewed (Wang et al., 2013). The over 200 publications covered present comprehensive and detailed studies, novel and simple ways to prepare electrode materials, a strong catalytic ability toward analytes, high sensitivity, and successful application of these glucose sensors, hence implying promising development and application potential. Anyway, the advancement may depend on research into graphene, metal, and layered double hydroxide-based materials, presently a challenge. Nevertheless, the technology involving those new materials is certain to rapidly develop in the near future, possibly leading to notably improved nonenzymatic glucose sensors.

4. RECENT DEVELOPMENTS: AUTOMATION AND FUTURE TRENDS The use of biosensors in food analysis and, in particular, in the determination of starch, is based on enzymatic methods and avoids sample pretreatment, apart from saving time and money in many cases. State-of-the-art biosensors utilize membranes which have the enzyme (usually oxidases) covalently immobilized. These membranes (10 mm diameter) are set in the measuring device by means of an o-ring. The sample is led into a chamber where the oxygen dissolved is transformed into hydrogen peroxide, which is then electrochemically oxidized (using a platinum electrode) into oxygen. The electrical current produced between the platinum anode and the silver cathode is amperometrically measured and is proportional to the analyte concentration. Calibration is carried out with the corresponding standards that are commercially available (Bucsis, 1998). In the case of starch analysis, after autoclaving the sample it is incubated with amyloglucosidase and the resulting glucose is analyzed using a suitable biosensor as described above. If free glucose occurs in the sample, a previous determination of glucose should take place, and thereafter the starch content is calculated. The whole procedure has been automated to a greater or lesser extent by some specialized firms. Biosensors are straightforward with important advantages, namely: (1) No sample pretreatment is normally required, which even allows for suspensions to be directly measured. This is due to the fact that amperometry, instead of


light absorption, is the measurement technique (2) high sensitivity and specificity, typical of enzymatic analysis (3) high flexibility and analysis frequency (c. 40 samples per hour). Bearing all that in mind, there is scarcely any doubt that the utilization of biosensors is expected to consolidate its leading position in starch analysis in foods. Enzyme electrodes should allow the continuous assay of this important component, not only for the purpose of classical at-line analysis but also with a view to online and inline monitoring of food production processes. The latter is already a well-established analytical procedure and the growing availability of commercial biosensors will open a promising path. Furthermore, the increasing stability of many enzymes that can be achieved by cross-linking and immobilization techniques will improve the determination of starch and derivatives. An accurate and rapid method for the quantification of starch in food samples based on direct potentiometry using a platinum redox electrode as a detector has recently been proposed (Sakac et al., 2013), along with a theoretical model based on potentiometric principles. The sensor’s working principle was based on the measured decrease in free triiodide ion after its complexation into a starchetriiodide complex. Sensor and analyte parameters were optimized, resulting in a significant reduction in analysis time and cost. The results obtained had good accuracy and precision and were well correlated with those obtained by a standard spectrophotometric method. A new procedure for the capillary zone electrophoretic separation of neutral carbohydrates with direct UV detection based on the enolization and chelation reactions with phosphates and copper (II), respectively, has recently been developed (Babar, 2012). Using two different electrolytes first consisting of sodium hydroxide and disodium hydrogenphosphate and second consisting of copper (II) sulfate and ammonia, UV absorption at 270 nm results from the enolization and chelation of phosphate and Cu (II) by the carbohydrates under strong alkaline conditions. This method has also been applied to detect polysaccharides such as starch. Some work is currently in progress concerning sonic spray ionization (SSI) ¨ zdemir et al., 2014), a liquid chromatography-mass spectrometry interfacing (O technique in which a liquid flow is sprayed from the tip of a capillary under atmospheric pressure with a gas flow coaxial to the capillary. In contrast to the electrospray or atmospheric pressure chemical ionization techniques, SSI forms charged droplets without heating the capillary or applying an electric field to the tip of the sprayer. Therefore, it is ideally suited for the analysis of thermally unstable compounds such as carbohydrates. Furthermore, ionization without additional high voltages increases the flexibility in the choice of mobile phases. SSI is proving to be very useful in the determination of small saccharides, oligo- and polysaccharides (among them starch), a very high sensitivity being achieved (Volmer, 2000). Additional work will probably be conducted in the future taking into account the promising results obtained so far in this field (Porcari et al., 2016).


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Electronic tongues are emerging as promising tools in food analysis, increasing attention being received in this field as shown in recent surveys in the literature (Escuder-Gilabert and Peris, 2010). They are analytical devices (groups of sensors) mainly employed to identify and classify the tastes of several chemical substances in beverages or liquid phase food samples, their mode of operation “imitating” the human sense of taste. The output of a nonspecific array of sensors shows different patterns for the different tastecausing chemical compounds and these data are statistically treated. A wide variety of chemical sensors are currently used in the design of e-tongues, the selection of the sensor array being carried out considering the chemical nature of the food samples analyzed. The application of e-tongues in the analysis of starch in foodstuffs is an indirect process, based on a previous hydrolysis of this polysaccharide and the subsequent determination of the resulting glucose by means of this “artificial sense.” In this sense, the development of an automated voltammetric e-tongue using a biosensor array (formed by three different enzymatic glucose oxidase electrodes) and the sequential injection analysis principle can be considered a primer in this field (Gute´s et al., 2006). Further approaches also include the recent amperometric bioelectronic e-tongue for glucose determination (Al-Issa et al., 2015); it contains eight sensor electrodes constructed using different metal electrodes, oxidoreductase enzymes, and membrane coatings. The response to varying concentrations of glucose was tested for two models, concentration determination by current density measurements at individual electrodes and concentration determination by a linear regression model for the entire electrode array. Finally, Expert Systems (ES) are currently being applied to a greater or lesser extent in various fields of analytical chemistry, including food analysis (Peris, 2002). The typical scheme consists of a centralized system, implemented around a personal computer, which (1) has the necessary sensors and actuators to connect itself to the process to be controlled and (2) runs the knowledge-based system. The outstanding role ESs play in the monitoring and control of food analysis processes is especially remarkable. Presumably, the determination of starch will soon take advantage of this technology, which can be of great utility in terms of a higher automation of the analysis process. For instance, ESs can easily control a flow injection system for the analysis of the glucose resulting from the hydrolysis of starch, as shown in the online determination of reducing sugars in the course of an alcoholic fermentation process using a rule-based system (Peris et al., 1997). Moreover, in the case of starch-containing foods, taking representative samples, handling them in such a way that no significant changes occur, and preparing the samples for starch analysis are of the utmost importance, since insufficient attention to these factors may very easily lead to distorted data and erroneous conclusions. Sample preparation is often the most critical and time-consuming step in the entire analytical process. For instance, when


determining the concentration of starch in peanut butter, the main difficulty is not the measurement procedure, but achieving an aqueous solution containing all (or a known fraction) of the starch in the sample. Therefore, a good sample preparation method leads to the removal of (potential) interferences and to a concentration of the compounds of interest. Thus, there is nothing strange in the fact that several attempts have recently been made to develop expert systems in this area, especially relevant in food analysis. Nevertheless, we believe that such efforts must be strongly encouraged, since the cornerstone of expert system applications in food analysis is still the analysis itself, preanalysis steps being somewhat left aside. The future of ES applications in food industries will probably bring exciting new approaches. The time will come when the use of expert systems will be an integral part of the practice of food analysis (and, in particular, starch analysis) in quality control laboratories. Expert systems will not replace food chemists, they will rather be very useful assistants which handle the details and allow the chemist to concentrate on the more challenging problems.

5. SOURCES OF FURTHER INFORMATION AND ADVICE The annual Starch Expo (International Starch & Starch Derivatives Exhibition) is the only specialized starch trade fair in Asia. It was launched in 2006 and has grown with the development of the starch industry in China since then. The show has developed into one of the most important events in starch industry, with a respected reputation not only in Asia but also in Europe and the United States. Therefore, it provides with an excellent opportunity to keep up to date with everything related to starch, including starch analysis. A detailed monograph “The Analytical Chemistry of Carbohydrates” (Scherz and Bonn, 1998) presents the whole field of qualitative and quantitative methods for the analysis of nearly all kinds of carbohydrates, including polysaccharides such as starch. Although it mainly focuses on the determination procedures of mono- and oligosaccharides, its importance lays on the fact that many methods for the analysis of starch involve the prior hydrolysis to glucose and determination of this monosaccharide. This book covers theoretical aspects as well as practical applications, with special emphasis on instrumental techniques such as chromatography, photometry, and electrophoresis. Another extensive and recent book, “Food analysis” (Nielsen, 2014) also deals with major methods and techniques utilized for the analysis of sugars and starch in food. It can be considered as an excellent review in this field and the reader is directed to this bibliographic source, especially if he/she is a beginner in the determination of food carbohydrates. “ASEAN Manual of Food Analysis” (Puwastien et al., 2011) is a recent handbook that reportsdamong other analysis proceduresdthe determination of starch in starch-containing foods, with a special mention to sampling


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procedures and sample pretreatment (acid digestion, acid hydrolysis, extraction procedures). Methods are described in a step-by-step approach and contain many practical details. Calculations are also carefully explained. The book is designed as a laboratory sourcebook with useful additional references. Considered for some food chemists as the “bible” of food analysis, “Handbook of Food Analysis” (Nollet and Toldra´, 2015) devotes an entire chapter to the analysis of carbohydrates and starch. Special attention is paid to the latter given its important presence in many foodstuffs. It covers all the stages in its analytical determination from sampling to the analysis itself, be it by classical methods or by modern instrumental techniques. Practical applications are also given along with the corresponding bibliographic references. Emphasizing effective, state-of-the-art methodology, the “Handbook of Food Analytical Chemistry” (Wrolstad et al., 2005) represents one of the most comprehensive resources of its kind. One of its chapters deals with the analysis of starch and starch derivatives in foodstuffs, including detailed instructions with annotated advisory comments, critical and troubleshooting notes, key references with annotations, time considerations, and anticipated results. In addition, useful appendices feature common abbreviations, laboratory stock solutions, equipment, and guidelines for commonly used techniques. “Starch” (Whistler, 2004), written by one of the leading authorities in the field of polysaccharides, deals with everything related to starch, including methods of analysis. It provides the analytical chemists with an excellent view of the complex world of starch and will help them with a great amount of information on the chemistry of this compound. Undoubtedly, a valuable tool in the chemical analysis of starch, most notably in foods. For over 40 years, the journal Starch/Sta¨rke (Sta¨rke is the German word for Starch) has focused on the most important carbohydratesdfrom a renewable resources point of viewdsuch as cellulose, starch, and sugars produced by photosynthesis. Comprehensive and topical, it publishes original articles dealing with fundamental and applied studies. Particular attention is given to recent studies on new analytical methods for starch, modified starches, starch derivatives, and starch saccharification products. Book reviews, an extensive documentation service, patent reviews, and previews of symposia complete the package. This journal is published monthly in English by John Wiley & Sons, Inc. Last but not least, since its inception in 1965 the journal Carbohydrate Research has gained a reputation for its high standard and wide scope. Articles published include all aspects of starch analytical chemistry and biochemistry. Normal length research papers, perspectives, notes, rapid communications, and book reviews, together with notices of relevant meetings can be found in this journal, which is published in English by Elsevier Science.


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Faithfull, N., 1990. Acid hydrolysis prior to automatic analysis for starch. Journal of the Science of Food and Agriculture 50, 419e421. Farhat, I.A., Oguntona, T., Neale, R.J., 1999. Characterisation of starches from West African yams. Journal of the Science of Food and Agriculture 79, 2105e2112. Figueira, J.A., Carvalho, P.H., Sato, H.H., 2011. Sugarcane starch: quantitative determination and characterization. Cieˆncia e Tecnologia de Alimentos 31 (3), 806e815. Forrest, B., 1992. Identification and quantification of hydroxypropylation of starch by FTIR. Sta¨rke (Starch) 5, 179e183. Gon˜i, I., Garcıá-Diz, L., Manãs, E., Saura-Calixto, F., 1996. Analysis of resistant starch: a method for foods and food products. Food Chemistry 56 (4), 445e449. Gu¨nzler, H., Williams, A., 2001. Handbook of Analytical Techniques. Wiley-VCH, Weinheim. Gute´s, A., Ibań˜ez, A.B., del Valle, M., Ce´spedes, F., 2006. Automated SIA e-tongue employing a voltammetric biosensor array for the simultaneous determination of glucose and ascorbic acid. Electroanalysis 18, 82e88. Hauffe, D., 1997. Analytik von Sta¨rke und komplexen Novel Food Kohlenhydraten durch HPLC. GIT Labor-Fachzeitschrift 41 (5), 460e467. Henshall, A., 1996. Analysis of starch and other complex carbohydrates by liquid chromatography. Cereal Foods World 41 (5), 419e424. Hermida, M., Rodrı´guez, N., Rodrı´guez-Otero, J.L., 2006. Determination of moisture, starch, protein, and fat in common beans (Phaseolus vulgaris L.) by near infrared spectroscopy. Journal of AOAC International 89 (4), 1039e1041. Herrero-Martıńez, J.M., Schoenmakers, P.J., Kok, W.T., 2004. Determination of the amyloseamylopectin ratio of starches by iodine-affinity capillary electrophoresis. Journal of Chromatography A 1053, 227e234. Holm, J., Bjo¨rck, I., Drews, A., Asp, N.G., 1986. A rapid method for the analysis of starch. Sta¨rke (Starch) 38, 224e226. Jarvis, C.E., Walker, J.R.L., 1993. Simultaneous, rapid, spectrophotometric determination of total starch, amylose and amylopectin. Journal of the Science of Food and Agriculture 63, 53e57. Karkalas, J., 1991. Automated enzymatic determination of starch by flow injection analysis. Journal of Cereal Science 14, 279e286. Larew, L.A., Mead, D.A., Johnson, D.C., 1988. Flow-injection determination of starch and total carbohydrate with an immobilized glucoamylase reactor and pulsed amperometric detection. Analytica Chimica Acta 204, 43e51. Latimer, G.W. (Ed.), 2012. Official Methods of Analysis of AOAC International. AOAC, Arlington, VA. Levine, L.H., Bauer, J., Levine, H.G., 2005. Critical Aspects of Starch Determination in Plant Tissues and a New Approach Utilizing HPAEC/PAD for the Quantification of Starch-derived Glucose. SAE Technical Papers. Li, B.W., 1996. Determination of sugars, starches, and total dietary fiber in selected highconsumption foods. Journal of AOAC International 79 (3), 718e723. Li, B.W., Zhao, Z., 1997. Determination of starches and dietary fiber polysaccharides in cooked dried beans: comparison of different temperatures and dimethyl sulfoxide treatments. Journal of Agricultural and Food Chemistry 45, 2598e2601. Lo´pez-Hernańdez, J., Gonza´lez-Castro, M.J., Va´zquez-Blanco, M.E., Va´zquez-Ode´riz, M.L., Simal-Lozano, J., 1994. HPLC determination of sugars and starch in green beans. Journal of Food Science 59 (5), 1048e1049.

280 PART j ONE Analyzing and Modifying Starch McCleary, B.V., Solah, V., Gibson, T.S., 1994. Quantitative measurement of total starch in cereal flours and products. Journal of Cereal Science 20 (1), 51e58. McCleary, B.V., Gibson, T.S., Mugford, D.C., 1997. Measurement of total starch in cereal products by amyloglucosidase-a-amylase method: collaborative study. Journal of AOAC International 80 (3), 571e579. McGrance, S.J., Cornell, H.J., Rix, C.J., 1998. A simple and rapid colorimetric method for the determination of amylose in starch products. Sta¨rke (Starch) 50 (4), 158e163. Meyers, R.A. (Ed.), 2000. Encyclopedia of Analytical Chemistry. Wiley, New York. Mitchell, G.A., 1990. Methods of starch analysis. Sta¨rke (Starch) 42 (4), 131e134. Morales, M.D., Escarpa, A., Gonza´lez, M.C., 1997. Simultaneous determination of resistant and digestible starch in foods and food products. Sta¨rke (Starch) 49 (11), 448e453. Morrison, W.R., Laignelet, B., 1983. An improved colorimetric procedure for determining apparent and total amylose in cereal and other starches. Journal of Cereal Science 1, 9e20. Nielsen, S. (Ed.), 2014. Food Analysis, fourth ed. Springer, Heidelberg. Nollet, L.M., Toldra´, F. (Eds.), 2015. Handbook of Food Analysis, third ed. CRC Press, Boca Raton, FL (Chapter 24). Ovando, M., Whitney, K., Simsek, S., 2013. Analysis of starch in food systems by highperformance size exclusion chromatography. Journal of Food Science 78, C192eC198. ¨ zdemir, A., Lin, J.L., Wang, S.H., Chen, C.H., 2014. A deeper look into sonic spray ionization. O RSC Advances 4, 61290e61297. Pare´, J.R., Be´langer, J.M. (Eds.), 1997. Instrumental Methods in Food Analysis. Elsevier, Amsterdam. Peris, M., 2002. Present and future of expert systems in food analysis. Analytica Chimica Acta 454, 1e11. Peris, M., Ors, R., Bonastre, A., Gil, P., Serrano, J., 1997. Advanced application of rule nets to the automation of chemical analysis systems. Analytica Chimica Acta 354, 249e253. Peris-Tortajada, M., Puchades, R., Maquieira, A., 1992. Determination of reducing sugars through the neocuproine method by flow injection analysis. Food Chemistry 43, 65e69. Porcari, A.M., Fernandes, G.D., Barrera-Arellano, D., Eberlin, M.N., Alberici, R.M., 2016. Food quality and authenticity screening via easy ambient sonic-spray ionization mass spectrometry. Analyst 141, 1172e1184. Puwastien, P., Siong, T.E., Kantasubrata, J., Craven, G., Feliciano, R.R., Judprasong, K. (Eds.), 2011. ASEAN Manual of Food Analysis, first ed. ASEAN Network of Food Data System, Bangkok. Rose, R., Rose, C., Omi, S., Forry, K., Durall, D., Big, W., 1991. Starch determination by perchloric acid vs enzymes: evaluating the accuracy of six colorimetric methods. Journal of Agricultural and Food Chemistry 39, 2e11. Sakac, N., Sak-Bosnar, M., Horvat, M., 2013. Direct potentiometric determination of starch using a platinum redox sensor. Food Chemistry 138, 9e12. Scherz, H., Bonn, G., 1998. The Analytical Chemistry of Carbohydrates. Thieme, Stuttgart. Skoog, D.A., West, D.M., Holler, F.J., Crouch, S.R., 2014. Fundamentals of Analytical Chemistry, ninth ed. Brooks Cole, Boston, MA. Teitelbaum, R.C., Ruby, S.L., Marks, T.J., 1980. A resonance Raman/iodine Mo¨ssbauer investigation of the starch-iodine structures. Aqueous solution and iodine vapor preparations. Journal of the American Chemical Society 102, 3322e3328. Trojanowicz, M., 2008. Advances in Flow Analysis. Wiley-VCH, Weinheim. Tsuge, H., Hishida, M., Iwasaki, H., Watanabe, S., Goshima, G., 1990. Enzymatic evaluation for the degree of starch retrogradation in foods and foodstuffs. Sta¨rke (Starch) 6, 213e216.


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Chapter 7

Chemical Modification of Starch Yu-Fang Chen, Lovedeep Kaur, Jaspreet Singh Massey University, Palmerston North, New Zealand

1. INTRODUCTION Starch is widely used in our daily life from centuries: Egyptians boiled wheat flour paste with diluted vinegar to cement strips of papyrus, while in ancient China, people coated the documents with the high-fluidity starch to prevent ink penetration (Normile, 1997). Starch was introduced in England and France during the mid-1500s for use in laundry or fashion (Murphy, 2007). By the 1930s, carbohydrate chemists started developing numerous starch products that greatly expanded starch utilities. Starches from different botanical sources, such as wheat, potato, rice, maize, and other tropical plants, are the main carbohydrates in human nutrition and offer a wide range of properties to achieve desired food product qualities. Food processors generally prefer starches with better behavioral characteristics because of the limitations of native starch characteristics, such as low shear resistance, thermal resistance, and thermal decomposition during processing. Therefore, different types of starch modifications are employed to optimize the structural characteristics and functional properties for targeted applications (Table 7.1). Different methods used to modify starch characteristics include enzymatic, physical, or chemical modification (Table 7.2). Starch can be either depolymerized or extended by using various enzymes. Enzymatic degradation by food-grade enzymes, such as amyloglucosidase, pullulanase, a-amylase, b-amylase, and isomerase, is used in the production of maltodextrin, modified starches, or glucose and fructose syrups. Physical methods of starch modification include hydrothermal treatments, pregelatinization, and nonthermal processes. Chemical modification is generally achieved through derivatization such as etherification, esterification, cross-linking, and grafting. The physicochemical properties of the modified starches, for instance, morphology, thermal characteristics, and pasting/rheological behavior, may be quantified by microscopy, differential scanning calorimetry (DSC), and Rapid Viscosity Starch in Food. https://doi.org/10.1016/B978-0-08-100868-3.00007-X Copyright © 2018 Elsevier Ltd. All rights reserved.

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TABLE 7.1 Some Properties and Applications of Modified Starches Types

Properties

Applications

Pregelatinization

Cold-water dispersibility

Useful in instant convenience foods

Partial acid or enzymatic hydrolysis

Reduced molecular weight polymers, exhibit reduced viscosity, increased retrogradation, and setback

Useful in confectionery, batters, and food coatings

Oxidation/ bleaching

Low viscosity, high clarity, and low temperature stability

Used in batters and breading for coating various food stuffs, in confectionery as binders and film formers, in dairy as texturizers

Pyroconversion (dextrinization)

Low to high solubility depending on conversion, low viscosity, high reducing sugar content

Used as coating materials for various foods, good film forming ability, and as fat replacers in bakery and dairy products

Etherification

Improved clarity of starch paste, greater viscosity, reduced syneresis, and freezee thaw stability

Used in wide range of food applications such as gravies, dips, sauces, fruit pie fillings, and puddings

Esterification

Lower gelatinization temperature and retrogradation, lower tendency to form gels, and higher paste clarity

Used in refrigerated and frozen foods, as emulsion stabilizers and for encapsulation

Cross-linking

Higher stability of granules toward swelling, high temperature, high shear and acidic conditions

Used as viscosifiers and texturizers in soups, sauces, gravies, bakery, and dairy products

Stability Dual modification

Stability against acid, thermal, and mechanical degradation and delayed retrogradation during storage

Used in canned foods, refrigerated and frozen foods, salad dressings, puddings, and gravies

Chemical Modification of Starch Chapter j 7

285

TABLE 7.1 Some Properties and Applications of Modified Starchesdcont’d Types

Properties

Applications

Grafting

Better biodegradability and thermal stability, higher hydrodynamic radius and hydrodynamic volume

Used for film making, during delivery, water-absorbing materials, and textile

Reproduced and modified from Singh, J., Kaur, L., McCarthy, O.J., 2007. Factors influencing the physico-chemical, morphological, thermal and rheological properties of some chemically modified starches for food applicationsda review. Food Hydrocolloids 21 (1), 1e22. http://doi.org/10.1016/j. foodhyd.2006.02.006, with permission from Elsevier.

Analyzer (RVA)/dynamic rheometer, respectively. As starch is the main source of our daily energy intake, the nutritional qualities of starch have attracted a lot of attention recently. A classification system of starch nutritional fractions was established to have better control on blood glucose after consuming starchbased foods (Gon˜i et al., 1997). Based on the human postprandial glucose responses for carbohydrate-based foods, different food products were classified into high-glycemic index (GI, >70), medium-GI (56 < GI < 69), and low-GI ( arabic gum > guar gum (Tester and Sommerville, 2003); the same effect has also been seen during b-amylase hydrolysis of wheat starch in the presence of xanthan, guar gum, and cellulose (Christianson et al., 1981). In starch-based systems, nonstarch polysaccharides are used as cryostabilizers by producing a freeze-concentrated matrix which limits molecular mobility or by restricting ice crystals growth. They significantly improve the freezeethaw stability of starch pastes and gels; even after several freezeethaw cycles the gels remain smooth because retrogradation and syneresis are considerably diminished and increases in gel hardness are moderated, e.g., in systems such as xanthan in sweet potato, yam, corn starches, or wheat flour (Sae-kang and Suphantharika, 2006), konjac glucomannan in rice starch (Charoenrein et al., 2011), b-glucan in rice starch (Satrapai and Suphantharika, 2007), galactomannan in sago and waxy maize starches (Ahmad and Williams, 2001), guar gum, alginate, and xanthan in sweet potato starch (Lee et al., 2002). The molecular weight of gums influence the short- and long-term retrogradation of corn starch (Funami et al., 2005c) and wheat starch (Funami et al., 2005a). The effect of pH on the freezeethaw stability of tapioca starchexanthan pastes have been evaluated, showing that at pH 7 the system is most stable than at alkaline conditions (pH 9), but at acidic conditions (pH 3) the system reaches the highest syneresis values and experiences disadvantageous textural changes due the starch hydrolysis. In these types of systems, starch (amylose) precipitates, which can be slowed down by increasing the viscosity of the continuous phase by xanthan. In nonstarch polysaccharides and starch systems, the addition of salt has different effects on the pasting properties, gelation, and retrogradation of starch, e.g., xanthan with sodium chloride, calcium chloride, sodium sulfate, and sodium phosphate decreases the cold paste viscosity of corn starch (Sudhakar et al., 1995b). Guar gum with calcium chloride and potassium chloride increases the cold paste viscosity of corn starch, while guar gum with sodium sulfate and sodium phosphate decreases it, but guar gum with sodium chloride does not change this viscosity at low salt concentrations but rather decreases it at higher concentrations (Sudhakar et al., 1996). Guar gum and xanthan with sodium chloride and calcium chloride increase the gelatinization temperature of rice starch (Samutsri and Suphantharika, 2012; Viturawong et al., 2008); xanthan and sodium chloride accelerates the aggregation and sedimentation of amylose (Mandala et al., 2004), Sugars (sucrose) are known to delay the gelatinization temperature, but this delay is diminished in the presence of gums such as xanthan and guar gum (Sudhakar et al., 1995a).

4.4 Interactions With Salts Salts are added to improve the quality (binder and nutrient source), acceptability (texture, flavor, and color enhancer), and shelf life (preservative) of

Starch Interactions With Native and Added Food Components Chapter j 20

787

foods by modifying the thermal and physicochemical properties of starch. Starchesalt systems depend mainly on the nature of the salt, i.e., ion valence and charge, salt concentration, presence of functional groups on starch such as sulfate and phosphate groups, and type of starch crystallinity: X-ray diffraction A-, B-, and C-pattern. For example, starches with A-pattern (corn and rice starches) are more resistant to sodium chloride and produce a higher Donnan potential than starches with B-pattern (potato and canna starches) while starches with C-pattern (lotus tuber starch) are in between (Lii and Lee, 1993). In general, saltewater, waterestarch and saltestarch interactions are important because they control the behavior of the whole system. Strongly hydrated ions tend to increase the order of the water structure, increase the viscosity of the system, and decrease the concentration of free water molecules, because anions are attracted to the hydrogen atoms of hydroxyl groups of starch with a subsequent deprotonation (Hþ), hence leaving the starch core negatively charged, which causes a stabilization of starch granules due to electrostatic repulsion forces between similarly charged molecules and aggregation of water around starch. On the other hand, cations are attracted to the hydroxyl groups of starch and destabilize starch granules. Deprotonation lowers the pH of the system. Weakly hydrated ions are unable to separate the protons on the starch molecule but are able to attract water dipoles; consequently, less water is available for hydration of starch and therefore solubility apparently decreases (Jane, 1993; Oosten, 1982). The effects of anions are higher than cations, at least for the first nontransition group (Lii et al., 2002), although the presence of cations is an important factor in starch gelatinization and recrystallization. The degree of granule swelling appears to follow the lyotropic series: the so-called salting-out ions inhibit swelling, perhaps due to electrostatic repulsion with the hydroxyl groups of the starch, e.g., sulfate in sago starch, whereas salting-in ions promote swelling, e.g., iodide and thiocyanate in sago starch (Ahmad and Williams, 1999). In wheat and corn starch systems, the addition of chloride and sulfate retard starch swelling, while iodide accelerates it (Medcalf and Gilles, 1966). Depending on the type of ion, the swelling process could proceed from the hilum to the periphery, from the periphery to the hilum, or be solubilize at low temperatures. The increase in storage modulus G0 in the presence of salting-out ions can be attributed to the aggregation of amylose, whereas salting-in ions inhibit it and therefore G0 decreases, e.g., in sago starch systems or in cassava starch systems (Jyothi et al., 2005). The most used salt in food industry is probably sodium chloride and therefore it has been tested in different food systems; however, today, it is being replaced by other types of salts such as potassium chloride or phosphates due to awareness of the negative effect of sodium on health. In general, in sodium chlorideestarch systems the gelatinization temperature increases to a maximum value (c. 6%e9%) and then decreases as concentration increases

788 PART j THREE Applications

further; the enthalpy of gelatinization decreases and the gelatinization process is delayed especially at low salt concentrations, e.g., in amaranth starch (Paredes-Lo´pez and Hernańdez-Lo´pez, 1991), rice flour and rice starch (Chungcharoen and Lund, 1987), wheat starch (Wootton and Bamunuarachchi, 1980), sago starch (Maaurf et al., 2001), and mung bean starch (Ahmed, 2012). The gelatinization enthalpy for potato starch is higher than for wheat starch, but diminishes as sodium chloride concentration increases, perhaps because of the length of the amylopectin branches that are longer in starches with an X-ray diffraction B-pattern and subsequent greater stability of the double helices and the fact that amylose forms complexes in wheat starch. Potato starch display a higher storage modulus G0 at the beginning of the cycle than wheat starch (Chiotelli et al., 2002). In potato starch, calcium chloride follows the same pattern as sodium chloride, but sodium sulfate and sodium hydrogen phosphate progressively increase the gelatinization temperature (Evans and Haisman, 1982). The rate and degree of starch recrystallization varies among starches and electrolytes. The hydrogen ions (Hþ) of starch hydroxyl groups seem to be displaced by salt cations and prevent the reorganization and realignment of amylose and amylopectin chains, leading to a lesser starch recrystallization and a lower melting enthalpy; divalent cations such as calcium and magnesium are more effective than univalent cations such as lithium, ammonium, sodium, and potassium. Perhaps, due to the larger size of cations compared to Hþ and their higher charge density which has an influence on the hydration energy and hence water activity, starch solubility might also change due to the ions’ presence. For example, the rate and degree of retrogradation decrease significantly in corn starch systems with different salts (Beck et al., 2011), and in rice starch gels with sodium chloride at different temperatures (Chang and Liu, 1991). In starch pressurized systems, at low sodium chloride concentrations, gelatinization is suppressed but with increasing salt content, the degree of gelatinization increases again. This phenomenon is more pronounced for potato starch than for tapioca and wheat starches which increase to a lesser extent. This might be caused by differences in the molecular structure between them due to the presence of phosphate groups in potato starch; in these starchpressurized systems, the effect of addition of chloride salts such as sodium, potassium, lithium, and calcium follow the lyotropic series and react in the same way when potassium salts such as thiocyanate, iodide, bromide, and chloride are added (Rumpold, 2005). In Section 4.3, the effect of salts in starch systems in the presence of nonstarch polysaccharides such as xanthan and guar gum has been discussed.

4.5 Interactions With Surfactants During food preparation, surfactants are usually added as emulsifier, stabilizer, and gel-maker to obtain desirable properties; these additives can be from


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natural sources, e.g., lecithin from egg yolk, proteins from milk, or can be synthetic. Surfactants are known to influence gelatinization and recrystallization of starch, depending mainly on its chemical structure, i.e., the nature of the hydrophilic head (polar) and hydrophobic tale (nonpolar) as well as the nature and presence of functional groups in starch. For example, the typical morphological changes in wheat starch occur at a low temperature when sodium dodecyl sulfate is added and at higher temperature when sodium stearoyl lactylate and glycerol monostearate are added (Eliasson, 1985). The pasting properties also vary among systems, e.g., monoglycerides have the greatest impact on wheat and tapioca starch systems in comparison to stearoyl2-lactylates and diacetyl tartaric acid esters of monoglycerides; however, tapioca and potato starches are less affected by these emulsifiers; both pasting temperature and peak viscosity are influenced by pH and ion concentration (Krog, 1973). Changes in the thermal and physicochemical properties of starches by the addition of surfactants have mainly been attributed to the inclusion complex formation between amylose and surfactants in a quite similar way than lipids, which was described in Section 4.1. Amylose aggregation could occur not only by complexation, but also by phase segregation. At low moderate surfactant concentrations, amylose aggregates into thicker and more rigid strands, but at higher concentrations forms spherical aggregates (Richardson et al., 2004). Surfactants interact not only with amylose but also with the exterior long chains of amylopectin and form inclusion complexes as well; however, chains need to have at least a specific chain length; below that length no binding could occur, e.g., sodium dodecyl sulfate is able to inhibit retrogradation with long exterior amylopectin chains (Lundqvist et al., 2002c). When nonionic surfactants known to form inclusion complexes with starch polysaccharides are added, e.g., polar lipids, they first bind and saturate amylose and thereafter start to bind to amylopectin, however, when ion surfactants are added and the starch polysaccharides have functional groups, e.g., hexadecyltrimethylammonium bromide in potato starch, the surfactant first binds amylopectin due to electrostatic interactions and afterward binds and saturates amylose, finally continuing to bind amylopectin until surfactant micelles start to form in the system (Lundqvist et al., 2002a,b). Due to electrostatic interactions, amylopectin could precipitate in neutrally charged systems.

4.6 Interactions With Polyols, Polyphenols, and Carboxylic Acids These types of compounds also influence the thermal and physicochemical properties of starch; they are usually added to impart some functional properties to starch food systems, such as flavor, aroma, and nutritional aspects or just to modify starch gelatinization and recrystallization. Amylose and amylopectin in starch granules are stabilized by hydrogen bonds, but during


heating in excess water these bonds can be ruptured and then granules can be disrupted. The hydroxyl groups of polyols, polyphenols, and carboxyl acids can form hydrogen bonds with starch polysaccharides during gelatinization, cooling and storage of starch gels and pastes, restraining in this way the recrystallization of starch chains. Polyols increase the starch gelatinization temperature and decrease starch retrogradation by lowering the hydrogen bond rupturing of the medium, lowering water concentration, and changing the water structure; effects rise as increasing the amount of hydroxyl groups present on the alcohol molecule and concentration, e.g., various alcohols in potato starch system (Smits et al., 2003), therefore polyhydric alcohols are better than monohydric or dihydric alcohols; in Section 4.3 there are some examples of the effects of sugar alcohols on starch systems. Polyphenols strongly decrease the gelatinization temperature and prevent starch retrogradation, which depend mainly on their molarity rather than on the amount of hydroxyl groups present in the polyphenol molecule, e.g., tea polyphenols in rice starch system (Wu et al., 2009); addition of polyphenols also improves the nutritional value of starch systems. Carboxylic acids decrease the gelatinization temperature and hinder retrogradation more than their corresponding salts at low concentrations because of action on hydrogen bonds of starch and water by nonpolar groups; the effect on the gelatinization temperature is reversed at higher concentrations by dimerization and clustering of water molecules by hydrophobic bonding, then the interaction of carboxyl groups and nonpolar groups is reduced (Gerlsma, 1970).

4.7 Interactions With Aroma and Flavor Compounds Sensory attributes are critical for the acceptance of food by consumers; therefore ligands such as alcohols, ketones, esters, aldehydes, and acids are often added and trapped in the starch matrix. Their interactions depend on the molecular weight, presence of functional groups, polarity, relative volatility, and physical state (Goubet et al., 1998). Starch, particularly amylose, forms inclusion complexes with ligands by noncovalent bonds and have a strong tendency to form supramolecular structures by association; complexes may induce to spherulitic crystallization or gelation of the starch system (Conde-Petit et al., 2006), although not all ligands complex with amylose (Arvisenet et al., 2002a). Ligands interact not only with amylose but also with amylopectin by adsorption (Arvisenet et al., 2002b) and are retained more effectively in the amorphous regions of starch than in the crystalline regions. The presence of lipids in some starches could assist in complex formation with ligands of low solubility (Tapanapunnitikul et al., 2008). If more than one ligand is added to the system, there could be competition among them to bind to starch (Guichard, 2002).


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5. FUTURE TRENDS Consumers are demanding food products with higher quality and unique properties, mainly related to the texture, appearance, mouthfeel, and taste, but also adapted to their lifestyle in which there is not much time left to prepare and cook food, e.g., precooked, cooked, frozen, extruded food. Considering that starch is always present in food systems and its interactions with other components influence the properties of the final food product, researches will certainly continue working on a better understanding of these interactions and developing novel and tailor-made food products that will fulfill consumers’ expectations. Food needs to be not only nutritious but also healthy and naturald consumers want to enjoy food without the risk of developing a disease related to it; therefore, there is a niche for starch to be studied. Certainly, starch will be tested in more food systems as a fat replacer due to its properties to mimic fat without jeopardizing food quality; new nano-sized compounds will be created, taking advantage of their ability to form inclusion complexes, especially amylose, for delivering functional and nutraceutical molecules into the digestive system of a human; foods with higher fiber content will be developed, considering starch’s ease of recrystallization and hence resistance to enzymatic hydrolysis. And unquestionably, starches from different sources than the commercial will be characterized and tested to identify exceptional properties and replace modified starches in systems in which they are used; starch is biodegradable and new goods can be developed, especially for food packing.

6. CONCLUSIONS In this chapter, the most important features of the main starch components, amylose and amylopectin, have been presented and it has been described how they interact with other major components such as proteins, lipids, and nonstarch polysaccharides as well as minor components such as sugars, salts, surfactants, alcohols, acids, and aroma/flavor compounds, in environments with different water content, pH and ionic strength, and different processing conditions as well as how these interactions modify the molecular structure at micro- and macroscale during the gelatinization and recrystallization of starch, providing specific properties which can be desirable or not, but ultimately defining the quality of the starch system. This knowledge is useful for a better control during food manufacturing, food quality improvement, innovation and development of novel foods, and processes that will respond in a better way to the today’s demands. Even though what it is known till now is important and useful for a better understanding of starch behavior in the presence of other components, more studies need to be done in more complex systems trying to approach more real


food systems, in which starch is added as a raw material or as an additive, since the interactions in multicomponent systems could differ from those reported in three or four components systems.

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796 PART j THREE Applications Hsieh, F., Peng, I.C., Huff, H.E., 1990. Effects of salt, sugar and screw speed on processing and product variables of corn meal extruded with a twin-screw extruder. Journal of Food Science 55 (1), 224e227. Huang, J.J., White, P.J., 1993. Waxy corn starch: monoglyceride interaction in a model system. Cereal Chemistry 70 (1), 42e47. Hug-Iten, S., Handschin, S., Conde-Petit, B., Escher, F., 1999. Changes in starch microstructure on baking and staling of wheat bread. Lebensmittel-Wissenschaft und-Technologie 32 (5), 255e260. Immel, S., Lichtenthaler, F.W., 2000. The hydrophobic topographies of amylose and its blue iodine complex. Sta¨rke (Starch) 52 (1), 1e8. Ito, A., et al., 2006. Regulatory effect of amino acids on the parting behavior of potato starch is attributable to its binding to the starch chain. Journal of Agricultural and Food Chemistry 54, 10191e10196. Jacobs, H., Delcour, J.A., 1998. Hydrothermal modifications of granular starch, with retention of the granular structure: a review. Journal of Agricultural and Food Chemistry 46 (8), 2895e2905. Jamilah, B., et al., 2009. Protein-starch interaction and their effect on thermal and rheological characteristics of a food system: a review. Journal of Food, Agriculture and Environment 7 (2), 169e174. Jane, J-l., 1993. Mechanism of starch gelatinization in neutral salt solutions. Sta¨rke (Starch) 45 (5), 161e166. Jane, J-l., 2006. Current understanding on starch granule structures. Journal of Applied Glycoscience 53, 205e213. Jane, J., et al., 1999. Effects of amylopectin branch chain length and amylose content on the gelatinization and pasting properties of starch. Cereal Chemistry 76 (5), 629e637. Jin, Z., Hsieh, F., Huff, H.E., 1994. Extrusion cooking of corn meal with soy fiber, salt and sugar. Cereal Chemistry 71 (3), 227e234. Jyothi, A., et al., 2005. Gelatinization properties of cassava starch in the presence of salts, acids and oxidising agents. Sta¨rke (Starch) 57, 547e555. Kalichevsky, M.T., Jaroszkiewicz, E.M., Blanshard, J.M.V., 1993. A study of the glass transition of amylopectin-sugar mixtures. Polymer 34 (2), 346e458. Karim, A.A., Norziah, M.H., Seow, C.C., 2000. Methods for the study of starch retrogradation (review). Food Chemistry 71, 9e36. Karkalas, J., Ma, S., Morrison, W.R., Pethrick, R.A., 1995. Some factors determining the thermal properties of amylose inclusion complexes with fatty acids. Carbohydrate Research 268, 233e247. Karkalas, J., Raphaelides, S., 1986. Quantitative aspects of amylose-lipid interactions. Carbohydrate Research 157, 215e234. Karlsson, M., 2005. Starch in Processed Potatoes. Media-Tryck Lund University, Lund. Kaur, K., Singh, N., 2000. Amylose-lipid complex formation during cooking of rice flour. Food Chemistry 71, 511e517. Kim, C.S., Walker, C.E., 1992. Changes in starch pasting properties due to sugars and emulsifiers as determined by viscosity measurement. Journal of Food Science 57 (4), 1009e1013. Kim, E.H.-J., et al., 2008. Effect of structural and physicochemical characteristics of the protein matrix in pasta on in vitro starch digestibility. Food Biophysics 3, 229e234. Kitahara, K., Suganuma, T., Nagahama, T., 1996. Susceptibility of amylose-lipid complexes to hydrolysis by glucoamylase from Rhizopus niveus. Cereal Chemistry 73 (4), 428e432.


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Klucinec, J.D., Keeling, P.L., 2006. Genetic modification of plant starches for food applications. In: Shetty, K., Paliyath, G., Pometto, A., Levin, R.E. (Eds.), Food Biotechnology. CRC Press; Taylor & Francis Group, Boca Raton, London, New York, pp. 687e720. Knutson, C.A., 1990. Annealing of maize starches at elevated temperatures. Cereal Chemistry 67 (4), 376e384. Kohyama, K., Nishinari, K., 1991. Effect of soluble sugars on gelatinization and retrogradation of sweet potato starch. Journal of Agricultural and Food Chemistry 39, 1406e1410. Kowblansky, 1985. Calorimetric investigation of inclusion complexes of amylose with long-chain aliphatic compounds containing different functional groups. Macromolecules 18, 1776e1779. Krog, N., 1973. Influence of food emulsifiers on pasting temperature and viscosity of various starches. Die Sta¨rke 25 (1), 22e27. Krueger, B.R., Walker, C.E., Knutson, C.A., Inglett, G.E., 1987. Differential scanning calorimetry of raw and annealed starch isolated from normal and mutant maize genotypes. Cereal Chemistry 64 (3), 187e190. Lalush, I., et al., 2005. Utilization of amylose-lipid complexes as molecular nanocapsules for conjugated linoleic acid. Biomacromolecules 6, 121e130. Le Corre, D., Bras, J., Dufresne, A., 2010. Starch nanoparticles: a review. Biomacromolecules 11, 1139e1153. Lee, M.H., et al., 2002. Freeze-thaw stabilization of sweet potato starch gel by polysaccharide gums. Food Hydrocolloids 16, 345e352. Lehmann, U., Robin, F., 2007. Slowly digestible starch e its structure and health implications: a review. Trends in Food Science and Technology 18, 346e355. Lii, C.-Y., Tomasik, P., Hung, W.-L., Lai, V.M.-F., 2002. Revised look at the interaction of starch with electrolyte: effect of salts of metals from the first non-transition group. Food Hydrocolloids 16, 35e45. Li, J.-Y., Yeh, A.-I., Fan, K.-L., 2007. Gelation characteristics and morphology of corn starch/soy protein concentrate composites during heating. Journal of Food Engineering 78, 1240e1247. Lii, C.Y., Lee, B.L., 1993. Heating A-, B-, and C-type starches in aqueous sodium chloride: effects of sodium chloride concentration and moisture content. Cereal Chemistry 70 (2), 188e192. Lim, S.-T., Lee, J.-H., Shin, D.-H., Lim, H.S., 1999. Comparison of protein extraction solutions for rice starch isolation and effects of residual protein content on starch pasting properties. Sta¨rke (Starch) 51 (4), 120e125. Lindahl, L., Eliasson, A.-C., 1986. Effects of wheat proteins on the viscoelastic properties of starch gels. Journal of the Science of Food and Agriculture 37, 1125e1132. Liu, H., Lelie`vre, J., 1991. A differential scanning calorimetry study of glass and melting transitions in starch suspensions and gels. Carbohydrate Research 219, 23e32. Liu, H., Yu, L., Xie, F.C.L., 2006. Gelatinization of cornstarch with different amylose/amylopectin content. Carbohydrate Polymers 65, 357e363. Luallen, T., 2004. Utilizing starches in product development. In: Eliasson, A. (Ed.), Starch in Food. Structure, Function and Applications. Woodhead Publishing Limited, CRC Press LLC, Cambridge, Boca Raton, pp. 403e434. Lundqvist, H., Eliasson, A.-C., Olofsson, G., 2002a. Biding of hexadecyltrimethylammonium bromide to starch polysaccharides. Part II. Calorimetric study. Carbohydrate Polymers 49, 109e120. Lundqvist, H., Eliasson, A.-C., Olofsson, G., 2002b. Binding of hexadecyltrimethylammonium bromide to starch polysaccharides. Part I. Surface tension measurements. Carbohydrate Polymers 49, 43e55.

798 PART j THREE Applications Lundqvist, H., Nilsson, G.S., Eliasson, A.-C., Gorton, L., 2002c. Changing the amylopectin-sodium dodecyl sulphate interaction by modifying the exterior chain length. Sta¨rke (Starch) 54, 100e107. Maaurf, A.G., et al., 2001. Gelatinisation of sago starch in the presence of sucrose and sodium chloride as assessed by differential scanning calorimetry. Carbohydrate Polymers 45, 335e345. Mandala, I.G., Bayas, E., 2004. Xanthan effect on swelling, solubility and viscosity of wheat starch dispersions. Food Hydrocolloids 18, 191e201. Mandala, I., Michon, C., Launay, B., 2004. Phase and rheological behaviors of xanthan/amylose and xanthan/starch mixed systems. Carbohydrate Polymers 58, 285e292. Mangala, S.L., Udayasankar, K., Tharanathan, R.N., 1999. Resistant starch from processed cereals: the influence of amylopectin and non-carbohydrate constituents in its formation. Food Chemistry 64, 391e396. Manin˜gat, C.C., Juliano, B.O., 1980. Starch lipids and their effect on rice starch properties. Sta¨rke (Starch) 32 (3), 76e82. Marshall, W.E., Chrastil, J., 1992. Interaction of food proteins with starch. In: Hudson, B.J.F. (Ed.), Biochemistry of Food Proteins. Springer ScienceþBusiness Media Dordrecht, Salem, pp. 75e97. Medcalf, D.G., Gilles, K.A., 1966. Effect of a lyotropic ion series on the pasting characteristics of wheat and corn starches. Die Sta¨rke 4, 101e105. Miles, M.J., Morris, V.J., Orford, P.D., Ring, S.G., 1985. The roles of amylose and amylopectin in the gelation and retrogradation of starch. Carbohydrate Research 135 (2), 271e281. Morrison, W.R., 1981. Starch lipids: a reappraisal. Sta¨rke (Starch) 33 (12), 408e410. Morrison, W.R., 1988. Lipids in cereal starches: a review. Journal of Cereal Science 8, 1e15. Morrison, W.R., et al., 1993. Swelling and gelatinization of cereal starches. IV. Some effects of lipid-complexed amylose and free amylose in waxy and normal barley starches. Cereal Chemistry 70 (4), 385e391. Morris, V.J., 1990. Starch gelation and retrogradation (review). Trends in Food Science and Technology 1, 2e6. Nishinari, K., Zhang, H., Ikeda, S., 2000. Hydrocolloid gels of polysaccharides and proteins. Current Opinion in Colloid and Interface Science 5, 195e201. Obiro, W.C., Ray, S.S., Emmambux, M.N., 2012. V-amylose structural characteristics, methods of preparation, significance, and potential applications. Food Reviews International 28, 412e438. Olkku, J., Rha, C., 1978. Gelatinisation of starch and wheat flour starch e a review. Food Chemistry 3, 293e317. Olsson, C., Langton, M., Hermansson, A.-M., 2002a. Dynamic measurements of b-lactoglobulin structures during aggregation, gel formation and gel break-up in mixed biopolymers systems. Food Hydrocolloids 16, 477e488. Olsson, C., Langton, M., Hermansson, A.-M., 2002b. Microstructures of b-lactoglobulin/amylopectin gels on different scales and their significance for rheological properties. Food Hydrocolloids 16, 111e126. Oosten, B.J., 1982. Tentative hypothesis to explain how electrolytes affect the gelatinization temperature of starches in water. Sta¨rke (Starch) 34 (7), 233e239. Paredes-Lo´pez, O., Hernańdez-Lo´pez, D., 1991. Application of differential scanning calorimetry to amaranth starch gelatinization. Sta¨rke (Starch) 43 (2), 57e61. Parker, R., Ring, S., 2001. Aspects of the physical chemistry of starch. Journal of Cereal Science 34 (1), 1e17. Pe´rez, S., Bertoft, E., 2010. The molecular structures of starch components and their contribution to the architecture of starch granules: a comprehensive review. Sta¨rke (Starch) 62 (8), 389e420.


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Perry, P.A., Donald, A.M., 2002. The effect of sugars on the gelatinisation of starch. Carbohydrate Polymers 49, 155e165. Prokopowich, D.J., Biliaderis, C.G., 1995. A comparative study of the effect of sugars on the thermal and mechanical properties of concentrated waxy maize, wheat, potato and pea starch gels. Food Chemistry 52, 255e262. Putseys, J.A., Lamberts, L., Delcour, J.A., 2010. Amylose-inclusion complexes: formation, identity and physico-chemical properties. Journal of Cereal Science 51, 238e247. Quiroga, C., 2007. Phase Segregation and Microstructural Changes in Starch e Protein Systems. Media-Tryck Lund University, Lund. Quiroga, C.C., Bergensta˚hl, B., 2007. Characterization on the microstructure of phase segregated amylopectin and b-lactoglobulin dry mixtures. Food Biophysics 2 (4), 172e182. Quiroga, C.C., Bergensta˚hl, B., 2008a. Phase segregation of amylopectin and b-lactoglobulin in aqueous system. Carbohydrate Polymers 72 (1), 151e159. Quiroga, C.C., Bergensta˚hl, B., 2008b. Rheological behavior of amylopectin and b-lactoglobulin phase-segregated aqueous system. Carbohydrate Polymers 74 (3), 358e365. Radley, J.A., 1976. Starch Production Technology. Applied Science Publishers, London. Ribotta, P.D., Colombo, A., Leoń, A.E., Anõń, M.C., 2007. Effects of soy protein on physical and rheological properties of wheat starch. Sta¨rke (Starch) 59, 614e623. Ribotta, P.D., Leoń, A.E., Anõń, M.C., 2003. Effect of freezing and frozen storage on the gelatinization and retrogradation of amylopectin in dough baked in a differential scanning calorimeter. Food Research International 36, 357e363. Richardson, G., Kidman, S., Langton, M., Hermansson, A.-M., 2004. Differences in amylose aggregation and starch gel formation with emulsifiers. Carbohydrate Polymers 58, 7e13. Riisom, T., Krog, N., Eriksen, J., 1984. Amylose complexing capacities of cis- and transunsaturated monoglycerides in relation to their functionality in bread. Journal of Cereal Science 2 (2), 105e118. Rojas, J.A., Rosell, C.M., de Barber, B., 1999. Pasting properties of different wheat flourhydrocolloid systems. Food Hydrocolloids 13, 27e33. Rumpold, B.A., 2005. Effect of salts and sugars on pressure-induced gelatinisation of wheat, tapioca, and potato starches. Sta¨rke (Starch) 57, 370e377. Ryan, K.J., Brewer, M.S., 2007. In situ examination of starch granule-soy protein and wheat protein interactions. Food Chemistry 104, 619e629. Sae-kang, V., Suphantharika, M., 2006. Influence of pH and xanthan gum addition on freeze-thaw stability of tapioca starch pastes. Carbohydrate Polymers 65, 371e380. Samutsri, W., Suphantharika, M., 2012. Effect of salts on pasting, thermal, and rheological properties of rice starch in the presence of non-ionic and ionic hydrocolloids. Carbohydrate Polymers 87, 1559e1568. Satrapai, S., Suphantharika, M., 2007. Influence of spent brewer’s yeast b-glucan on gelatinization and retrogradation of rice starch. Carbohydrate Polymers 67, 500e510. Seneviratne, H., Biliaderis, C.G., 1991. Action of a-amylases on amylose-lipid complex superstructures. Journal of Cereal Science 13, 129e143. Shewry, P.R., Halford, N.G., 2002. Cereal seed storage proteins: structures, properties and role in grain utilization. Journal of Experimental Botany 53 (370), 947e958. Shi, X., BeMiller, J.N., 2002. Effects of food gums on viscosities of starch suspensions during pasting. Carbohydrate Polymers 50, 7e18. Singh, N., et al., 2003. Morphological, thermal and rheological properties of starches from different botanical sources. Food Chemistry 81, 219e231.

800 PART j THREE Applications Slade, L., Levine, H., 1987. Recent advances in starch retrogradation. In: Stivala, S.S., Crescenzi, V., Dea, I.C.M. (Eds.), Industrial Polysaccharides. The Impact of Biotechnology and Advanced Methodologies. Gordon and Breach Science, New York, pp. 387e430. Smits, A.L.M., Kruiskamp, P.H., van Soest, J.J.G., Vliegenthart, J.F.G., 2003. The influence of various small plasticisers and malto-oligosaccharides on the retrogradation of (partly) gelatinised starch. Carbohydrate Polymers 51, 417e424. Song, J.-Y., et al., 2006. Pasting properties of non-waxy rice starch hydrocolloids Mixtures. Sta¨rke (Starch) 58, 223e230. Sopade, P.A., Halley, P.J., Junming, L.L., 2004. Gelatinisation of starch in mixtures of sugars. II. Application of differential scanning calorimetry. Carbohydrate Polymers 58, 311e321. Sudhakar, V., Singhal, R.S., Kulkarni, P.R., 1995a. Effect of sucrose on starch-hydrocolloid interactions. Food Chemistry 52, 281e284. Sudhakar, V., Singhal, R.S., Kulkarni, P.R., 1995b. Studies on starch-hydrocolloid interactions: effect of salts. Food Chemistry 53, 405e408. Sudhakar, V., Singhal, R.S., Kulkarni, P.R., 1996. Effect of salts on interactions of starch with guar gum. Food Hydrocolloids 10 (3), 329e334. Swinkels, J.J.M., 1985a. Composition and properties of commercial native starches. Sta¨rke (Starch) 37 (1), 1e5. Swinkels, J.J.M., 1985b. Sources of starch, its chemistry and physics. In: van Beynum, G.M.A., Roels, J.A. (Eds.), Starch Conversion Technology (Food Science and Technology). Marcel Dekker, New York, pp. 15e46. Szczodrak, J., Pomeranz, Y., 1992. Starch-lipid interactions and formation of resistant starch in high-amylose barley. Cereal Chemistry 69 (6), 626e632. Taggart, P., 2004. Starch as an ingredient: manufacture and applications. In: Eliasson, A. (Ed.), Starch in Food. Structure, Function and Applications. Woodhead Publishing Limited, CRC Press LLC, Cambridge, Boca Raton, pp. 373e402. Takeuchi, I., 1969. Interaction between protein and starch. In: AACC-AOCS Joint Meeting, Washington, DC. Tang, M.C., Copeland, L., 2007. Analysis of complexes between lipids and wheat starch. Carbohydrate Polymers 67 (1), 80e85. Tapanapunnitikul, O., Chaiseri, S., Peterson, D.G., Thompson, D.B., 2008. Water solubility of flavor compounds influences formation of flavor inclusion complexes from dispersed highamylose maize starch. Journal of Agricultural and Food Chemistry 56, 220e226. Techawipharat, J., Suphantharika, M., BeMiller, J.N., 2008. Effects of cellulose derivatives and carrageenans on the pasting, paste, and gel properties of rice starches. Carbohydrate Polymers 73, 417e426. Tester, R.F., Debon, S.J., 2000. Annealing of starch e a review. International Journal of Biological Macromolecules 27 (1), 1e12. Tester, R. f., Karkalas, J., Qi, X., 2004. Starch-composition, fine structure and architecture. Journal of Cereal Science 39, 151e165. Tester, R.F., Morrison, W.R., 1990. Swelling and gelatinization of cereal starches. I. Effects of amylopectin, amylose, and lipids. Cereal Chemistry 67 (6), 551e557. Tester, R.F., Sommerville, M.D., 2003. The effects of non-starch polysaccharides on the extent of gelatinisation and swelling and a-amylase hydrolysis of maize and wheat starches. Food Hydrocolloids 17, 41e54. Tolstoguzov, V.B., 1986. Functional properties of protein-polysaccharide mixtures. In: Mitchell, J.R., Ledward, D.A. (Eds.), Functional Properties of Food Macromolecules. Elsevier Applied Science, London, pp. 385e416.


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Part Four

Starch and Health


Chapter 21

Starch Digestion and Applications of Slowly Available Starch Marion G. Priebe, Coby Eelderink, Renate E. Wachters-Hagedoorn, Roel J. Vonk University of Groningen, Groningen, The Netherlands

1. INTRODUCTION Maintenance of plasma glucose levels within narrow limits is of vital importance for the body to provide energy for the various organs and tissues. Abnormal elevation of postprandial plasma glucose (hyperglycemia), as observed in diabetic patients, can be harmful for cells, tissues, and organs. In addition, frequent high-postprandial glucose concentrations could be a risk factor for the development of type 2 diabetes and/or cardiovascular disease in susceptible persons. Starchy products can play a role in the prevention of these hyperglycemic events, if starch in these products is slowly digested and/or starch-derived glucose is released into the circulation in a more slow and attenuated manner, which will be referred to as “slowly available starch.” In this chapter, we discuss the mechanism and determinants of intestinal starch digestion as well as absorption of starch-derived glucose, various methods of its measurement and the application of slowly available starch in the prevention of postprandial hyperglycemia. First, we give a brief overview of the mechanisms that operate to maintain normal plasma glucose levels and of the causes and consequences of hyperglycemia. Secondly, possible targets to slow down the rate at which starch becomes available for absorption in the gastrointestinal tract, as well as the need for the development of new technical approaches to achieve this goal, are discussed in more detail. To evaluate these new approaches accurate tools to monitor the digestion and absorption of starch are indispensable. In the third section we give an outline of the methods to monitor starch digestion in vitro and in vivo. In the fourth section the current (nutritional) strategies to prevent hyperglycemia are discussed. We conclude with a short description of possible future developments. Starch in Food. http://dx.doi.org/10.1016/B978-0-08-100868-3.00021-4 Copyright © 2018 Elsevier Ltd. All rights reserved.

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2. PREVENTION OF POSTPRANDIAL HYPERGLYCEMIA: THE ROLE OF STARCH 2.1 Regulation of Postprandial Plasma Glucose The plasma glucose concentration is tightly controlled. Despite wide fluctuations in glucose influx into the circulation (e.g., during feeding) and efflux out of the circulation (e.g., during fasting and exercise), the plasma glucose concentration is normally maintained within a relatively narrow range, between approximately 3.3 and 8.3 mmol/L. Maintenance of relatively stable plasma glucose concentrations requires dynamic regulation of glucose influx and efflux. The diet is normally the major source of glucose. Between meals or during fasting, plasma glucose levels are maintained mainly by hepatic breakdown of glycogen and by gluconeogenesis. In most people during fasting, glycogen stores in the liver are sufficient to prevent hypoglycemia for 8e12 h, but this time period will be shorter if glucose demand is increased during exercise or when glycogen stores are depleted during starvation or illness. The prevention or correction of hypoglycemia is critical to survival, because glucose is the predominant metabolic fuel for the brain and the brain itself can neither synthesize glucose nor store more than a few minutes supply as glycogen. Thus, it is not surprising that physiological mechanisms have evolved that very effectively prevent or correct hypoglycemia. When glucose levels approach the hypoglycemic range, counterregulatory hormone responses are induced. In healthy persons, the peak postprandial plasma glucose, which occurs normally within the first hour after the start of the meal, seldom exceeds 8.3 mmol/L. The postprandial increase in glucose rarely lasts beyond 120 min. In response to this rise, the pancreas increases its secretion of insulin and suppresses the release of glucagon, thereby limiting hepatic glucose production and promoting the uptake of glucose by muscle and fat tissue. Increased insulin levels effectively deposit a large proportion of glucose into these tissues if receptor sensitivity to insulin is normal. The degree of postprandial hyperglycemia is controlled by the balance between glucose influx from the small intestine and glucose removal out of the blood stream. When glucose removal exceeds glucose influx, plasma glucose returns to fasting levels.

2.2 Hyperglycemia and Persons at Risk 2.2.1 Patients With Type 2 Diabetes Diabetes mellitus is a group of metabolic diseases characterized by an elevated blood glucose level (hyperglycemia) resulting from defects in insulin secretion and/or in insulin action. In type 1 diabetes there is an absolute insulin deficiency, whereas in type 2 diabetes (T2DM) insulin deficiency is relative. Insulin levels are normal or, more commonly, elevated in T2DM, but the

Starch Digestion and Applications of Slowly Available Starch Chapter j 21

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responses of tissues to insulin are inadequate. Chronic hyperglycemia causes damage and dysfunction of various cells, tissues, and organs. Macrovascular complications (e.g., ischemic heart disease and stroke) and microvascular complications (e.g., retinopathy, neuropathy, and nephropathy) can occur. These complications have been shown to be associated to a greater extent to postprandial hyperglycemia than to high fasting glucose (DiNicolantonio et al., 2015). Several mechanisms have been described to explain hyperglycemia-induced damage: (1) increased glucose flux via the polyol pathway; (2) increased advanced glycation endproduct (AGE) formation; (3) increased expression of the receptor for AGEs; (4) activation of protein kinase C isoforms; (5) increased glucose flux via the hexosamine pathway. All these five mechanisms seem to be caused by one single event which is the overproduction of reactive oxygen species by the mitochondrial electron-transport chain, due to hyperglycemia (Brownlee, 2001; Giacco and Brownlee, 2010). The main aim of the treatment is to regulate the blood glucose level and by consequence to prevent complications. People with type 1 diabetes are usually dependent on daily insulin injections for survival. Drug therapy of T2DM consists mainly of oral antidiabetic agents and sometimes of insulin. The nonpharmacological treatment of T2DM consists of optimization of physical activity and diet. The amount and type of carbohydrates as well as the rate and extent of digestion are the leading determinants of the rate of absorption and the postprandial glucose response. Replacement of foods with rapidly available starch with foods containing slowly available starch may contribute to the reduction of postprandial plasma glucose excursions.

2.2.2 Persons With Prediabetes Prediabetes precedes overt T2DM for at least 4e7 years (Harris et al., 1992) and is the stage in which blood glucose concentrations are higher than normal but not high enough to be classified as T2DM. The definition of prediabetes includes persons with either high-risk glycated hemoglobin (HbA1c) concentration or impaired fasting glucose or impaired glucose tolerance or a combination of the last two conditions (Faerch et al., 2014). HbA1c reflects the average blood glucose level of the preceding 6e8 weeks (Scheen, 2003). Persons with prediabetes are mostly not aware of their abnormal glycemia because this does not cause symptoms. About 70% of persons with prediabetes will progress to T2DM (Faerch et al., 2014). It was shown that 45% and 50% of the American population aged 45e64 years and 65 years, respectively, has prediabetes (Menke et al., 2015). In Europe, the prevalence of persons with impaired glucose tolerance is 31% and 52% in persons aged 40e59 years and 60e79 years, respectively (Valensi et al., 2005). From the moment that T2DM manifests itself with symptoms and is diagnosed, pancreatic b-cells often have already been irreversibly damaged due to the toxic effect of hyperglycemia. Furthermore, repeated elevated postprandial glycemia is associated with higher risk of cardiovascular disease in persons with prediabetes


(DiNicolantonio et al., 2015). These facts demonstrate the need that also in persons with prediabetes frequent postprandial hyperglycemia is avoided.

2.2.3 Healthy Persons There are indications that reduction of postprandial glycemia could also be beneficial for the general population since some epidemiological and mechanistic studies suggest that also postchallenge (2 h after an oral glucose tolerance test) hyperglycemia in the nondiabetic range is an indicator of cardiovascular risk (Blaak et al., 2012).

3. TARGETS TO INFLUENCE POSTPRANDIAL GLYCEMIA The postprandial glucose response is not only a reflection of intestinal glucose absorption, but the combined response of several physiological processes. Blood glucose in the venous system is supplied with glucose from the intestinal tract and with glucose produced by the liver. Furthermore, glucose is continuously taken up by the tissues, mainly the muscle and the brain. So, the postprandial blood glucose concentration after ingestion of starchy food is the result of (1) the systemic appearance of starch-derived (exogenous) glucose, (2) the systemic appearance of endogenously produced glucose, and (3) the glucose removal from the blood stream. The rate of appearance of exogenous glucose is determined by processes in the gastrointestinal tract and generally considered as the main factor influencing postprandial glucose response (which will be further discussed below). The determinants of the rate of appearance of starch-derived glucose include gastric emptying, starch digestion, absorption of starch-derived glucose, and intestinal transit time. Understanding which food characteristics can influence these processes enables the development of starchy products of which the appearance of starch-derived glucose is delayed.

3.1 Intestinal Starch Digestion Starch is a polysaccharide consisting of amylose and amylopectin. Amylose is made out of a-1-4 glucose chains with very few branches, whereas amylopectin has many branches due to a-1-6 bonds. Amylose and amylopectin can be hydrolyzed by salivary and pancreatic a-amylase. Salivary a-amylase is inactivated in the acid environment of the stomach. Alpha-amylase hydrolyzes the internal a-1,4-glycosidic bonds but not the a-1,6-glycosidic linkages at the branching sides in amylopectin. The glucose oligosaccharides containing uncleaved a-1,6-glycosidic bonds are the so-called a-limit dextrins. After starch hydrolysis by a-amylase, the remaining products, maltose, maltotriose and the a-limit-dextrins, are hydrolyzed into glucose by the enzyme complexes sucroseeisomaltase and maltaseeglucoamylase located in the brush border membrane (Lin et al., 2012). Alpha-amylase, sucraseeisomaltase, and


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maltaseeglucoamylase are the so-called a-glucosidases. By inhibition of intestinal a-glucosidases the digestion of carbohydrates to absorbable monosaccharides can be delayed. The rate and extent of starch digestion depends on the accessibility of starch to a-glucosidases. This accessibility can vary depending on the type and characteristics of starch as well as on the physical form of the food matrix. In addition, there are compounds which are able to interact with digesting enzymes causing delayed starch digestion. Examples of these “a-glucosidase inhibitors” are the antidiabetic drugs acarbose, miglitol, and voglibose. Alpha-glucosidase inhibitors can also be food-derived, examples being polyphenols (Coe and Ryan, 2016) or glycoproteins from common white bean (Phaseolus vulgaris) (Barrett and Udani, 2011). Furthermore, the presence of water-soluble or viscous fiber can interfere with starch digestion as they can hinder the contact between starch and the digestive enzymes (Dikeman and Fahey, 2006).

3.2 Absorption in the Small Intestine Glucose enters the enterocytes by active transport mediated by the sodiumglucose cotransporter (SGLT-1). The energy is provided by the sodium gradient across the brush border membrane, maintained by the enzyme NaþKþ-ATPase in the basolateral membrane. Glucose leaves the intestinal cells down its concentration gradient via the facilitative glucose transporter (GLUT2). Adjacent to the brush border membrane lays the unstirred layer that acts as diffusion barrier for high-permeability compounds and is able to affect the rate of transport. The rate of absorption of starch-derived glucose can be delayed by the presence of viscous fiber by means of hindering transport of glucose to the brush border membrane and by thickening the unstirred layer (Dikeman and Fahey, 2006).

3.3 Transit Time 3.3.1 Gastric Emptying Rate The rate of gastric emptying is a major determinant of the rate of nutrient delivery to the small intestine. Delaying gastric emptying decreases the rate of digestion and absorption of carbohydrates. Several factors are known to influence the gastric emptying rate. Meal-related factors involved are the size, composition, osmolarity, caloric content, and viscosity of a meal. The glucoselowering effect of viscous fibers for example is partly explained by their ability to reduce gastric emptying (Dikeman and Fahey, 2006). Besides the intrinsic characteristics of a meal also small intestinal factors affect the gastric emptying rate. This is mediated via modulation of the neurohumoral and central nervous system. Several peptide hormones, like cholecystokinin (CCK), glucagon-like peptide-1 (GLP-1), and peptide YY (PYY) are thought to be involved in the regulation of gastric emptying.


3.3.2 Intestinal Transit Time The contact time between the nutrients and the intestinal mucosa is another factor affecting the rate and extent of intestinal carbohydrate digestion and absorption (Caspary, 1992). The absorption rate of nutrients could increase when intestinal transit is slowed down since a delay in small bowel transit increases the time of contact between the luminal contents and the absorptive epithelium (Huge et al., 1995). The orocecal transit time (OCTT) is the time needed for food to pass from the mouth into the cecum and is frequently used as an indicator of small intestinal transit time. Only few studies have investigated the relation between the OCTT and the digestion and absorption of carbohydrates. In ileostomy patients, Chapman et al. showed that the absorption of starch is directly related to the small intestinal transit time (Chapman et al., 1985). Intestinal transit time is influenced by viscosity and particle size of the digesta, whereas larger particles seem to accelerate (McIntyre et al., 1997) and higher viscosity seems to slow down (Dikeman and Fahey, 2006) the passage. In both humans and dogs, intraileal infusion of nutrients has inhibitory effects on gastric emptying, duodenal and jejunal motility, and proximal intestinal transit. This phenomenon is called the “ileal brake,” a negative feedback response from the distal to the proximal gut. The regulating mechanism of the ileal brake is incompletely characterized, but there is evidence that it is at least in part hormonally mediated.

3.4 Gastrointestinal Hormones Gastrointestinal hormones are peptide hormones secreted by endocrine cells, which are widely distributed throughout the mucosa of the gastrointestinal tract. These hormones regulate intestinal and pancreatic functions, by affecting secretion, motility, absorption, digestion, and cell proliferation (Thomas et al., 2003). Therefore, these hormones could be challenging targets to affect the rate of starch digestion and absorption of starch-derived glucose. The complex mechanisms that regulate the secretion of gastrointestinal hormones are not completely understood yet. CCK is produced primarily in the duodenum and the jejunum. The physiological roles of CCK are the stimulation of pancreatic secretion, the contraction of the gall-bladder and reducing the gastric emptying rate (Zietek and Daniel, 2015). This hormone is triggered mainly by protein and fat intake (Gribble, 2012). PYY is released mostly from endocrine L-cells of the ileum, colon, and rectum. The most studied biological function of PYY is its potency to reduce appetite and food intake. In addition, it inhibits pancreatic secretion, stimulates the contraction of the gallbladder, and slows down gastric emptying as well as intestinal motility. The most potent nutritional stimulators of PYY secretion are fat and protein, whereas intake of carbohydrates elicits a lower response.


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Effects of dietary fiber on postprandial PYY response are controversial (Cooper, 2014). GLP-1 is secreted from L-cells that are found in high density in the ileum and colon but were also reported to be present in the duodenum in comparable amounts as K-cells (Theodorakis et al., 2006). It stimulates postprandial insulin secretion, decreases the secretion of glucagon, and delays gastric emptying. Ingestion of protein and fat stimulates GLP-1 release (Carr et al., 2008) as well as presence of carbohydrates in the more distal small intestine (Little et al., 2006). A late and prolonged increase in plasma concentrations of GLP-1 has been shown after ingestion of slowly digestible uncooked corn starch (Wachters-Hagedoorn et al., 2006) and when digestion of sucrose was delayed by the a-glucosidase inhibitor acarbose (Qualmann et al., 1995; Seifarth et al., 1998). But when sugar and starch are ingested in complex food matrices the effect on stimulation of GLP-1 secretion is less clear (Eelderink et al., 2016; Karhunen et al., 2010; Klosterbuer et al., 2012). Other stimuli of GLP-1 secretion seem to be short-chain fatty acids produced by the colonic microbiota upon fermentation of dietary fiber (Kaji et al., 2014; Zietek and Daniel, 2015), which awaits confirmation in human studies (Bodinham et al., 2013). Glucose-dependent insulinotropic polypeptide (GIP) is produced by the K-cells located in the proximal intestine. Like GLP-1, GIP is an incretin hormone which potentiates glucose-stimulated insulin release. Its secretion is triggered by absorbable carbohydrates, proteins, and lipids (Deacon and Ahren, 2011). Schirra et al. (1996) demonstrated that GIP release is proportional to glucose delivery in the duodenum using duodenal glucose perfusion.

3.5 Food Characteristics Influencing Postprandial Glycemia The rate of digestion of starch in different foods is highly variable. Whereas starch in some food is very rapidly digested and results in a blood glucose response comparable with that after ingestion of glucose, other starchy foods release glucose in a very slow manner. This variability can be explained by differences in the physicochemical characteristics of starch and the physical form of the food, which are determined by the botanical origin as well as by processing and the presence of other food components (food composition).

3.5.1 Starch Characteristics Starches from different botanical origin vary in their proportion of amylose and amylopectin. Starches with higher amylose content are less susceptible to hydrolysis by a-glucosidases mainly because amylose has a larger molecular size than amylopectin and has not a branched but a linear structure (Biliaderis, 1991). Raw starch, dependent on its source, is either slowly and completely digestible (most cereal starches) or (partly) resistant to digestion (potato and legume starch) (Cummings and Englyst, 1995). Starch granules that are fully


gelatinized are easily digestible. The degree of gelatinization can be influenced by the water content of the product, the temperature, the duration of heating, and the pressure (Bjorck et al., 1994). During cooling and aging of gelatinized starch recrystallization occurs, this is termed retrogradation. Retrogradation takes place most rapidly in amylose and results in poor digestibility.

3.5.2 Physical Form of Food Starch is slowly digested if the physical form of food (particle size, compactness) hinders the accessibility of the pancreatic enzyme a-amylase (e.g., in breads with intact grains, legumes, and spaghetti). Disruption of the tissue or cell structure by, e.g., grinding increases the availability of starch (Bjorck et al., 1994; Cummings and Englyst, 1995). Several studies demonstrated that incorporation of boiled intact cereal grains into breads (either 40% or 80% grains and 60% or 20% white flour, respectively) could decrease the postprandial glucose response as compared to white wheat bread (Holm and Bjorck, 1992; Jenkins et al., 1986; Liljeberg et al., 1992; Liljeberg and Bjorck, 1994). These grains were from barley, rye, and wheat, whereas oat grains were not able to decrease postprandial glycemia (Liljeberg et al., 1992). Substituting white wheat flour by parboiled cracked wheat grains (bulgur, raw weight 67 g per portion) in bread resulted also in decreased glycemia compared to white bread (Jenkins et al., 1986). However, bread in which 85% of flour was substituted with broken and soaked wheat kernels could not decrease glycemia compared to a fiber-rich wheat bread (Eelderink et al., 2016). A more compact food matrix is also expected to decrease starch digestion by hindering amylolysis. Pasta, made from white flour, has the property to reduce postprandial glycemia compared to bread made from the same ingredients (Granfeldt et al., 1992). Pasta, with added wheat bran, however, did not result in lower postprandial glycemia, but lower insulinemia compared to a fiber-rich wheat bread (Eelderink et al., 2012b). This discrepancy between the glucose and insulin response was also observed after intake of certain types of breads. Several rye breads (Juntunen et al., 2002, 2003; Leinonen et al., 1999) and differently processed wheat bread (Rizkalla et al., 2007) elicited lower insulin but similar glucose responses compared to white wheat bread, which seemed to be due to a firmer structure and/or a more rigid texture of these breads. A possible explanation of these observations and their relevance will be discussed below. 3.5.3 Food Composition Several components in food are described that might influence the postprandial glucose response to starch. Fat, for example, has the potency to delay gastric emptying. By this mechanism the reduced glucose response to starch by coingestion of fat can be explained (Collier and O’Dea, 1983; Normand et al., 2001).


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Presence of amylase-inhibiting phytochemicals or plant extracts in food could have the potency to decrease postprandial glycemia. A great number of studies assessed the effect of polyphenol-rich food, like berries, tea, or purple wheat, but results are inconsistent (Coe and Ryan, 2016; Eelderink et al., 2012a). Extracts from white beans are also widely studied and their efficacy depends on the technique with which it is processed and extracted (Obiro et al., 2008). The presence of viscous fibers, like guar gum, psyllium, arabinoxylan, and b-glucan in starchy foods also leads to reduced postprandial glycemia (Boers et al., 2016; El et al., 2012; Gibb et al., 2015; Giulia et al., 2016). This is thought to be due to delayed gastric emptying and a delayed absorption of glucose in the small intestine by a reduction in diffusion rate through the intraluminal bulk phase of the small intestine (Jenkins et al., 1978, 1987).

4. TECHNIQUES FOR MONITORING STARCH DIGESTION Several methods are available to evaluate attempts to lower the rate and extent of starch digestion or the intestinal absorption of starch-derived glucose. These methods vary in invasivity as well as in accuracy.

4.1 In Vitro Approaches 4.1.1 Starch Fractions Starch can be classified as rapidly digestible, slowly digestible, and resistant starch. In vitro approaches have been developed to quantify these starch fractions. The most widely applied approach is developed by Englyst et al. (1992). According to these procedures, using controlled enzymatic hydrolysis with pancreatin and amyloglucosidases, the total amount of starch and the rapidly digestible, slowly digestible, and resistant starch fractions can be measured. The released glucose is analyzed by colorimetry. 4.1.2 Rate of Hydrolysis Granfeldt et al. (1992) published a method to measure the rate of in vitro starch digestion. After the test substrate has been chewed under standardized conditions the starch is incubated with pepsin. The incubate is transferred to a dialysis tube and incubated with pancreatic a-amylase. Samples of the dialysate are taken at different time intervals and the degree of hydrolysis can be calculated.

4.2 In Vivo Approaches 4.2.1 Hydrogen Breath Test Starch that escapes digestion and absorption in the small intestine is fermented in the colon by bacteria to short-chain fatty acids (acetate, propionate, and butyrate) and gases (hydrogen, methane, carbon dioxide). The hydrogen breath


test is not suitable to study the rate of starch digestion but can give an indication whether all starch is digested or not. Hydrogen excretion in breath can be used to mark the arrival of undigested carbohydrates in the colon (Wang et al., 2008).

4.2.2 13C-Labeled Starch 13 C, a stable isotope of carbon, can be used to study the digestion, absorption, and metabolism of carbohydrates in vivo. A stable isotope is an element having the same number of nuclear protons but possessing a different number of nuclear neutrons. Stable isotopes are not radioactive and are harmless for application in humans. 13C-labeled starch or starchy products can be derived from plants which are naturally enriched with 13C (e.g., corn) or obtained by growing cereals under an atmosphere enriched with 13CO2 (Verbeke et al., 2010). 4.2.2.1

13

CO2 Breath Test

After hydrolysis of ingested 13C-labeled starch, 13C-glucose is absorbed, oxidized, and exhaled as 13CO2. 13CO2 is also produced in the colon during fermentation of undigested 13C-enriched starch and is thereafter partly excreted in breath. 13 CO2 in breath samples can be quantified using isotope ratio mass spectrometry (IRMS). The 13CO2 breath test can be used to evaluate the digestion and fermentation of various 13C-starch preparations. Using mathematical models it is possible to distinguish between 13CO2 produced by oxidation of absorbed glucose and that produced by fermentation (Christian et al., 2002). In combination with the results of the hydrogen breath test, it is a noninvasive method to quantitatively assess digestion and fermentation of starch in different food products (Wang et al., 2008). 4.2.2.2

13

C-Enriched Plasma Glucose

13

The C-enrichment of plasma glucose can be analyzed in blood samples that are collected after intake of 13C-labeled starch. Measurement of 13C-glucose in plasma allows more specific study of the bioavailability of starch than does the total blood glucose response (see Fig. 21.1).

4.2.3 Dual Isotope Method By administering two substrates labeled with stable isotopes (D-[6,6-2H2] glucose and 13C-starch), it is possible to estimate the systemic appearance rates of both hepatic and starch-derived (intestinal) glucose. In the dual isotope method D-[6,6-2H2]glucose (deuterium labeled) glucose is infused continuously to be able to determine the rate at which glucose appears in the systemic circulation from exogenous (intestinal) and endogenous (mainly hepatic) sources. After a steady state is reached, in which the deuterium enrichment of the glucose in the systemic circulation stays constant, 13C-labeled


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FIGURE 21.1 Postprandial plasma glucose concentrations (upper panel) and 13C-enrichment of plasma glucose (lower panel) in healthy men after ingestion of 132 g 13C-enriched wheat bread.

carbohydrates are ingested. Due to the insulin response the concentration of deuterium-labeled glucose decreases which enables calculation of insulinstimulated glucose uptake into tissue (see Fig. 21.2). Distinction between endogenous and exogenous glucose is possible based on the 13C enrichment in plasma glucose. Based on the rate of appearance of glucose and the plasma 13 C-glucose concentration fluxes of plasma glucose derived from both endogenous sources and intestine can be calculated. Thus, using the dual isotope method, the intestinal glucose absorption can be quantified.

4.2.4 Ileostomy Model An ileostomy is a surgical created opening from the ileum through the abdominal wall. The main reasons to perform an ileostomy are cancer of the colon or ulcerative colitis. After ingestion of a test meal the small bowel transit time and the unabsorbed starch in the ileostomy effluent can be determined. A disadvantage of this model is the possibility of considerable bacterial overgrowth, increasing the risk of overestimation of the carbohydrate absorption, due to bacterial degradation of the starches.


FIGURE 21.2 Deuterium (H2)-enrichment of postprandial plasma glucose in healthy men after ingestion of 132 g 13C-enriched wheat bread and during a continuous H2-glucose infusion (0.07 mg glucose/kg body weight/min).

4.2.5 Intubation Studies After intestinal intubation with a triple lumen tube, ileal contents can be aspirated. Proximal to the aspiration port a recovery marker is perfused at a determined concentration rate. A meal marker is used to confirm that the total test meal is passed through the ileum. Based on the dilution of the recovery marker in the aspirated sample, the flow rate through the ileum is calculated. The perfusion-aspiration method allows assessment of nutrients flowing through the ileum. It is, however, a very invasive method. Another disadvantage is the disturbance of the normal gastrointestinal functioning, like gastrointestinal motility.

5. CURRENT (NUTRITIONAL) STRATEGIES TO PREVENT POSTPRANDIAL HYPERGLYCEMIA 5.1 Administration of a-Glucosidase Inhibitors 5.1.1 Patients With T2DM Alpha-glucosidase inhibitors are antidiabetic drugs acting by competitively inhibiting a-glucosidases. These a-glucosidase inhibitors delay carbohydrate digestion and therefore the postprandial glucose absorption resulting in an attenuation of postprandial plasma glucose and insulin levels in healthy volunteers and diabetic patients. Acarbose is the most extensively studied and most widely used a-glucosidase inhibitor. The main indication of acarbose is the treatment of T2DM. Several controlled studies have demonstrated the beneficial effects of acarbose in T2DM (for an overview see Scheen, 2003; Van de Laar et al., 2005). Application of acarbose induces a reduction of HbA1c level by 0.2%e1.4% (Scheen, 2003) or a mean of 0.8% (meta-analysis, Van de Laar et al., 2005) in comparison with a placebo. The most common


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adverse side effects of acarbose are gastrointestinal disturbances, like flatulence, diarrhea, and abdominal distension (Balfour and McTavish, 1993).

5.1.2 Persons With Prediabetes Application of a-glucosidase inhibitors reduces the progression to T2DM in persons with prediabetes by 22% or 60% compared to placebo or diet and exercise respectively (Van de Laar et al., 2006). However, for the authors it is not clear whether this can be interpreted as prevention, delay, or masking of diabetes (Van de Laar et al., 2006). Application of acarbose is also able to reduce the occurrence of major cardiovascular events in prediabetics (Chiasson et al., 2003). To date other pharmacological or lifestyle interventions showed reduction of risk factors for CVD but could not demonstrate reduction of cardiovascular events (Faerch et al., 2014). The mechanism by which acarbose may reduce the progression to T2DM or cardiovascular effects is largely unknown. Thus, acarbose, a therapeutic agent for diabetes, seems to be of benefit in persons with prediabetes by reducing the risk of developing T2DM and cardiovascular disease. However, the high costs and the adverse side effects could limit the large-scale applicability of acarbose (Scheen, 2003). Furthermore, it is debatable whether drug therapy is preferable to lifestyle intervention. Therefore, it seems necessary to facilitate adherence to a diet that restricts the occurrence of episodes with postprandial hyperglycemia as much as possible.

5.2 Use of the Glycemic Index The glycemic index (GI) concept has been introduced by Jenkins et al. (1981) to enable comparison of foods based on their glycemic response. The GI is defined as the incremental area under the blood glucose response curve of a 50 g carbohydrate portion of a test food expressed as percent of the response to the same amount of carbohydrate from a reference food taken by the same subject (Wolever et al., 1991). However, there is some disagreement about the usefulness of the application of the GI for a “healthy” food choice in the prevention or treatment of T2DM. In 1997, an expert consultation from the Food and Agriculture Organisation (FAO) and the World Health Organisation (WHO) about the importance of dietary carbohydrates in human nutrition and health was published (Food and Agriculture Organization of the United Nations/World Health Organization, 1998). In this report the use of the GI to decide better food choices is recommended, particularly for those people who have problems controlling blood glucose levels. This was in general confirmed by a scientific update in 2007 (Mann et al., 2007). This statement is also in agreement with the opinion of The European Association for the Study of Diabetes which promotes the use of low-GI food as a tool, among many, that can be used in the management of diabetes (Mann et al., 2004).


In contrast, the GI concept is not supported by the ADA (American Diabetes Association) for the treatment of T2DM (Evert et al., 2014). This organization regards the total amount of carbohydrates in the diet and the available insulin as more important factors influencing the glycemic response than the source or type of carbohydrates (Evert et al., 2014). Even though the GI concept is supported by many organizations, it is recognized that putting it into practice requires some attention. One aspect is that by choosing carbohydrate foods not only the GI should be considered, but also the overall nutritional composition of the product. Consumption of low-GI products high in fat is not recommended (Mann et al., 2007). Furthermore, it is advised that the GI is used to compare products of similar composition within food groups, like various types of fruit or various cereal products (Mann et al., 2004). Another aspect is the availability of a sufficient large choice of low-GI alternatives for high-GI products to ensure dietary variety. The need to develop low-GI products with a high-fiber content and low in energy is therefore stressed by the FAO/WHO (Food and Agriculture Organization of the United Nations/World Health Organization, 1998).

5.3 The Glycemic Index and Slowly Available Starch Generally it is assumed that a low-postprandial blood glucose response or low GI is reflecting the slow release characteristic of starch in food. However, there are several stable isotope studies which indicate that this is not necessarily the case.

5.3.1 High-Postprandial Glycemia Despite Slow Appearance of Starch-Derived Glucose In a dual isotope study, glucose kinetics after ingestion of 13C-labeled fiberrich wheat pasta and bread (Eelderink et al., 2012a,b) was explored. The cross-over study in 10 healthy males showed that total blood glucose after both products was the same, despite the fact that the rate of appearance of exogenous glucose was lower after pasta than after bread. The endogenous glucose production was slightly more suppressed after pasta than after bread and could therefore not explain the difference in glycemia. However, after pasta the uptake of glucose into tissue was much slower than after wheat bread and by that “counterbalanced” the slow rate of appearance of exogenous glucose resulting in the similar glycemic response. The low-glucose uptake which led to the same total glucose response despite slower rate of exogenous glucose appearance was caused by a lower insulin response (Eelderink et al., 2012b). This lower insulin response was accompanied by a lower GIP response and it seems plausible that the low-GIP concentrations are responsible for the low insulin response, as GIP is known to potentiate glucose-induced insulin release (Eelderink et al., 2012b). In dual isotope studies consistently strong correlations between the rate of appearance


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of starch-derived glucose and GIP concentration were observed (Eelderink et al., 2012b, 2015, 2016; Peronnet et al., 2015; Wachters-Hagedoorn et al., 2006) which is in accordance with findings of duodenal glucose perfusion studies (Schirra et al., 1996). This means that due to the low GIP and insulin response the slow-release characteristics of starch can be masked.

5.3.2 Low-Postprandial Glycemia Despite Rapid Appearance of Starch-Derived Glucose In a single label study, Schenk et al. (2003) explored the glucose fluxes of two breakfast cereals with different GI and wheat fiber content: corn flakes (GI 131.5 33.0, 1.6 g total fiber/portion) and bran flakes (GI: 54.5 7.2, 38.5 g total fiber/portion) in a cross-over manner in six healthy males. Unexpectedly, they did not find a difference in the rate of appearance of total glucose but an early increase of the rate of disappearance of total glucose after bran flakes, explaining the difference in total blood glucose response. The 180-min areas under the curves of insulin were not different but an early (t ¼ 0e20 min) and marked insulin response after the bran flakes was observed which strongly correlated with this early increase in glucose disappearance. This effect was hypothesized to be due to the higher wheat protein content of bran flakes (15.4 vs. 4.3 g). Lower glycemia has also been demonstrated to be caused by increased glucose disposal in an overnight study. In a dual isotope study, 10 healthy males received barley kernels or white wheat bread as evening meal and 13Clabeled glucose (50 g) as standard breakfast (Priebe et al., 2010). The plasma glucose response after the glucose load in the morning was 29% lower after the barley evening meal. The endogenous glucose production and the insulin response was the same, whereas the uptake of glucose into the tissue was 30% higher after the barley evening meal, indicating higher peripheral insulin sensitivity. Hydrogen excretion in the breath, an indicator of colonic carbohydrate fermentation, as well as plasma butyrate concentrations were higher in the morning after the barley evening meal indicating that colonic fermentation might be involved in this overnight second meal effect (Priebe et al., 2010). This means that other factors than the digestive starch characteristics can determine postprandial glycemia. In addition, starchy products with a high GI could be a more heterogeneous group as generally assumed, comprising also products with slowly available starch. Furthermore, due to the difference in postprandial glucose kinetics, these products have differential effects on other metabolic parameters, like the insulin and GIP response.

5.4 Addition of Dietary Fiber As described above, soluble dietary fiber can delay gastric emptying and hinder absorption of glucose in the small intestine. Indeed, a number of acute studies demonstrated that foods enriched with soluble fiber are able to


decrease postprandial glucose response in normo- and hyperglycemic subjects. The most-studied soluble fiber are guar gum, a gel-forming galactomannan, psyllium, which is also called ispaghula husk (Gibb et al., 2015; Sierra et al., 2001, 2002) and b-glucans, which are mostly derived from barley or oats (Alminger and Eklund-Jonsson, 2008; El et al., 2012; Juntunen et al., 2002; Tapola et al., 2005). Recently, the glucose-lowering potential of arabinoxylan, isolated from the endosperm of wheat kernels, has been demonstrated (Garcia et al., 2007; Giulia et al., 2016; Hartvigsen et al., 2014a,b; Lu et al., 2000). Several clinical studies investigated the long-term effect of soluble fiber on parameters related to glycemic control in patients with T2DM. A study of Chandalia et al. (2000) showed improvement of 24-h glucose and insulin concentrations after 6 weeks of a high-fiber diet (25 g insoluble and 25 g soluble fiber). Furthermore, 1 month consumption of 20 g insoluble fiber (unspecified) ameliorated fasting and 2-h glucose as well as insulin concentrations (Chen et al., 2016). Gibb et al. (2015) summarized the results of trials investigating the addition of psyllium to the diet and found improved fasting blood glucose concentrations and improved HbA1c in T2DM patients. Psyllium was ingested before 2e3 meals per day and the amount varied between 3.5 and 5.1 g per meal. In addition, beneficial effects were observed in persons with prediabetes, but they were less pronounced than in diabetes patients (Gibb et al., 2015).

6. FUTURE TRENDS 6.1 Extending the Choice of Foods With Slowly Available Starch As discussed in the previous sections, the choice of foods with slowly available carbohydrates is limited. Extending the range of those products would increase the dietary variety for patients with T2DM and persons with prediabetes. There are different strategies to manipulate the rate of starch digestion or the intestinal absorption of starch-derived glucose. One approach is to adapt the manufacturing process in such a way that gelatinization of the starch granules is limited. Two dual isotope studies have shown the potency of this processing method to reduce the rate of appearance of starch-derived glucose as well as postprandial glucose by manufacturing biscuits with slowly available starch and comparing them with extruded cereals (Peronnet et al., 2015; Vinoy et al., 2013). It is of interest to investigate the applicability of this manufacturing process in other starchy food products. Another challenging approach would be the addition of food-derived a-glucosidase inhibitors to starchy foods. Here, more knowledge is needed as to the appropriate dose, the bioavailability and the stability of the compounds during processing or extraction (Coe and Ryan, 2016; Obiro et al., 2008).


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6.2 Characterization of Slowly Available Starch The GI is a suitable indicator of the blood glucose raising potential of a given food, but it does not always provide information with regard to the digestive characteristics of starch, which is needed to evaluate strategies to manipulate starch digestion and/or absorption of starch-derived glucose. As discussed above, dual isotope studies have shown that it is possible that products with slowly available starch elicit relative high-postprandial glycemia. However, in these cases, the postprandial insulin and GIP response is reduced (Eelderink et al., 2012b, 2015), suggesting that these parameters are more sensitive to reflect more subtle differences in availability of starch.

6.3 Stimulating Secretion of Gastrointestinal Hormones Advances in the understanding of the complex regulating mechanisms of the secretion of gastrointestinal hormones may help to identify novel targets to manipulate the digestion and absorption of carbohydrates. Ingested nutrients or nutrient-derived compounds are, among other factors, involved in the regulating mechanism of the secretion of gastrointestinal hormones. GLP-1 for example would be an interesting target because of its retarding effect on gastric emptying. So far, it has been demonstrated that the GLP-1 response can be stimulated by the presence of carbohydrates in the distal small intestine. However, the effect of sugars or starch in a complex food matrix is less clear. In addition, the GLP-1-stimulating potency of short-chain fatty acids is interesting and should be explored in humans. Perhaps, in the future, it will be possible to develop functional foods that stimulate the secretion of specific gastrointestinal hormones resulting in a more gradually intestinal digestion and absorption of carbohydrates.

REFERENCES Alminger, M., Eklund-Jonsson, C., 2008. Whole-grain cereal products based on a high-fibre barley or oat genotype lower post-prandial glucose and insulin responses in healthy humans. European Journal of Nutrition 47, 294e300. Balfour, J.A., McTavish, D., 1993. Acarbose. An update of its pharmacology and therapeutic use in diabetes mellitus. Drugs 46, 1025e1054. Barrett, M.L., Udani, J.K., 2011. A proprietary alpha-amylase inhibitor from white bean (Phaseolus vulgaris): a review of clinical studies on weight loss and glycemic control. Nutrition Journal 10, 24. Biliaderis, C.G., 1991. The structure and interactions of starch with food constituents. Canadian Journal of Physiology and Pharmacology 69, 60e78. Bjorck, I., Granfeldt, Y., Liljeberg, H., Tovar, J., Asp, N.G., 1994. Food properties affecting the digestion and absorption of carbohydrates. The American Journal of Clinical Nutrition 59, 699Se705S. Blaak, E.E., Antoine, J.M., Benton, D., Bjorck, I., Bozzetto, L., Brouns, F., et al., 2012. Impact of postprandial glycaemia on health and prevention of disease. Obesity Reviews 13, 923e984. Bodinham, C.L., Al-Mana, N.M., Smith, L., Robertson, M.D., 2013. Endogenous plasma glucagon-like peptide-1 following acute dietary fibre consumption. The British Journal of Nutrition 110, 1429e1433.

822 PART j FOUR Starch and Health Boers, H.M., MacAulay, K., Murray, P., Seijen Ten, H.J., Hoogenraad, A.R., Peters, H.P., et al., 2016. Efficacy of different fibres and flour mixes in South-Asian flatbreads for reducing postprandial glucose responses in healthy adults. European Journal of Nutrition. Brownlee, M., 2001. Biochemistry and molecular cell biology of diabetic complications. Nature 414, 813e820. Carr, R.D., Larsen, M.O., Winzell, M.S., Jelic, K., Lindgren, O., Deacon, C.F., et al., 2008. Incretin and islet hormonal responses to fat and protein ingestion in healthy men. American Journal of Physiology. Endocrinology and Metabolism 295, E779eE784. Caspary, W.F., 1992. Physiology and pathophysiology of intestinal absorption. The American Journal of Clinical Nutrition 55, S299eS308. Chandalia, M., Garg, A., Lutjohann, D., von, B.K., Grundy, S.M., Brinkley, L.J., 2000. Beneficial effects of high dietary fiber intake in patients with type 2 diabetes mellitus. The New England Journal of Medicine 342, 1392e1398. Chapman, R.W., Sillery, J.K., Graham, M.M., Saunders, D.R., 1985. Absorption of starch by healthy ileostomates: effect of transit time and of carbohydrate load. The American Journal of Clinical Nutrition 41, 1244e1248. Chen, C., Zeng, Y., Xu, J., Zheng, H., Liu, J., Fan, R., et al., 2016. Therapeutic effects of soluble dietary fiber consumption on type 2 diabetes mellitus. Experimental and Therapeutic Medicine 12, 1232e1242. Chiasson, J.L., Josse, R.G., Gomis, R., Hanefeld, M., Karasik, A., Laakso, M., 2003. Acarbose treatment and the risk of cardiovascular disease and hypertension in patients with impaired glucose tolerance: the STOP-NIDDM trial. JAMA 290, 486e494. Christian, M.T., Amarri, S., Franchini, F., Preston, T., Morrison, D.J., Dodson, B., et al., 2002. Modeling 13C breath curves to determine site and extent of starch digestion and fermentation in infants. Journal of Pediatric Gastroenterology and Nutrition 34, 158e164. Coe, S., Ryan, L., 2016. Impact of polyphenol-rich sources on acute postprandial glycaemia: a systematic review. Journal of Nutritional Science 5, e24. Collier, G., O’Dea, K., 1983. The effect of coingestion of fat on the glucose, insulin, and gastric inhibitory polypeptide responses to carbohydrate and protein. The American Journal of Clinical Nutrition 37, 941e944. Cooper, J.A., 2014. Factors affecting circulating levels of peptide YY in humans: a comprehensive review. Nutrition Research Reviews 27, 186e197. Cummings, J.H., Englyst, H.N., 1995. Gastrointestinal effects of food carbohydrate. The American Journal of Clinical Nutrition 61, S938eS945. Deacon, C.F., Ahren, B., 2011. Physiology of incretins in health and disease. The Review of Diabetic Studies 8, 293e306. Dikeman, C.L., Fahey, G.C., 2006. Viscosity as related to dietary fiber: a review. Critical Reviews in Food Science and Nutrition 46, 649e663. DiNicolantonio, J.J., Bhutani, J., O’Keefe, J.H., 2015. Acarbose: safe and effective for lowering postprandial hyperglycaemia and improving cardiovascular outcomes. Open Heart 2, e000327. Eelderink, C., Moerdijk-Poortvliet, T.C., Wang, H., Schepers, M., Preston, T., Boer, T., et al., 2012a. The glycemic response does not reflect the in vivo starch digestibility of fiber-rich wheat products in healthy men. The Journal of Nutrition 142, 258e263. Eelderink, C., Noort, M.W., Sozer, N., Koehorst, M., Holst, J.J., Deacon, C.F., et al., 2015. The structure of wheat bread influences the postprandial metabolic response in healthy men. Food and Function 6, 3236e3248. Eelderink, C., Noort, M.W., Sozer, N., Koehorst, M., Holst, J.J., Deacon, C.F., et al., 2016. Difference in postprandial GLP-1 response despite similar glucose kinetics after consumption of wheat breads with different particle size in healthy men. European Journal of Nutrition.


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Eelderink, C., Schepers, M., Preston, T., Vonk, R.J., Oudhuis, L., Priebe, M.G., 2012b. Slowly and rapidly digestible starchy foods can elicit a similar glycemic response because of differential tissue glucose uptake in healthy men. The American Journal of Clinical Nutrition 96, 1017e1024. El, K.D., Cuda, C., Luhovyy, B.L., Anderson, G.H., 2012. Beta glucan: health benefits in obesity and metabolic syndrome. Journal of Nutrition and Metabolism 2012, 851362. Englyst, H.N., Kingman, S.M., Cummings, J.H., 1992. Classification and measurement of nutritionally important starch fractions. European Journal of Clinical Nutrition 46, S33eS50. Evert, A.B., Boucher, J.L., Cypress, M., Dunbar, S.A., Franz, M.J., Mayer-Davis, E.J., et al., 2014. Nutrition therapy recommendations for the management of adults with diabetes. Diabetes Care 37 (Suppl. 1), S120eS143. Faerch, K., Vistisen, D., Johansen, N.B., Jorgensen, M.E., 2014. Cardiovascular risk stratification and management in pre-diabetes. Current Diabetes Reports 14, 493. Food and Agriculture Organization of the United Nations/World Health Organization, 1998. Carbohydrates in Human Nutrition. FAO/WHO Expert Consultation. Food and Agriculture Organization of the United Nations, Geneva. Garcia, A.L., Otto, B., Reich, S.C., Weickert, M.O., Steiniger, J., Machowetz, A., et al., 2007. Arabinoxylan consumption decreases postprandial serum glucose, serum insulin and plasma total ghrelin response in subjects with impaired glucose tolerance. European Journal of Clinical Nutrition 61, 334e341. Giacco, F., Brownlee, M., 2010. Oxidative stress and diabetic complications. Circulation Research 107, 1058e1070. Gibb, R.D., McRorie Jr., J.W., Russell, D.A., Hasselblad, V., D’Alessio, D.A., 2015. Psyllium fiber improves glycemic control proportional to loss of glycemic control: a meta-analysis of data in euglycemic subjects, patients at risk of type 2 diabetes mellitus, and patients being treated for type 2 diabetes mellitus. The American Journal of Clinical Nutrition 102, 1604e1614. Giulia, F.A., Grecchi, I., Muggia, C., Palladini, G., Perlini, S., 2016. Effects of a bioavailable arabinoxylan-enriched white bread flour on postprandial glucose response in normoglycemic subjects. Journal of Dietary Supplements 13, 626e633. Granfeldt, Y., Bjorck, I., Drews, A., Tovar, J., 1992. An in vitro procedure based on chewing to predict metabolic response to starch in cereal and legume products. European Journal of Clinical Nutrition 46, 649e660. Gribble, F.M., 2012. The gut endocrine system as a coordinator of postprandial nutrient homoeostasis. The Proceedings of the Nutrition Society 71, 456e462. Harris, M.I., Klein, R., Welborn, T.A., Knuiman, M.W., 1992. Onset of NIDDM occurs at least 4e7 yr before clinical diagnosis. Diabetes Care 15, 815e819. Hartvigsen, M.L., Gregersen, S., Laerke, H.N., Holst, J.J., Bach Knudsen, K.E., Hermansen, K., 2014a. Effects of concentrated arabinoxylan and beta-glucan compared with refined wheat and whole grain rye on glucose and appetite in subjects with the metabolic syndrome: a randomized study. European Journal of Clinical Nutrition 68, 84e90. Hartvigsen, M.L., Laerke, H.N., Overgaard, A., Holst, J.J., Bach Knudsen, K.E., Hermansen, K., 2014b. Postprandial effects of test meals including concentrated arabinoxylan and whole grain rye in subjects with the metabolic syndrome: a randomised study. European Journal of Clinical Nutrition 68, 567e574. Holm, J., Bjorck, I., 1992. Bioavailability of starch in various wheat-based bread products: evaluation of metabolic responses in healthy subjects and rate and extent of in vitro starch digestion. The American Journal of Clinical Nutrition 55, 420e429.

824 PART j FOUR Starch and Health Huge, A., Weber, E., Ehrlein, H.J., 1995. Effects of enteral feedback inhibition on motility, luminal flow, and absorption of nutrients in proximal gut of minipigs. Digestive Diseases and Sciences 40, 1024e1034. Jenkins, D.J., Jenkins, A.L., Wolever, T.M., Collier, G.R., Rao, A.V., Thompson, L.U., 1987. Starchy foods and fiber: reduced rate of digestion and improved carbohydrate metabolism. Scandinavian Journal of Gastroenterology. Supplement 129, 132e141. Jenkins, D.J., Wolever, T.M., Jenkins, A.L., Giordano, C., Giudici, S., Thompson, L.U., et al., 1986. Low glycemic response to traditionally processed wheat and rye products: bulgur and pumpernickel bread. The American Journal of Clinical Nutrition 43, 516e520. Jenkins, D.J., Wolever, T.M., Leeds, A.R., Gassull, M.A., Haisman, P., Dilawari, J., et al., 1978. Dietary fibres, fibre analogues, and glucose tolerance: importance of viscosity. British Medical Journal 1, 1392e1394. Jenkins, D.J., Wolever, T.M., Taylor, R.H., Barker, H., Fielden, H., Baldwin, J.M., et al., 1981. Glycemic index of foods: a physiological basis for carbohydrate exchange. The American Journal of Clinical Nutrition 34, 362e366. Juntunen, K.S., Laaksonen, D.E., Autio, K., Niskanen, L.K., Holst, J.J., Savolainen, K.E., et al., 2003. Structural differences between rye and wheat breads but not total fiber content may explain the lower postprandial insulin response to rye bread. The American Journal of Clinical Nutrition 78, 957e964. Juntunen, K.S., Niskanen, L.K., Liukkonen, K.H., Poutanen, K.S., Holst, J.J., Mykkanen, H.M., 2002. Postprandial glucose, insulin, and incretin responses to grain products in healthy subjects. The American Journal of Clinical Nutrition 75, 254e262. Kaji, I., Karaki, S., Kuwahara, A., 2014. Short-chain fatty acid receptor and its contribution to glucagon-like peptide-1 release. Digestion 89, 31e36. Karhunen, L.J., Juvonen, K.R., Flander, S.M., Liukkonen, K.H., Lahteenmaki, L., Siloaho, M., et al., 2010. A psyllium fiber-enriched meal strongly attenuates postprandial gastrointestinal peptide release in healthy young adults. The Journal of Nutrition 140, 737e744. Klosterbuer, A.S., Thomas, W., Slavin, J.L., 2012. Resistant starch and pullulan reduce postprandial glucose, insulin, and GLP-1, but have no effect on satiety in healthy humans. Journal of Agricultural and Food Chemistry 60, 11928e11934. Leinonen, K., Liukkonen, K., Poutanen, K., Uusitupa, M., Mykkanen, H., 1999. Rye bread decreases postprandial insulin response but does not alter glucose response in healthy Finnish subjects. European Journal of Clinical Nutrition 53, 262e267. Liljeberg, H., Bjorck, I., 1994. Bioavailability of starch in bread products. Postprandial glucose and insulin responses in healthy subjects and in vitro resistant starch content. European Journal of Clinical Nutrition 48, 151e163. Liljeberg, H., Granfeldt, Y., Bjorck, I., 1992. Metabolic responses to starch in bread containing intact kernels versus milled flour. European Journal of Clinical Nutrition 46, 561e575. Lin, A.H., Lee, B.H., Nichols, B.L., Quezada-Calvillo, R., Rose, D.R., Naim, H.Y., et al., 2012. Starch source influences dietary glucose generation at the mucosal alpha-glucosidase level. The Journal of Biological Chemistry 287, 36917e36921. Little, T.J., Doran, S., Meyer, J.H., Smout, A.J., O’Donovan, D.G., Wu, K.L., et al., 2006. The release of GLP-1 and ghrelin, but not GIP and CCK, by glucose is dependent upon the length of small intestine exposed. American Journal of Physiology. Endocrinology and Metabolism 291, E647eE655. Lu, Z.X., Walker, K.Z., Muir, J.G., Mascara, T., O’Dea, K., 2000. Arabinoxylan fiber, a byproduct of wheat flour processing, reduces the postprandial glucose response in normoglycemic subjects. The American Journal of Clinical Nutrition 71, 1123e1128.


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Mann, J., Cummings, J.H., Englyst, H.N., Key, T., Liu, S., Riccardi, G., et al., 2007. FAO/WHO scientific update on carbohydrates in human nutrition: conclusions. European Journal of Clinical Nutrition 61 (Suppl. 1), S132eS137. Mann, J.I., De, L.I., Hermansen, K., Karamanos, B., Karlstrom, B., Katsilambros, N., et al., 2004. Evidence-based nutritional approaches to the treatment and prevention of diabetes mellitus. Nutrition, Metabolism, and Cardiovascular Diseases 14, 373e394. McIntyre, A., Vincent, R.M., Perkins, A.C., Spiller, R.C., 1997. Effect of bran, ispaghula, and inert plastic particles on gastric emptying and small bowel transit in humans: the role of physical factors. Gut 40, 223e227. Menke, A., Casagrande, S., Geiss, L., Cowie, C.C., 2015. Prevalence of and trends in diabetes among adults in the United States, 1988e2012. JAMA 314, 1021e1029. Normand, S., Khalfallah, Y., Louche-Pelissier, C., Pachiaudi, C., Antoine, J.M., Blanc, S., et al., 2001. Influence of dietary fat on postprandial glucose metabolism (exogenous and endogenous) using intrinsically 13C-enriched durum wheat. The British Journal of Nutrition 86, 3e11. Obiro, W.C., Zhang, T., Jiang, B., 2008. The nutraceutical role of the Phaseolus vulgaris alphaamylase inhibitor. The British Journal of Nutrition 100, 1e12. Peronnet, F., Meynier, A., Sauvinet, V., Normand, S., Bourdon, E., Mignault, D., et al., 2015. Plasma glucose kinetics and response of insulin and GIP following a cereal breakfast in female subjects: effect of starch digestibility. European Journal of Clinical Nutrition 69, 740e745. Priebe, M.G., Wang, H., Weening, D., Schepers, M., Preston, T., Vonk, R.J., 2010. Factors related to colonic fermentation of nondigestible carbohydrates of a previous evening meal increase tissue glucose uptake and moderate glucose-associated inflammation. The American Journal of Clinical Nutrition 91, 90e97. Qualmann, C., Nauck, M.A., Holst, J.J., Orskov, C., Creutzfeldt, W., 1995. Glucagon-like peptide 1 (7-36 amide) secretion in response to luminal sucrose from the upper and lower gut. A study using alpha-glucosidase inhibition (acarbose). Scandinavian Journal of Gastroenterology 30, 892e896. Rizkalla, S.W., Laromiguiere, M., Champ, M., Bruzzo, F., Boillot, J., Slama, G., 2007. Effect of baking process on postprandial metabolic consequences: randomized trials in normal and type 2 diabetic subjects. European Journal of Clinical Nutrition 61, 175e183. Scheen, A.J., 2003. Is there a role for alpha-glucosidase inhibitors in the prevention of type 2 diabetes mellitus? Drugs 63, 933e951. Schenk, S., Davidson, C.J., Zderic, T.W., Byerley, L.O., Coyle, E.F., 2003. Different glycemic indexes of breakfast cereals are not due to glucose entry into blood but to glucose removal by tissue. The American Journal of Clinical Nutrition 78, 742e748. Schirra, J., Katschinski, M., Weidmann, C., Schafer, T., Wank, U., Arnold, R., et al., 1996. Gastric emptying and release of incretin hormones after glucose ingestion in humans. Journal of Clinical Investigation 97, 92e103. Seifarth, C., Bergmann, J., Holst, J.J., Ritzel, R., Schmiegel, W., Nauck, M.A., 1998. Prolonged and enhanced secretion of glucagon-like peptide 1 (7-36 amide) after oral sucrose due to alpha-glucosidase inhibition (acarbose) in type 2 diabetic patients. Diabetic Medicine 15, 485e491. Sierra, M., Garcia, J.J., Fernandez, N., Diez, M.J., Calle, A.P., 2002. Therapeutic effects of psyllium in type 2 diabetic patients. European Journal of Clinical Nutrition 56, 830e842. Sierra, M., Garcia, J.J., Fernandez, N., Diez, M.J., Calle, A.P., Sahagun, A.M., 2001. Effects of ispaghula husk and guar gum on postprandial glucose and insulin concentrations in healthy subjects. European Journal of Clinical Nutrition 55, 235e243.

826 PART j FOUR Starch and Health Tapola, N., Karvonen, H., Niskanen, L., Mikola, M., Sarkkinen, E., 2005. Glycemic responses of oat bran products in type 2 diabetic patients. Nutrition, Metabolism, and Cardiovascular Diseases 15, 255e261. Theodorakis, M.J., Carlson, O., Michopoulos, S., Doyle, M.E., Juhaszova, M., Petraki, K., et al., 2006. Human duodenal enteroendocrine cells: source of both incretin peptides, GLP-1 and GIP. American Journal of Physiology. Endocrinology and Metabolism 290, E550eE559. Thomas, R.P., Hellmich, M.R., Townsend Jr., C.M., Evers, B.M., 2003. Role of gastrointestinal hormones in the proliferation of normal and neoplastic tissues. Endocrine Reviews 24, 571e599. Valensi, P., Schwarz, E.H., Hall, M., Felton, A.M., Maldonato, A., Mathieu, C., 2005. Pre-diabetes essential action: a European perspective. Diabetes and Metabolism 31, 606e620. Van de Laar, F.A., Lucassen, P.L., Akkermans, R.P., Van de Lisdonk, E.H., De Grauw, W.J., 2006. Alpha-glucosidase inhibitors for people with impaired glucose tolerance or impaired fasting blood glucose. The Cochrane Database of Systematic Reviews CD005061. Van de Laar, F.A., Lucassen, P.L., Akkermans, R.P., Van de Lisdonk, E.H., Rutten, G.E., van Weel, C., 2005. Alpha-glucosidase inhibitors for patients with type 2 diabetes: results from a Cochrane systematic review and meta-analysis. Diabetes Care 28, 154e163. Verbeke, K., Ferchaud-Roucher, V., Preston, T., Small, A.C., Henckaerts, L., Krempf, M., et al., 2010. Influence of the type of indigestible carbohydrate on plasma and urine short-chain fatty acid profiles in healthy human volunteers. European Journal of Clinical Nutrition 64, 678e684. Vinoy, S., Normand, S., Meynier, A., Sothier, M., Louche-Pelissier, C., Peyrat, J., et al., 2013. Cereal processing influences postprandial glucose metabolism as well as the GI effect. Journal of the American College of Nutrition 32, 79e91. Wachters-Hagedoorn, R.E., Priebe, M.G., Heimweg, J.A., Heiner, A.M., Englyst, K.N., Holst, J.J., et al., 2006. The rate of intestinal glucose absorption is correlated with plasma glucosedependent insulinotropic polypeptide concentrations in healthy men. The Journal of Nutrition 136, 1511e1516. Wang, H., Weening, D., Jonkers, E., Boer, T., Stellaard, F., Small, A.C., et al., 2008. A curve fitting approach to estimate the extent of fermentation of indigestible carbohydrates. European Journal of Clinical Investigation 38, 863e868. Wolever, T.M., Jenkins, D.J., Jenkins, A.L., Josse, R.G., 1991. The glycemic index: methodology and clinical implications. The American Journal of Clinical Nutrition 54, 846e854. Zietek, T., Daniel, H., 2015. Intestinal nutrient sensing and blood glucose control. Current Opinion in Clinical Nutrition and Metabolic Care 18, 381e388.

Chapter 22

Development of Foods High in Slowly Digestible and Resistant Starch Luis A. Bello-Perez, Javier D. Hoyos-Leyva Instituto Politećnico Nacional, Yautepec, Mexico

1. INTRODUCTION Glycemic index (GI) is a variable related to nutritional quality of foods. Differences in glycemic and insulinemic responses to dietary starch are related to the starch fractions digestion. Recently, development of starchy functional ingredients and foods are trends in carbohydrate research. Slowly digestible (SDS) and resistant starch (RS) are two fractions of starch digestion described around 25 years ago (Englyst et al., 1992). These fractions have been widely studied due to its benefits on human health. Both were involved in a concept named “nutraceutical starch.” Some researchers reported SDS uses for control and prevention of hyperglycemic diseases such as type 2 diabetes, obesity, cardiovascular diseases (Cook et al., 2003; Giugliano et al., 2006; Hu et al., 2004; Jenkins et al., 2002; Zhang and Hamaker, 2009). RS is neither digested nor absorbed in the small intestine, with low caloric supply; RS is fermented in the large bowel with production of short-chain fatty acids, which are related to colon cancer prevention, decreased cholesterol production, inhibited fat accumulation, and increased absorption of minerals (Annison and Topping, 1994). Currently, quantification of starch fractions (rapidly digestible starch (RDS), SDS, and RS) is an issue under discussion because diverse research works are based on measuring the rate of starch hydrolysis and others on prediction of glycemic index (pGI); however, Englyst’s test (1992) is the common method for quantification of RDS, SDS, and RS fractions. Englyst’s method is based on the physiological processes occurring in the hydrolysis, degradation, and assimilation of starch by digestive enzymes, mainly in the small intestine, where higher starch hydrolysis occurs by the action of both a-amylase and amyloglucosidase enzymes. The validation of these Starch in Food. http://dx.doi.org/10.1016/B978-0-08-100868-3.00022-6 Copyright © 2018 Elsevier Ltd. All rights reserved.

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quantification methods has sought scientific basis by interlaboratory studies as well as in vivo studies, which has ratified the reported values from the use of in vitro methods. The findings of the health beneficial effects of SDS are supporting to produce starches and foods with high SDS content, which should be analyzed using Englyst’s test. Perhaps in the future, these starch fractions could be used to nutritional claims proposal. Until today, RS is included in the total dietary fiber content, and perhaps in the future, RS content should be determined and included in the claims. The production of SDS and RS include starch isolation and thereafter starch modification. Native starch from different botanical sources presents specific molecular structure, organization, and composition (amylose/amylopectin ratio); these characteristics are important in starch modification to produce SDS and RS. Different botanical sources and modifications (chemical, physical, enzymatic, and combinations of these) have been investigated to obtain high amounts of SDS and RS fractions. Native starches from potato, unripe banana, and plantains are resistant to digestion in the upper intestine and they are fermented in the colon. However, cooking of those native starches decrease SDS and RS fractions with the concomitant increase in RDS (Zhang et al., 2006a,b). In this sense, there is interest to modify starch with the objective to maintain high SDS and RS content even after cooking, and they can be used in the formulation of diverse foods. Processing of starchy foods, such as bakery products, snacks, breakfast cereals, pasta, can modify the starch structure in the food matrix and change its digestibility. This is other interest area in the food industry.

2. MOLECULAR AND PHYSICOCHEMICAL CHARACTERISTICS OF FUNCTIONAL STARCHES Starch is organized in a granular structure of glycosidic chains named amylose and amylopectin. Amylose is linear glucose chains, while amylopectin is highbranched chains. SDS and RS are related to the size of the starch granules and its surface area (Colonna et al., 1992); granules of potato starch, with a large surface area and a smooth surface, coupled with their specific supramolecular properties, explaining its resistance to enzymatic digestion. Amylose/amylopectin also has an important role in the starch digestibility, since the presence of a-1,6 bonds decrease enzymatic hydrolysis (Park and Rollings, 1994); also, high-amylose starches are resistant to the hydrolysis by digestive enzymes due to the low water absorption, producing low gelatinization when are used in cooked low-moisture foods (Brumovsky and Thompson, 2001; Hasjim et al., 2010; Jane, 2006). The crystalline arrangement of type A and/or B has a great influence on starch digestibility, as these types of crystals differ between the arrangements of the double helix of linear chains of amylopectin and water content. The A-type arrangement is more susceptible to enzymatic hydrolysis because they have higher amount of short chains than the B-type that shows

Development of Foods High in SDS and RS Chapter j 22

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higher amount of long chains. The chain-length distribution of starches is related to enzymatic susceptibility due to which B-type presents higher levels of RS than A-type (Jane et al., 1997). Marsden and Gray (1985) reported that the hydrolysis with a-amylase occurs predominantly in the amorphous regions of the granule. When starch is gelatinized a disarrangement of the starch components in the lattice occurred (Mangala et al., 1999), which facilitates the enzymatic action (Perera et al., 2001). Zhang et al. (2008a,b) investigated the relationship between digestion properties and molecular structure of different varieties of maize starch under cooking conditions. The results in vitro digestion of corn flour showed that RDS value ranged between 70.1% and 98.9%, for SDS between 0.2% and 20.3%, and for the RS between 0% and 13.7%. Cooking of the corn flour is responsible of the high RDS due to which disorganization of starch structure increases enzymatic susceptibility (Zhang et al., 2006a). A significant correlation (R ¼ 0.76) between the RS and amylose content was found; after cooking (disorganization of starch structure) this essentially linear component is reorganized, producing a structure that is not recognized by digestive enzymes and not hydrolyzed in the upper intestine. Likewise, in this study they reported a parabolic relationship between SDS and the weight ratio of short chains (SC) and long chains (LC) of amylopectin, suggesting that the amylopectin with high proportion of short chains or high proportion of long chains was responsible for the high SDS content. They divided the samples into two groups: group I with an SC/LC ratio of 0.166 and 0.525, and group II with high values of SC/LC (0.52 and 0.78). Based on the analysis of correlations between the content of SDS and SC/LC ratios of the two groups, it was found that the SDS content is mainly determined by the long chains of amylopectin. A relationship between the RS content and amylose chains (LC) in the first group of samples was found due to which high levels of LC are present in this group. Two representative samples of Groups I and II with high content of SDS showed variability in the peaks of the products; this difference is attributed to the degree of polymerization (DP) of the samples that influenced the enzymatic hydrolysis.

3. SLOWLY DIGESTIBLE AND RESISTANT STARCH PRODUCTION AS INGREDIENT FOR FOOD INDUSTRY The increasing interest in SDS and RS due to the health benefits associated with their consumption has been conducted to produce both fractions using chemical, physical, enzymatic, and combinations of them. The production of SDS and RS is due to interest in the development foods with high slow starch digestion (no glycemic peaks) and indigestible carbohydrate content (dietary fiber). Uncooked cereal starches showed high SDS and RS content, but with higher SDS than RS; potato starch showed an inverse pattern than cereal starches due to the high RS content (Zhang et al., 2006a). When those starches


(cereals and tuber) were cooked, SDS and RS decreased with the concomitant increase in RDS (Zhang et al., 2006b). The early studies of both starch fractions were focused on classification of the different RS types. The first classification included four RS types and thereafter one type more was incorporated. RS1 is defined as starch granules embedded in nonedible plant material, physically inaccessible, usually observed in seeds and unprocessed whole grains; RS2 are native starch granules that present a crystalline arrangement that is inaccessible to enzymatic hydrolysis, e.g., unripe banana and plantain, potato; RS3 is retrograded starch, which is a product of internal reorganization of the starch granule when it is cooked and cooled; RS4 is a chemically modified starch (esterification, acetylation, oxidation, cross-linking, etc.), the introduction of functional groups in the starch structure is not recognized by digestive enzymes (Cummings and Stephen, 2007); RS5 is a complex between amylose chains and lipids that is not hydrolyzed by enzymes and is a thermally stable (Hasjim et al., 2010). When the starch has cooked in excess of water, gelatinization occurs and high quantity of RS and SDS are converted to RDS. To maintain high SDS and RS levels, diverse treatments to starch have been achieved, all of them with the objective to produce structural changes in the starch that reduce resistant enzymatic hydrolysis.

3.1 Starch Modifications With Nutritional Purpose To increase SDS and RS content, starch can be modified by chemical, physical, and enzymatic treatments. The starch modification treatments have been done single, dual, cyclic, and some researchers have focused to combine two or more treatments mentioned above to modify starch digestibility. The base of these modifications is the structural change in both granular structure and molecular arrangement of starch components, as the enzymes that hydrolyzed starch are very specific.

3.1.1 Physical Modifications Physical modifications are treatments that involve temperature, moisture, pressure, conductivity, and/or other physical variables. These physical variables can be exploited to structural modifications within starch granules. Jacobs and Delcour (1998) describe hydrothermal treatments as physical changes occurring at a temperature above the glass transition, but not above the gelatinization temperature of the starch granules; such changes can only occur when the starch polymers in the amorphous phase is in a mobile gelatinous state. Most employee hydrothermal treatments are annealing (ANN) and heat-moisture treatment (HMT). The first involves incubation of the starch granules in excess or medium water content (40%e55%) to temperature above glass transition but below gelatinization temperature; HMT is realized with lower water content (between 10% and 35%) and heating at


831

temperatures between 90 and 120 C above gelatinization temperature, for a period of time which may vary between 15 min and 16 h (Chung et al., 2009b). Diverse studies are focused in digestibility and physicochemical changes induced by hydrothermal treatments achieved on isolated starches and flours from different botanical sources (De La Rosa-Millan et al., 2015; Hoyos-Leyva et al., 2015; Klein et al., 2013; Lee and Moon, 2015; Lee et al., 2012; Rocha et al., 2012; Sui et al., 2014; Vamadevan et al., 2013; Wang et al., 2016). The effects of ANN and HMT on starch digestibility are related to treatment conditions (moisture, temperature, time, and number of cyclesd when two or more hydrothermal treatments are carried out) and native starch characteristics (amylose content, length of polysaccharides chains). In general, the SDS and RS formation during these processes may be due to phenomena associated with the reorganization of the structure of starch (Ozturk et al., 2009). The changes induced by HMT treatment have been attributed to increased crystallinity, reduced water molecules in the starch structure, increased interaction between amylose and amylopectin chains, strengthening intramolecular bonds, the formation amyloseelipid complexes, and changes in the arrangement of the crystalline regions of the granule. As intramolecular interactions are strengthened by HMT, modified starch requires more heat than native starch for structure disorganization and granule breakdown, and finally gel formation (Olayinka et al., 2008). Overall, the changes induced by hydrothermal treatments are related to reinforce intramolecular bonds, which results in change in its digestibility (Table 22.1); moreover, HMT treatment has been used to produce boiling stable RS. Different from HMT, ANN produces a molecular mobility within the crystalline regions of the granule and the organization of a dense packing of the crystalline regions. This treatment is related to reduction of voids within the starch granule (amorphous areas), producing changes in their physicochemical properties such as gelatinization temperature, pasting formation, and water absorption capacity (Jayakody and Hoover, 2008; Vamadevan et al., 2013). HMT and ANN produce intramolecular strengthening that has effect on a reduction of amylose leaching, which is most evident in high-amylose starches, because additional interactions between amyloseeamylose, amylopectineamylopectin, and amyloseeamylopectin are promoted, which result in a reduction of amylose leaching and decreasing of retrogradation (Chung et al., 2009a).

3.1.2 Enzymatic Modifications The modifications can be achieved with debranching enzymes such as pullulanase and isoamylase (Shin et al., 2004). Both enzymes hydrolyzed a-1-6 linkages of amylopectin to produce linear chains. The DP of the linear chains depends of the botanical source of starch. The linear chains obtained by debranching of starch are submitted to heatingecooling cycles to produce reorganization named starch retrogradation, which depends of the DP of linear


TABLE 22.1 Production of Slowly Digestible (SDS) and Resistant (RS) Starch by Hydrothermal Treatments Starch Fractions Digestion Native Starch Source

Treatment

Modified

SDS (%)

RS (%)

SDS (%)

RS (%)

45.5

22.1

54.6

18.1

49.3

16.5

References

Digestion of Raw Starch Waxy rice

HMT ANN þ HMT

Zeng et al. (2015a,b)

“Morado” banana

HMT

6.5

89.4

13.6

59.9

Hoyos-Leyva et al. (2015)

Black bean

HMT

53.62

33.46

34.09

39.23

Fatima bean

HMT

77.29

10

63.86

2.21

Ambigaipalan et al. (2014)

Plantain flour

ANN

5.9

66.5

3.7

68.8

De La RosaMillań et al. (2014)

Waxy potato

HMT

8.1

75.8

39.8

41.3

Lee et al. (2012)

Potato

HMT

5.4

78.5

35.1

40.1

Lee et al. (2011)

Lentil

HMT

70.1

9.1

23.6

14.7

Pea

ANN

62.9

10

34.4

11.2

Chung et al. (2009a,b)

Sweet potato

ANN

15.6

67.3

31

43.4

Shin et al. (2005)

Van Hung et al. (2016)

Digestion of Gelatinized Starch Highamylose rice

ANN

3.7

6.3

10.7

19.5

Rice

ANN

14

6.5

19

22.6

Waxy rice

ANN

12.8

10.2

16.5

26.9

Maize

HMT

6.2

2.1

18.9

11.7

Highamylose maize

HMT

7.4

6.6

18.8

29.5

Wheat

HMT

4.87

2.27

1.08

14.32

Wang et al. (2016)

Chen et al. (2015)


833

TABLE 22.1 Production of Slowly Digestible (SDS) and Resistant (RS) Starch by Hydrothermal Treatmentsdcont’d Starch Fractions Digestion Native

Modified

Starch Source

Treatment

SDS (%)

RS (%)

SDS (%)

RS (%)

References

Buckwheat

ANN

47

4

50

3

Liu et al. (2015)

57.5

3.5

HMT Wheat flour

HMT

14.02

1.09

4.98

13.28

Chen et al. (2015)

“Morado” banana

HMT

7.5

12.7

12.1

30.7

Hoyos-Leyva et al. (2015)

Plantain flour

ANN

10.9

9.9

15.4

6.8

De La RosaMillań et al. (2014)

Black bean

ANN

34.98

36.15

24.48

43.26

Pinto bean

ANN

27.16

41.61

22.27

58.13

Simsek et al. (2012)

Lentil

HMT

0.8

5.3

5.5

15.7

Pea

ANN

0.6

5.2

1.8

10.5

Chung et al. (2009a,b)

ANN, annealing; HMT, heat moisture treatment.

chains. Digestive enzymes do not recognize retrograded starch structure because it is considered RS. Patent was reported in starch with amylose up to 40%. This process involves starch gelatinization, enzymatic debranching of starch with pullulanase, deactivation of the debranching enzyme, and separation of the RS, using drying, extrusion, or crystallization by addition of salt (Chiu et al., 1994). We used plantain starch to prepared RS by debranching with pullulanase at different times and autoclaving. Five hours of debranching were enough to produce the highest RS content (18%). However, this RS-rich powder can be added to products where cooking is not necessary, such as smoothies, yogurt, fruits, breakfast cereals (Gonza´lez-Soto et al., 2004). Debranching of starch with pullulanase, using high concentration and short reaction time was achieved; after debranching the starch solution was fast cooling and storage, showing an increase in SDS content (Miao et al., 2009). Waxy sorghum starch was debranched with isoamylase for 8 h followed by storage for 3 days at 1 C; these were the optimal conditions to obtain the maximum SDS content. The authors reported that the structure of SDS is formed of mainly imperfect crystalline regions containing small portions of double helices and amorphous regions (Shin et al., 2004).


Starch structure can be modified with the use of enzymes such as a-amylase, b-amylase, and transglucosidase. Normal maize with A-type crystalline arrangement (high amount of short chains) was modified with b-amylase, the enzyme that hydrolyzes the second a(1e4) linkage from the nonreducing end of amylose and amylopectin chains, producing maltose and a-dextrins, and transglucosidase which catalyzes both hydrolysis and transfer reactions to produce new a(1e6) linkages. An increase in SDS content was recorded in the enzymatic-treated starch compared with its native counterpart. The increase in SDS was attributed to a higher level of short chains and the increase in the number of a(1e6) linkages (Zhang et al., 2008a). Mango and plantain starches were gelatinized and enzymatic treatment with b-amylase and transglucosidase. The treatment with both enzymes produced higher increase of SDS content in plantain than mango starch. The treatment with b-amylase showed a reduction of SDS content in plantain starch, but an increase of 6.3%e22.3% was determined in mango starch. This pattern is related with the higher short chains present in mango than plantain starch (Espinosa-Solis et al., 2011). However, the treatment with both enzymes produced an increase in RS content, but it was higher in mango than plantain starch. A reduction in structural variables such as gyration radius and molar mass were determined in starches after the enzymatic treatments. An increase in the short chains level of amylopectin (DP 5e12) and decrease in the crystallinity percentage was found in the enzyme-modified starches. H1-nuclear magnetic resonance results showed a significant increase of a(1e6) linkages in starches modified with both enzymes (Casarrubias-Castillo et al., 2012). Potato and arrowroot starch were gelatinized, hydrolyzed with by a-amylase or b-amylase, and debranched by pullulanase. After debranching for 10 h, the starch solutions were equilibrated at room temperature and stored at 4 C for 16 h to enhance starch retrogradation. Gelatinized starches (potato and arrowroot) showed low RS level, with arrowroot showing a higher percentage (1.1%); debranched starch showed an increase in RS content due to starch retrogradation, but the hydrolysis by a-amylase or b-amylase and after debranching increased the RS amount more. Production of linear fragments of low molecular weight with greater mobility increased the tendency toward retrogradation. The increase in SDS content was not important in the diverse treatments (Villas-Boas and Franco, 2016). Other enzymes such as 4-a-glucanotransferase (Jiang et al., 2014) and amylosucrase (AS) (Kim et al., 2014) have been used to produce an RS structure (Table 22.2). 4-a-glucanotransferase catalyzes the transfer of a segment of a 1,4-a-D-glucan to a new position in an acceptor carbohydrate such as glucose or other 1,4-a-D-glucans (Takaha and Smith, 1999); in this sense, the glucanotransferase sculpt starch structure to modify starch digestion (Jiang et al., 2014). Normal corn starch dispersion (8% w/w) was cooked and treated with 4-a-glucanotransferase for different times. The product of the

TABLE 22.2 Production of Slowly Digestible (SDS) and Resistant (RS) Starch by Enzymatic Modifications Starch Fractions Native Starch Source

Modified

Modification

SDS (%)

RS (%)

SDS (%)

RS (%)

Sweet potato

BE (BE 48 U/mL)

40.2

42.4

6.4

17.8

34.8

41.3

Waxy rice

PULs

36.2

34.7

46.1

38.4

References

Raw Starcha

BE-AS (BE 48 U/mL) 45.5

22.1

PULs þ crystallization

Jo et al. (2016) Zeng et al. (2015a)

Sweet potato

PULs þ HMT

8

72

31.6

58

Huang et al. (2015)

Corn

4-a-GTs

9.4

10.52

20.92

17.63

Jiang et al. (2014)

Water yam

IS

0.3

99.5

32.9

51

2

96.5

Trinh et al. (2013)

Waxy sorghum

IS

IS þ boiling þ HMT 7.1

11.2

27

22.1

Shin et al. (2004)

Gelatinized Starchb Adlay

AS

58.3

10.4

24

51.5

Kim et al. (2016)

Potato

a-AMY þ PULs

18

8.1

27.8

36.8

11.3

36.3

Villas-Boas and Franco (2016)

b-AMY þ PULs Arrowroot

a-AMY þ PULs

4.7

1.1

11.3

53.4

Waxy rice

PULs

13.2

11.4

27.6

10.9

31.4

16.3

PULs þ crystallization Corn

4-a-GTs

Water yam

IS

Plantain

b-AMY

Jiang et al. (2014) 9

23

IS þ boiling þ HMT 10.96

14.09

b-AMY þ TGs Mango

b-AMY b-AMY þ TGs

Zeng et al. (2015a)

6.37

19.34

25

49

4

80

7.15

32.74

18.53

18.05

22.32

16.84

11.78

32.92

Trinh et al. (2013) CasarrubiasCastillo et al. (2012)

4-a-GTs, 4-a-glucanotransferase; AS, amylosucrase; BE, glycogen branching enzyme (48 U/mL); HMT, heat moisture treatment; IS, isoamylase; PULs, pullulanase; a-AMY, a-amylase; b-AMY, b-amylase; b-AMY þ TGs, b-amylase-transglucosidase. a The samples were analyzed in raw state. b Before digestion analysis, the samples were subjected to a gelatinization step as boiling in excess of water or autoclaving.


hydrolysis was freeze-dried and analyzed. The amylose content in the samples decreased with increase in the hydrolysis time produced by a preferential hydrolysis on amylose molecules. The fraction of high molecular weight (amylopectin) decreased with the concomitant increase of the fraction of low molecular weight. These low-molecular weight fractions such as smaller amylopectin cluster and cyclic glucans likely originated from the degradation of amylopectin and amylose by 4-a-glucanotransferase through cleaving and reorganization of starch molecules. Increase in the hydrolysis time by 4-a-glucanotransferase produced an increase in SDS and RS content compared with the control sample (gelatinized starch). Rearrangement of branched chains in amylopectin due to 4-a-glucanotransferase reaction produced changes in the crystalline structure and consequently in the starch hydrolysis pattern (Jiang et al., 2014). Waxy maize starch was gelatinized and treated by AS for different times. AS uses sucrose as substrate, producing a(1e4)-linked glucan and releasing fructose. Increase in the reaction time produced decrease in short chains (DP 6e12) and increase in long chains (DP 25e36); the X-ray diffraction pattern showed an amorphous pattern prone to a crystalline structure with increase in the reaction time. The most significant change was shown in the SDS content due to an increase in the level with an increase in the reaction time with concomitant reduction in the RDS content, but after 6 h of reaction, no changes in the SDS content was found. The RS content did not change with the reaction time and was lower than the SDS content. This pattern could be related to changes in the amylopectin structure during the reaction with AS. The authors commented that more studies are necessary to understand the mechanism of SDS formation by AS. Starches with different amylose/ amylopectin ratio, different crystalline structure (polymorphism), and chainlength distribution can give more information (Kim et al., 2014). Recently, waxy starch from a nonconventional botanical source (adlay, Coix lachrymajobi var. ma-yuen Stapf.) was modified using AS for 40 h of reaction to change the digestion properties. The raw starch showed the highest SDS content that decreased after gelatinization, but increase in this fraction was found in the starch treated by AS. The AS-modified starch presented the highest RS content (51%) followed by the gelatinized sample and raw starch. The AS-modified starch showed similar RDS and SDS (24%) content, suggesting that this starch could produce health benefits after consumption due to the presence of the two fractions (SDS and RS) considered with functional characteristics, and the AS-modified starch could be used to prepare low glycemic foods (Kim et al., 2016).

3.1.3 Chemical Modifications Chemical modification involves the introduction of functional groups into the starch molecule. The common chemical starch modifications are etherification, esterification, cross-linking, and grafting of starch or combinations of them.


837

Chemical modification has been done to increase SDS and RS fractions in starch. Cross-linking, hydroxypropylation, acetylation, esterification, and combinations of them reduced the starch digestion, increasing SDS and RS fractions (Han and BeMiller, 2007). In the modification of waxy maize starch, cross-linking combined with hydroxypropylation produced a major increase in the SDS fraction (24%) than cross-linking followed by acetylation (13%); the RS level was 14% and 24%, respectively, showing lower RS content by the dual modification than hydroxypropylation. Some authors reported differences in chemical modifications due to reagent concentrations, differences in the type and amount of swelling-inhibiting salt used, and the sequence of modifications when dual modification was achieved (Carlos-Amaya et al., 2011; Han and BeMiller, 2007; Villwock and BeMiller, 2005). In a comparative study of glucose response in humans by raw consumption of different RS types, cross-linked (RS4) starch intake resulted in a significantly decreased glucose response; the RS content in cross-linked starch was 83%, while RS2 had an RS content of 46% (Haub et al., 2010). Table 22.3 shows the starch digestibility of chemically modified starches from different sources. The more common starch chemical modification has been the esterification with anhydrous octenyl succinic, which gives to the starch a hydrophobic character and is used for emulsion stabilization (Tesch et al., 2002). The fractions of SDS and RS can be increased using the esterification of isolated starch from diverse botanical sources (Simsek et al., 2015). Han and BeMiller (2007) reported higher increase of RS content in waxy corn, potato, and tapioca starches using single and dual chemical modifications. The higher RS level (40.7%) for modified waxy corn starch was found with hydroxypropylation followed by an esterification treatment, while SDS content was higher raised with the esterification of tapioca starch (up to 100%). The main reason for higher SDS and RS values in chemically modified starches is due to the high specificity of digestive enzymes (a-amylase and amyloglucosidase) that do not recognize the chemically modified structure of amylose and amylopectin chains, which results in high values of nonhydrolyzed starch fractions.

3.2 Commercial Functional Starches There are diverse commercial starches that can be used in food industry as functional ingredients; they have high RS content and can be named RS-rich powder. ActiStar RM resistant starch (Cargill, Minneapolis, USA) is a butyrogenic RS, added to improve texture in bakery products, snacks, bars, and breakfast cereals; its functionality in food processing includes less viscous than high amylose-derived products, very bland taste and smooth mouth feel, stable to low pH and temperatures up to 120 C, depending on the application. ActiStar RT (Cargill, Minneapolis, USA) is a resistant modified starch isolated from tapioca, recommended for supply partially other bakery flours with good

TABLE 22.3 Production of Slowly Digestible (SDS) and Resistant (RS) Starch by Chemical Modifications. Starch Fractions Digestions Were Determined After Cooking Starch Fractions Digestion Native

Modified

Starch Source

Modification

SDS (%)

Corn

ESTR

15.35

5.36

20.03

9.4

Tapioca

ESTR

15.36

4.02

22.58

13.93

Rice

ESTR

16.32

2.35

14.13

9.04

Potato

ESTR

30.55

7.32

36.02

8.13

Wheat

ESTR

12.96

6.11

19.14

9.33

Canna

CL AC

13.7

32.2

HP

14.2

37.6

ESTR

12.7

49.4

24.48

44.29

25.84

44.67

17.76

43.02

27.1

44.6

15.13

19.45

21.32

24.99

9.61

29.14

0.05

19.5

0.02

7

Pinto bean

AC

Banana

CL

34.98

36.15

Ozonated 27.16

41.61

Ozonated 12.75

21.49

ESTER CL-ESTER Corn

HP

0.01

7.3

CL Waxy corn

RS (%)

25.4

AC

18.1

SDS (%)

17.9

Black bean

2

RS (%)

CL (without salt)

12.2

0

19.9

12.9

CL (without salt)-AC

12.9

24.2

CL (without salt)-HP

20.9

13.9

HP-CL

20.6

25.6

HP-ESTER

22.7

40.7

Potato

ESTER

17.8

3.9

26.9

32.8

Tapioca

ESTER

13.8

1.1

31.5

27.8

References Simsek et al. (2015)

Juansang et al. (2012)

Simsek et al. (2012)

Carlos-Amaya et al. (2011)

Chung et al. (2008) Han and BeMiller (2007)

AC, acetylated starch; CL, cross-linked starch; ESTER, esterified starch; HP, hydroxypropylated starch.


839

sensorial properties. These RS have low water-binding capacity, which permit high inclusion levels with few formulation changes while maintaining a desirable finished texture. The enterprise Ingredion (Ingredion Incorporated, Westchester, IL) has a wide series of resistant starch ingredients for the food industry that include HIMAIZE 260 which is a high-amylose resistant starch (RS content between 40% and 60%), HYLON VII is a RS2 type, obtained from high-amylose hybrid corn with RS content of 70%; owing to its high-amylose content, this product can form more rigid and stronger gels and films. NOVELOSE 330 is a nongranular, retrograded starch (RS3) from corn starch with RS content up to 30%; it is recommended specially for extruded food products. In addition of being a source of dietary fiber, these products have functional properties for their use in different food matrices. Its low water-holding capacity, neutral taste, smooth mouth feel, and relative thermal stability (as in case of HYLON VII that under atmospheric cooking temperatures required heating above 154 C for gelatinization) allow the use of these RS ingredients in a wide variety of food products. There are RS4-type ingredients commercially available. Fibersym RW (85% of total dietary fiber) and FiberRite RW (MPG Ingredients Inc., Atchison, KS) are RS4 type produced by cross-linking starch with sodium trimethaphosphate. These starches are characterized by their low swelling capacity; however, the water absorption is similar to wheat flour, which makes it possible for the partial replacement of this ingredient for Fibersym RW in bakery products without change in water retention.

3.3 Functional Properties of RS-Rich Powders RS-rich powders have desirable physicochemical properties (Fausto et al., 1997) such as swelling, viscosity increase, gel formation, and water-binding capacity, making it useful in a variety of foods. RS has a small particle size, white appearance, bland flavor, and provides good handling in processing and crispness, expansion, and improves texture in the final product (Fuentes-Zaragoza et al., 2010; Sajilata et al., 2006). RS generally have high gelatinization temperature, good extrusion and film-forming qualities, and lower water-holding properties than traditional fiber products. Compared with traditional high-fiber products, RS allow the formation of low bulk with improved texture, appearance, and mouth feel. RS increase coating crispness of products and the bowl life of breakfast cereals. The lower water-holding capacity of RS-rich powder produced that the dough where is added has lower dough water absorption than dough made with other fiber sources; this characteristic is closest to the commercial white bread dough (Sajilata et al., 2006). Also, cross-linked starch (RS4) had similar water-holding capacity to wheat starch, so that no change in bakery products as water absorption and baking time were found (Maningat and Seib, 2010).


4. DEVELOPMENT OF FOODS WITH FUNCTIONAL STARCH INGREDIENTS SDS and RS powders have been added to some food matrixes to reduce the caloric charge and increase the indigestible carbohydrate content. As we reviewed in this chapter, SDS and RS can be obtained by natural starchy botanical sources or by starch modifications. Many products had been formulated with these starches to reduce caloric content and postprandial insulinemic response after consumption of the foodstuff. RS is considered as a prebiotic and its use has regulated within fiber ingredient normativity; actually in the food industry there are many RS products that can be used for preparation of breads, pancakes, waffles, muffins, cookies, breakfast cereals, pasta, noodles, nutritional bars, low-fat fermented milks, ultraheat-treated flavored milk drinks, ready to use powdered mixes such as instant soups, chocolate drinks (Aparicio-Saguilań et al., 2007). The European Food Safety Authority (EFSA) defined a cereal high in SDS content when the cereal contained at least 55% of available carbohydrates as starch of which at least 40% is SDS (EFSA, 2011). Bread elaborated with banana flour showed 6.7% of RS content, significantly higher compared to control sample, which had 1% of this starch fraction (Juarez-Garcia et al., 2006); in agreement with the RS content, the hydrolysis indices was lower (65.1%) than control bread (81.9%); the starch hydrolysis is associated with starch gelatinization degree and other nonstarchy ingredients in food formulation such as proteins, lipids, and fiber that may encapsulate starch granules and reduce their hydrolysis. RS3 (Novelose 330) was used for the production of batter-fried foodstuffs as it can withstand the frying conditions (185 C) (Sanz et al., 2008). This RSrich powder, used as an ingredient, increases the RS content in products such as biscuits, producing higher proportions of stiffer doughs and less consumer acceptance (Laguna et al., 2011). Biscuits were elaborated with different level of RS2 (Laguna et al., 2011). This RS-rich powder was used to replace 20% of the wheat flour, showing the highest acceptability by consumers; the RS content in the biscuit was 3.92% higher than in the control biscuit, which showed 0.49% of RS. RS2 has been used as a prebiotic in sausage (Amini Sarteshnizi et al., 2015). Amylose content showed a negative effect on cooking yield of the sausage because the RS used was the high-amylose RS2 type. High-amylose content in starch granules produced a reduction in swelling ability, high gelatinization temperatures, retrogradation ability, and amylose leaching during heating. The effect of cross-linked RS4 added to an extruded ready-to-eat breakfast cereal ring was reported by Miller et al. (2011). The addition of RS4 levels between 5% and 10% did not alter the size and textural characteristics of the cereal ring. The increase of RS4 up to 15% or 20% decreased the cereal ring diameter; however, this level of RS showed extended shelf-life. In agreement with the increase of RS4 added to the cereal formulation, the total dietary fiber


841

content in the extruded cereal was higher (20%) than in control cereal (5.6%). An in vivo study of the consumption of RS4 in nutrition bars (Al-Tamimi et al., 2010) demonstrated that this kind of RS decreased the postprandial insulin and glucose responses; this low response was also observed when high-glycemic ingredients were eaten, which suggests that the abstinence from consumption of high-caloric content products for maintenance of the blood glucose levels when RS4 is included in the diet is not necessary (Al-Tamimi et al., 2010). The use of RS in gluten-free products has been widely investigated. The effect of RS on foodstuffs depends of RS type, process conditions, and food type. Bread supplemented with RS was characterized by softer crumb in comparison to control bread (Korus et al., 2009), reflected in a more elastic structure, reduced creep and recovery compliance, and elevated zero shear viscosity. RS4 has a positive influence on dough development and bread yield; however, increase in the RS level decreased bread volume; also, the increase in RS level could increase the swelling power, solubility, water-binding capacity in cakes matrices, but decrease the pasting viscosities and change the color of cookies, depending of the amount added (Ren and Shin, 2013). Pregelatinized starch allow batterlike doughs less susceptible to shear during processing, increase the bread volume and crumb softness (Pongjaruvat et al., 2014). The addition of RS-rich powder in gluten-free formulations produced cake batters that are less elastic and thinner; however, the volume of the cakes increased with RS content about 15% (Tsatsaragkou et al., 2014). Others characteristics that can be change with RS addition to cookies are a decrease in surface porosity but increase in average pore diameter. Hydrothermal modified starch was used in gluten-free bread, where decreasing in crumb hardness and retrogradation enthalpy was found, indicating that the RS-rich powder retarded the hardness of the bread (Krupa et al., 2010). Table 22.4 shows the SDS and RS content in some foodstuffs added with flours or starches with different RS type. The products in which was added the RS-rich powders are mainly breads, cookies, pasta, and breakfast cereals, prepared under extrusion, baking, or boiling water conditions. All these products showed higher level of RS than control products; however, the addition of RS as an ingredient is limited by quality changes that may not be accepted for the consumers. The replacement of traditional starches or flours varies between 10% and 85% in the formulation according to each product.

4.1 Chemical and Physical Changes That Induce SDS and RS Formation in Different Food Matrixes The SDS and RS content could be influenced by the presence of diverse ingredients in food formulations. Proteins, lipids, sugars, and organic acids present in the food matrixes have effect on starch digestion (Escarpa et al.,


TABLE 22.4 Starch Fraction Digestion in Foodstuffs Enriched With Resistant Starch (RS) Ingredients Starch Fraction

Food Product

Starch Source

SDS

RS

Observations

References

Noodles

RS3 (15%)

21.65

52.11

RS3 (10%)

17.08

43.33

Low glycemic index (54.58) and low cooking loss, high sensory scores (4.23) for appearance, flavor, and mouth feel.

Menon et al. (2015)

Fried glutenfree snacks

RS2 (UPF)

21.07

3.01

Extrusion and frying processes can have an effect on the SDS and RS content in the final product, as low values for RS were reported in the formulations with higher additions of unripe plantain flour.

Flores-Silva et al. (2015)

Spaghetti

RS2 (UPF25%)

4.14

Gluten-free spaghettis shown higher cooking loss than control spaghetti (with semolina).

Flores-Silva et al. (2014)

Pasta

RS2 (uncooked)

10.99

RS can be used up to 20% in pasta formulations without affecting the cooking quality, sensory properties, and textural characteristics.

Aravind et al. (2013)

SDS content increase with higher amounts of unripe banana flour added to cookies formulation. The bakery conditions were 180 C for 25 min.

AgamaAcevedo et al. (2012)

9.82

RS2 (cooked)

Cookies

RS3 (uncooked)

10.23

RS3 (cooked)

11.85

Control

8.3

2.3

RS2 (UBF15%)

8.7

5.1

RS2 (UBF30%)

9.3

5.4

RS2 (UBF50%)

10.9

8.4


843

TABLE 22.4 Starch Fraction Digestion in Foodstuffs Enriched With Resistant Starch (RS) Ingredientsdcont’d

Food Product

Starch Source

Biscuits

Starch Fraction RS

Observations

References

RS2 (60%)

11.23

Replacement of 20% of wheat flour by RS not affect the dough rheology.

Laguna et al. (2011)

Extruded breakfast cereal

RS4 (crosslinked)

9.4a

RS4 levels up to 10% did not affect the expansion during extrusion. When this RS was added in levels of 15% and 20% the cereal rings exhibited a crisper texture, smaller in size, and extended bowl life.

Miller et al. (2011)

Muffins

RS2

e

Increasing the RS content decrease elastic properties of the muffin batter.

Baixauli et al. (2008)

Batterfried squid rings

RS3

13.2

RS3 did not affect the viscoelastic properties, increase the hardness, fragility in the crispness profile, and increase the golden brown color in the final product.

Sanz et al. (2008)

Cookies

Lintnerized UPF

8.42

Predicted glycemic index was 60.53. Addition of 85% of lintnerized flour has high sensory scores (3.0). Bakery conditions were 200 C for 20 min.

AparicioSaguilań et al. (2007)

a

SDS

e

RS was reported as total dietary fiber; UPF, unripe plantain flour.


1997). During processing and storage of starchy products, starch is subject to gelatinization and retrogradation, and the presence of those diverse ingredients mentioned above restricted swelling of starch granules and reorganization of starch components disorganized during heating. The events mentioned are involved in the increase or decrease of SDS and RS content in the foods. The addition of organic acids such as lactic acid in a wheat bread matrix influenced amylopectin hydrolysis or partial debranching that implied a significant increase of the RS content (Liljeberg et al., 1996). During storage of foods, the SDS and RS content may be change, mainly due to the retrogradation process. The increase of retrograded RS fraction is related to storage time, temperature, presence of lipids and proteins, and starch type. The starch characteristics include amylose/amylopectin ratio, chainlength distribution (Eerlingen and Delcour, 1995). Dundar and Gocmen (2013) reported that autoclaving at high temperature (145 C) showed higher yield in RS production (25%) than lower temperature (140 C) (22%). Moreover, the RS content increases linearly with storage time; when the storage was prolonged at 72 h the RS content increases between 4% and 5% more for both autoclaving treatments (140 and 145 C, respectively). The hydrothermal cooking of starch induces changes in its crystalline structure due to which the exposed hydroxyl groups of amylose and amylopectin become linked to water molecules, increasing granule solubility and swelling. The availability of water determines the starch digestibility through enzymatic hydrolysis; during starch hydrolysis Km and Vmax showed an increase when starch is in excess of water (Singh et al., 2010; Slaughter et al., 2002). Changes in starch digestibility also can be observed in extruded starches and foodstuffs (Flores-Silva et al., 2015; Kim et al., 2006; Manrique-Quevedo et al., 2007; Zhang et al., 2016). In cooking by extrusion, feed moisture, feed rate, screw speed, and extrusion temperatures can affect the production of SDS and RS fractions. Kim et al. (2006) reported the effect of extrusion conditions and storage time on RS formation from pastry wheat flour. Feed moisture produced the main effect, 20% of moisture had 0.50% of RS content, while 60% increase RS up to 2.6%. The extruded pastry wheat flour that was stored for 14 days, showed an RS increase from 2.54% to 4.07% (feed moisture 60%). Zhang et al. (2016) reported changes in SDS and RS content in extruded maize flour related to starch amylose content; extruded normal maize flour showed 5.5% and 5.4% of SDS and RS content, respectively, while extrusion of high-amylose maize flour (feed moisture 12.47%) results in 2.70% and 7.75% of SDS and RS content, respectively. The feed moisture content during extrusion of high-amylose maize flour was evaluated. Extrusion produced a decrease in SDS and RS when was compared with its native counterpart. The effect of moisture content in the feed (12.5% and 19%) was evaluated, but RS content did not change, and a slight increase in SDS content was determined in the flour extruded with 19% moisture (2.7% for 12.5% and 5.0% for 19%).


845

This pattern could be due to crystallinity degree transformation under extrusion (Zhang et al., 2016). Cooking time and particle size define the cooking stage of some starchy food products such as noodles (Ye and Sui, 2016) or spaghettis. When spaghettis are subjected to hydrothermal cooking for a long time, the synergistic action of excess water and boiling temperature affect the final characteristics of the product, reducing the texture quality. Long-time cooking (low temperature-long time) can induce SDS and RS formation in foodstuffs; this effect is more notable with high-amylose starches (Liljeberg et al., 1996). The cooking time increases the starch digestibility of Chinese noodles (Ye and Sui, 2016); the reduction of SDS and RS were observed as cooking time increased up to 12 min. Before the digestion test, the noodles were ground and grouped in different particle sizes (3.2e5.0; 2.0e3.2; 1.0e2.0; 0.5e1.0 mm) mimicking the chewing. The particle size showed a significant effect on SDS content; particles of 3.2e5.0 mm with 12 min of cooking had an SDS content of 14.4%, while 0.5e1.0 mm particles at the same cooking time showed an SDS content of 0.7%. In this product, the cooking time increased the RDS content of noodles, while SDS was the fraction with high losses.

4.2 Raw or Cooked Starchy Foods Preparation and Changes in Starch Digestion In general, starch is consumed after processing, where their physicochemical properties and nutritional characteristics are affected by conditions such as temperature, heating time, humidity, and number of cycles of heatingecooling (Sagum and Arcot, 2000). There are many cooking processes that includes pressure cooking, autoclaving, reautoclaving, roasting, baking, frying, toasting, puffing, extrusion, traditional hydrothermal cooking (100 C in excess of water during several minutes). In vitro studies of starch digestion after cooking process are scarce.

4.2.1 SDS and RS as Raw Ingredients Actual trends of healthy food products open new opportunities in the marketing of some raw materials like fiber and others prebiotic products such as high SDS and RS. Raw materials for consumption imply preparations such as beverages of nutritional supplements or a mixture with cereals and fiber. In this case, SDS and RS can produced high benefits on human health. Beverages with SDS and RS addition have low calorie content, low GI, improve colon health, control glucose assimilation, reduce blood lipid levels, and increase the satiety. The controlled release of glucose is an energy source for persons who do not consume regular meals. The native starches classified as RS2 (potato, banana, plantain, high-amylose starches, etc.) are materials that can be eaten


raw, due to their characteristic of low starch digestion. The consumption of these materials can be as flours or isolated native starch powders that can be mixed in salad dressings, sauces, smoothies, cereals, etc. as a rich source of SDS and RS. These products do not require complex unit operations for their production.

4.2.2 Thermal Stability of SDS and RS Due to heating treatment of cooking and moisture content within most starchy products, heat-or thermal-stable SDS and RS have been researched. It is well known that gelatinization induce reduction of SDS and RS fractions. The thermal stability has related with retrogradation process or major crystalline arrangement within starch granules induced by starch modifications. RS3 is considered thermally stable (Eerlingen and Delcour, 1995; Gruchala and Pomeranz, 1993) and its production is mainly from high-amylose starches. Starch gelatinization produced lixiviation of amylose chains from starch granules toward the continuous, with high tendency to starch reorganization (retrogradation) during cooling. Thermally stable RS3 was produced by retrogradation of partial debranched banana starch and HMT treatments (Lehmann, 2002). The retrogradation process induce high thermal stability in modified starches; the onset temperature in native banana starch was 73 C while in retrograded modified starch was up to 145e147 C. The authors reported that HMT increase the thermal stability of RS structures, due to which thermal transitions around 103 and 162 C were observed. HMT treatment is recognized for its effect on the generation of boiling-stable RS. The yield of boiling-stable granular RS by HMT was investigated using high-amylose maize starch (70%) after a partial acid hydrolysis with 1% HCl at different reaction times (6, 30, 78 h of incubation) (Brumovsky and Thompson, 2001). HMT cause the production of major boiling-stable RS (63.2%) compared to ANN treatment (32.7%). The acid treatment hydrolyses firstly the amorphous regions of the granule, providing potential freedom for chains to form double helices and for double helices association. The hydrothermal treatment would allow the potential mobility of the chains and form more highly ordered structures (Brumovsky and Thompson, 2001). Hoyos-Leyva et al. (2015) reported the digestibility of HMT modified “Morado” banana starch. HMT treatment induced the formation of boilingstable RS and SDS, which were higher (30.7% and 12.1%, respectively) than native “Morado” banana starch (12.7% and 7.5%, respectively). HMT increased the phase transition temperature and enthalpy, as well as the crystallinity level; this is due to the rearrangement of the double helices packing within crystalline lamella. The thermal stability was evidenced for the increases of both crystalline percentage and double helices rearrangement, these results may be related to the concept that enzyme resistance is associated to the formation of crystalline material (Eerlingen and Delcour, 1995) or to the formation of double helices (Gidley et al., 1995).


847

4.3 SDS and RS in Gluten-Free Products The development of starches high in SDS and RS content allowed that different native and modified starches could be used in the design of functional foods for people with celiac disease. This disease is characterized by the restriction or prohibition of the consumption of foods with gluten. Gluten is found mainly in wheat that is highly used in the food industry for the production of foodstuffs such as bread, biscuits, snacks, and pasta. The search of new botanical starch sources with high content of SDS and RS has led to the design of diverse gluten-free food products with low caloric content. FloresSilva et al. (2014) designed extruded gluten-free spaghettis using unripe plantain, chickpea, and normal maize flours in different levels. Spaghettis formulated with 25%e30% of unripe plantain flour showed high quantities of RS (3.54%e4.14%) compared to control spaghetti (RS ¼ 2.02%), which was done with 100% semolina. In spaghetti, additional to formulation effect on starch digestion (protein, fat, and other organic compounds that alter starch digestion), extrusion produces changes in molecular organization within starch granules, which depends on cooking conditions in excess of water and heat. The quality of spaghettis is evaluated from cooking loss, firmness, and sensory panels. The use of flours with high-amylose content (up to 30%) results in higher cooking loss, due to amylose solubilization during cooking process (Flores-Silva et al., 2014; Hernandez-Nava et al., 2009). The addition of nongluten flours in the fabrication of spaghetti produces a weak network which interrupt and weaken the spaghetti structure (Hernandez-Nava et al., 2009; Rayas-Duarte et al., 1996). Modified starches are used in gluten-free foodstuffs which can influence rheological and thermal properties of the dough, such as water absorption, texture, bread staling (Ferreira et al., 2014; Witczak et al., 2010; Ziobro et al., 2012).

5. FUTURE TRENDS The main objective of the addition of RS-rich powders in foods is to increase the indigestible carbohydrate content (dietary fiber). However, the addition could modify some functional and sensory characteristics of the products (e.g., texture). Although diverse studies have shown the health benefits of the SDS fraction and many studies have shown how to modify starch structure to increase it, the reported values are still not enough for its inclusion as a functional ingredient regulated within the nutritional claim. Some studies reported that starch ingredients with 40% of the SDS fraction have a beneficial effect on the reduction of GI and controlled release of glucose in the small gut. The liberation of glucose from starch hydrolysis in the ileum produce signals in the brain that is associated with satiety. However, there is no official method for the determination of SDS fraction. On the other hand, processing and storage of cooked foods can induce the formation of SDS and RS fractions, but


the effect of process conditions and ingredients used in the formulation matrix related to the increase of both functional starch fractions is not well understood.

6. CONCLUSIONS There is interest of the food industry to develop starchy ingredients rich in SDS and RS fractions to modify the starch digestibility and increase the indigestible carbohydrates. Those SDS and RS powders can be used as ingredients in foods with health benefits for consumers. The addition of RS as a functional ingredient has been accepted in the development of functional foods mainly with low caloric content and high fiber content, due to the consumer’s requirement about healthy foodstuffs, but the role of SDS and the effect on human health is under discussion. On the other hand, how processing and storage conditions (moisture and temperature) can affect the production of SDS and RS in food to produce beneficial effects is a topic that deserves more investigation.

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852 PART j FOUR Starch and Health Liu, H., Guo, X., Li, W., Wang, X., Lv, M., Peng, Q., Wang, M., 2015. Changes in physicochemical properties and in vitro digestibility of common buckwheat starch by heat-moisture treatment and annealing. Carbohydrate Polymers 132, 237e244. Mangala, S.L., Udayasankar, K., Tharanathan, R.N., 1999. Resistant starch from processed cereals: the influence of amylopectin and non-carbohydrate constituents in its formation. Food Chemistry 64, 391e396. Maningat, C.C., Seib, P.A., 2010. Understanding the physicochemical and functional properties of wheat starch in various foods. Cereal Chemistry 87, 305e314. Manrique-Quevedo, N., Gonza´lez-Soto, R., Othman-Abu-Hardan, M., Garcıá-Sua´rez, F.J., BelloPe´rez, L.A., 2007. Caracterizacioń de mezclas de almidones de mango y pla´tano pregelatinizados mediante diferentes condiciones de extrusioń. Agrociencia 41, 637e645. Marsden, W.L., Gray, P.P., 1985. Enzymatic hydrolysis of cellulose in lignocellulosic materials. Critical Reviews in Biotechnology 3, 235e276. Menon, R., Padmaja, G., Sajeev, M.S., 2015. Cooking behavior and starch digestibility of NUTRIOSEÒ (resistant starch) enriched noodles from sweet potato flour and starch. Food Chemistry 182, 217e223. Miao, M., Jiang, B., Zhang, T., 2009. Effect of pullulanase debranching and recrystallization on structure and digestibility of waxy maize starch. Carbohydrate Polymers 76, 214e221. Miller, R.A., Jeong, J., Maningat, C.C., 2011. Effect of RS4 resistant starch on extruded ready-toeat (RTE) breakfast cereal quality. Cereal Chemistry 88, 584e588. Olayinka, O.O., Adebowale, K.O., Olu-Owolabi, B.I., 2008. Effect of heat-moisture treatment on physicochemical properties of white sorghum starch. Food Hydrocolloids 22, 225e230. Ozturk, S., Koksel, H., Kahraman, K., Ng, P.K.W., 2009. Effect of debranching and heat treatments on formation and functional properties of resistant starch from high-amylose corn starches. European Food Research and Technology 229, 115e125. Park, J.T., Rollings, J.E., 1994. Effects of substrate branching characteristics on kinetics of enzymatic depolymerization of mixed linear and branched polysaccharides: I. Amylose/ amylopectin a-amylolysis. Biotechnology and Bioengineering. Perera, C., Lu, Z., Sell, J., Jane, J., 2001. Comparison of physicochemical properties and structures of sugary-2 cornstarch with normal and waxy cultivars. Cereal Chemistry 78, 249e256. Pongjaruvat, W., Methacanon, P., Seetapan, N., Fuongfuchat, A., Gamonpilas, C., 2014. Influence of pregelatinised tapioca starch and transglutaminase on dough rheology and quality of glutenfree jasmine rice breads. Food Hydrocolloids 36, 143e150. Rayas-Duarte, P., Mock, C.M., Satterlee, L.D., 1996. Quality of spaghetti containing buckwheat, amaranth, and lupin flours. Cereal Chemistry 73, 381e387. Ren, C., Shin, M., 2013. Effects of cross-linked resistant rice starch on the quality of Korean traditional rice cake. Food Science and Biotechnology 22, 697e704. Rocha, T.S., Felizardo, S.G., Jane, J.L., Franco, C.M.L., 2012. Effect of annealing on the semicrystalline structure of normal and waxy corn starches. Food Hydrocolloids 29, 93e99. Sagum, R., Arcot, J., 2000. Effect of domestic processing methods on the starch, non-starch polysaccharides and in vitro starch and protein digestibility of three varieties of rice with varying levels of amylose. Food Chemistry 70, 107e111. Sajilata, M.G., Singhal, R.S., Kulkarni, P.R., 2006. Resistant starchda review. Comprehensive Reviews in Food Science and Food Safety 5, 1e17. Sanz, T., Salvador, A., Fiszman, S.M., 2008. Resistant starch (RS) in battered fried products: functionality and high-fibre benefit. Food Hydrocolloids 22, 543e549. Shin, S.I., Choi, H.J., Chung, K.M., Hamaker, B.R., Park, K.H., Moon, T.W., 2004. Slowly digestible starch from debranched waxy sorghum starch: preparation and properties. Cereal Chemistry 81, 404e408.


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854 PART j FOUR Starch and Health Zeng, F., Ma, F., Kong, F., Gao, Q., Yu, S., 2015b. Physicochemical properties and digestibility of hydrothermally treated waxy rice starch. Food Chemistry 172, 92e98. Zhang, G., Ao, Z., Hamaker, B.R., 2006a. Slow digestion property of native cereal starches. Biomacromolecules 7, 3252e3258. Zhang, G., Ao, Z., Hamaker, B.R., 2008a. Nutritional property of endosperm starches from maize mutants: a parabolic relationship between slowly digestible starch and amylopectin fine structure. Journal of Agricultural and Food Chemistry 56, 4686e4694. Zhang, G., Hamaker, B.R., 2009. Slowly digestible starch: concept, mechanism, and proposed extended glycemic index. Critical Reviews in Food Science and Nutrition 49, 852e867. Zhang, G., Sofyan, M., Hamaker, B.R., 2008b. Slowly digestible state of starch: mechanism of slow digestion property of gelatinized maize starch. Journal of Agricultural and Food Chemistry 56, 4695e4702. Zhang, G., Venkatachalam, M., Hamaker, B.R., 2006b. Structural basis for the slow digestion property of native cereal starches. Biomacromolecules 7, 3259e3266. Zhang, X., Chen, Y., Zhang, R., Zhong, Y., Luo, Y., Xu, S., Liu, J., Xue, J., Guo, D., 2016. Effects of extrusion treatment on physicochemical properties and in vitro digestion of pregelatinized high amylose maize flour. Journal of Cereal Science 68, 108e115. Ziobro, R., Korus, J., Witczak, M., Juszczak, L., 2012. Influence of modified starches on properties of gluten-free dough and bread. Part II: quality and staling of gluten-free bread. Food Hydrocolloids 29, 68e74.

Chapter 23

Starch: Physical and Mental Performance, and Potential Health Problems Suzanne Hendrich

Iowa State University, Ames, IA, United States

1. INTRODUCTION 1.1 Types of Starch, Including Digestion-Resistant Starches Starches are glucose chains readily digested by mammalian amylases. Native food starches are composed of amylose (long glucose chains with a-(1-4) linkages) and amylopectin (branched glucose polymers with a-(1-4) (1-6) linkages). Starches as functional food additives may be modified in several ways. Pregelatinization allows for more rapid cooking of starchy foods. Dextrinization by heating can create a variety of useful products such as starch with greatly increased water solubility. Esterification to form carboxymethylstarch can improve freezeethaw stability. Cross-linking produces starches with greater resistance to retrogradation (pudding mixes) (Tharanathan, 2005). However, dietary intake of modified starches is probably generally less than 100 mg/day based on a survey of intake in the UK (Co, 2005). This review will focus on starches according to their physiological effects, characterized as rapidly digestible (RDS), slowly digestible (SDS), or resistant to mammalian enzymatic digestion, resistant starch (RS) (Englyst et al., 1992).

1.2 Starch Digestion and Glucose Bioavailability, Starch Residue Fermentation and Products Maintaining adequate blood glucose normally depends on the dynamic effects of insulin and glucagon in response to feeding of glucose sources (mainly dietary starches) and glucose storage as glycogen. As the body digests starch, blood glucose increases, which lead to increased blood insulin, facilitating tissue uptake of glucose for its use for energy from glycolysis. Ingested glucose may also be stored as glycogen, especially in muscle and liver. Starchy Starch in Food. http://dx.doi.org/10.1016/B978-0-08-100868-3.00023-8 Copyright © 2018 Elsevier Ltd. All rights reserved.

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foods typically predominate in RDS, which results in a postprandial fluctuation in blood glucose, with peak blood glucose occurring 30e60 min after a meal and declining to fasting state within w2 h in individuals with normal glucose metabolism. Ingestion of SDS blunts and slows this pattern of glucose metabolism. Ingestion of RS further blunts blood glucose response, depending on the RS:RDS proportion in the meal (Birt et al., 2013). RS also provides fermentable glucose to gut bacteria in the lower intestine. Increased short chain fatty acids are a major product of this fermentation, with butyrate especially increased as shown by in vitro anaerobic human fecal incubations with resistant-starch residues (Li et al., 2015). Butyrate is a key substrate that seems to maintain normal colonocyte function. Butyrate metabolism in the gut may have implications for physical and cognitive performance but such investigations are in early stages. The health of our brains and our muscles may depend on dietary starches; nuances discovered or engineered into starch structures may be important in optimizing key physiological functions.

1.3 Major Theories or Hypotheses Regarding Effects of Starches on Physical Performance Whereas the brain relies on the liver to provide its steady source of glucose, muscle takes up glucose, stores it as glycogen, and cannot release this glucose to other tissues, which indicates the importance of glucose as a muscle fuel. During very high-intensity physical activity such as a sprint, glucose is the fuel source. Generally, glycogen stores vanish after about 3 h of moderate intensity physical activity; energy comes increasingly from circulating fatty acids during and after this time. The merits of SDS versus RDS during and before long duration physical activity is under investigation. Supplying carbohydrates that require minimal to no digestion (sucrose or glucose) at 15e20 min intervals during exercise may benefit performance (Rodriguez et al., 2009). The merits of supplying carbohydrate as a mixture of glucose and other sugars rather than glucose alone to permit most rapid absorption of carbohydrate energy source is also under study. The hypothesis is that because other sugars use different transporters than glucose, availability of glucose energy is sped by ingestion of mixed sugars over ingestion of glucose alone. Marathon runners or others performing long-endurance events may benefit from building extra muscle glycogen stores in the short term before an event. The practice of carbohydrate loading, usually from dietary starches has moderate research support (Rodriguez et al., 2009). Depleting glycogen stores before carbohydrate loading by eating a diet lower in carbohydrates, substituting dietary carbohydrate energy for fat may further build glycogen stores before a longendurance event. Availability of glucose before, during, and in recovery from exercise, and therefore the role of various forms of dietary carbohydrates including starches in exercise performance is a topic of great interest analyzed in this review.

Starch: Physical and Mental Performance Chapter j 23

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1.4 Major Theories or Hypotheses Regarding Effects of Starches on Mental Performance After meals and overnight, liver glycogen stores mobilize with the help of glucagon to maintain a steady supply of glucose for the brain, which requires glucose for energy. Cognitive function requires relatively constant availability of glucose. Cognitive impairment in people with type 1 diabetes (T1D) is associated with hypoglycemia after insulin injections (Tonoli et al., 2014). Carbohydrate intake before activities requiring mental effort may benefit performance, but all macronutrients benefit postmeal cognitive function (Greenwood, 2003). Impaired glucose tolerance is associated with cognitive impairment (Messier and Gagnon, 2000), but the causes of this phenomenon are not fully understood. This review will examine studies relating dietary starches and various aspects of cognitive function.

1.5 Major Theories or Hypotheses Regarding High Starch DietsdAcute and Long-Term Adverse Effects Dietary starches fuel the brain and muscles and may affect performance of both organs. Discovery and engineering of novel starches may have implications for the physiological functions of starch, for benefit in optimizing body fuel under a range of conditions of environment and other stresses. To assure such benefits and prevent unanticipated harm, the toxicity of novel starches must be assessed. “The dose makes the poison” (as paraphrased from Paracelsus). This is a central tenet of toxicology, and even seemingly benign or beneficial molecules such as glucose, the digestion product of conventional starches, may be toxic. Human glucose toxicity may occur during hyperglycemia from poorly controlled diabetes. Hyperglycemia increases advanced glycation end products (AGEs) that arise from interactions between glucose and free amines in body proteins. AGEs may also arise from oxidative stress reactions prominent in aging. AGEs interact with their cellular receptors (RAGE) to cause inflammation damaging small blood vessels especially in kidney and retina, leading to severe renal disease and blindness (Wautier et al., 2016). Dietary starches may contribute to such pathologies. This review will attempt to evaluate that statement and examine how starch modifications, especially modifications to starch digestibility influence the ability of starches to contribute to hyperglycemia.

2. DIETARY STARCHES AND PHYSICAL PERFORMANCE 2.1 Effects on Physical PerformancedAnimal Studies Dietary starches may benefit endurance exercise in rodents. Male BALB/C mice ingested water, glucose, maltodextrin, or natural or synthetic glycogen (SG) in solutions. Blood glucose response was significantly less from 0 to


60 min after ingestion of glycogen than after ingestion of glucose. Glucose and SG showed similar blood glucose responses from 60 to 120 min after dosing. Insulin response was blunted after glycogen ingestion compared with glucose. Muscle glycogen was similar after 2 h of exercise in mice given glucose or SG at 20 min intervals during exercise. The low osmotic pressure exerted by SG allowed it to more rapidly empty the stomach than glucose. This work suggested that SG might tend to support sustained physical performance better than glucose (Inagaki et al., 2011). Highly branched cyclic dextrin (HBCD), a synthetic starch, increased swimming endurance and caused blunter blood glucose and insulin response in male mice given the treatments 10 min after beginning exercise. If given 30 min after exercise, endurance effects were the same between glucose and HBCD (Takii et al., 1999); 500 mg/kg doses were needed for the enhanced endurance effect of HBCD, which would correspond to a human dose of 50 mg/kg or 3.5 g/70 kg adult according to the 10-fold greater surface area/kg of mice compared with humans. Gavage of male ICR mice with glutinous rice (GR) or GR amylopectin for 7 days at 7.5 or 15 g starch/kg increased blood lactate dehydrogenase activity similarly after a forced swim test, indicating improved lactic acid metabolism by the starch which, in theory, should reduce fatigue (Zhang et al., 2014). Male Wistar rats fed high-fructose/fat diet (HFHF) versus corn starch-based diet showed diminished running time after 16 weeks on the diet. The HFHF rats developed impaired glucose tolerance and obesity, signs of metabolic syndrome (Cameron et al., 2012). This suggests that a starch-based diet supports better health and improved exercise performance in comparison with a diet that might simulate human diets containing lots of fats and sweets. Another rodent model showed benefits of RS. Male Wistar rats selected for ability to gain weight on a diet containing 46% of energy from fat were fed this diet for 16 weeks to create obesity. Then the rats were calorie-restricted for 2 weeks to achieve w17% weight loss and randomized to a diet with 40% of energy from fat with/without 5.9% RS and with/without 30 min of treadmill exercise per day. Exercise lessened weight regain more than RS, the combination was only as effective as exercise alone (Higgins et al., 2011). To achieve a similar effect in postobese humans might require increasing typical RS intake in the United States by 50%; a serving per day of legumes might suffice (Hendrich, 2010), but it is prudent not to extrapolate too directly from rats to humans.

2.2 Effects on Physical PerformancedHuman Studies When carbohydrates and exercise are linked, many people first think of carbohydrate loading, a process of enhancing glycogen stores in preparation for long-term endurance activity that must rely to a significant extent on energy from glucose. Many, but not all, studies of carbohydrate loading support the benefit of this practice, and specific recommendations are made by the American College of Sports Medicine regarding how best to accomplish


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carbohydrate loading when necessary (Rodriguez et al., 2009). Eight endurance-trained cyclists consumed 10.5 g carbohydrate/kg (as potato starch) or their typical 6 g carbohydrate/kg for 3 days before a 3 h cycling trial. Increased dietary carbohydrates increased muscle glycogen measured before exercise, power output, and distance traveled per hour were enhanced after carbohydrate loading (Rauch et al., 1995). No recent studies have examined the effect of starch type on carbohydrate loading efficacy, but seemingly, starches that yield the greatest available glucose would be of the most benefit. The time course of the digestion and uptake of such starches (e.g., RDS vs. SDS) might not matter very much if at all to the ultimate accumulation of muscle glycogen during a few days of carbohydrate loading and rest. Human studies are practically more useful than animal models in showing the comparative efficacy of starches in exercise performance. Six endurancetrained male cyclists showed slower oxidation of a 22-unit glucose polymer than of soluble starch (the longest chains of glucose (with some branching) that are soluble in water) during 90 min of exercise. Both forms of glucose were similar in gastric emptying, so probably the soluble starch was more rapidly digested than the glucose polymer (Hawley et al., 1991). The implication of this finding is that soluble starch may be a better energy source during long-term exercise than a glucose polymer, but many other variants of starch structure need further study. Eight male cyclists exercised for 60 min to deplete muscle glycogen, then ate 3000 kcal over 12 h with 65% of energy from glucose, maltodextrin, amylopectin, or amylose (RS). Muscle glycogen replenishment at 24 h after the 60 min exercise bout was 50% less from ingesting amylose than from the other glucose sources, but 30 min time trial performance at this 24 h time point did not differ with dietary treatment (Jozsi et al., 1996). Long-term exercise performance might be impaired with lesser muscle glycogen stores, but this dietary condition of consuming only amylose as dietary carbohydrate source would be unlikely to occur in reality. Twenty-four males of similar fitness exercised for 2 h on a cycle ergometer ingesting drinks with 15 g maltodextrin or HBCD at 1 h. The HBCD treatment significantly lessened rating of perceived exertion (RPE) during the second hour of exercise compared with maltodextrin, perhaps due to more rapid gastric emptying and availability of HBCD (Furuyashiki et al., 2014). In a cross-over design, seven male triathletes completed 5 km running, 40 km cycling, and 5 km running, repeated after a month, ingesting HBCD or glucose drinks during the event. Plasma noradrenalin and urinary IL-8, IL-10, and IL-12p40 concentrations were significantly less after HBCD than glucose treatment, suggesting a benefit of HBCD to postexercise stress (Suzuki et al., 2014). Nine male cyclists completed 150 min of exercise and a bout of cycling to exhaustion after ingesting 1 g/kg of either hydrothermally modified low glycemic index starch (HMS) or maltodextrin, then ingested the same doses of the two starches at the beginning of their recovery from exercise (Roberts et al., 2011). The HMS


treatment caused greater utilization of fatty acids during exercise and recovery than did maltodextrin, suggesting that HMS may have had a glycogen-sparing effect compared with maltodextrin, but the starches did not differ in effects on physical performance in this study. Another study of 10 male cyclists showed that HMS ingested during exercise (60 min cycling followed by higher intensity intervals and then maximal sprints) was similar in effects on performance to a commercial sucrose/glucose beverage ingested isocalorically compared with HMS (Baur et al., 2016). HMS increased gastrointestinal distress during sprints compared with the commercial beverage. Although HMS showed increased fat oxidation during exercise compared with commercial beverage, it did not relatively enhance performance. Comparing starch types (amylopectin vs. amylose vs. other starch modifications) with glucose during exercise and in exercise recovery after even longer exercise periods with greater numbers of participants, especially including women, would seem to be important areas of further study about starch and exercise. Greater focus on performance results related to starch such as distance/time and RPE will aid discernment of the practical benefits of starch ingestion to exercise. Researchers have begun to study effects of starches on exercise during diabetes. Seven participants with T1D ingested glucose or waxy barley starch (WBS, high in amylopectin) 2 h before running on a treadmill at intervals of increasing intensity (5 min 4 min bouts over 26 min) and a 10 min running trial (Gray et al., 2015). WBS increased carbohydrate oxidation during rest compared with glucose and increased running distance in the last 2.5 min of the running trial, suggesting a modest performance benefit in this population. This research reinforces the feasibility of exercise without adverse effects in people with T1D. Another study compared a vegetarian diet with 60% carbohydrate with a conventional diet (50% carbohydrate) for 12 weeks at 500 kcal restriction of daily energy requirement in people with type 2 diabetes (T2D) along with personalized daily exercise programs. The vegetarian diet improved fitness of these individuals (N ¼ 37/group), but the conventional diet did not (Veleba et al., 2016). This suggests that a diet higher in complex carbohydrates, as the vegetarian diet was likely to be, might benefit people with T2D, as improved physical fitness may tend to help people persist with increased physical activity.

2.3 Summary Much work remains to determine what starch types might optimize exercise performance, but some studies indicate possible performance benefits, sparing glycogen and enhancing exercise function with specific starches, compared with glucose. Overall dietary content of carbohydrates and what mixtures of carbohydrates benefit health, exercise, and mitigate chronic diseases in the long term remain topics worthy of further study, based on a few intriguing recent studies.


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3. DIETARY STARCHES AND MENTAL PERFORMANCE 3.1 Effects of Starches on Mental PerformancedAnimal Studies Sensory response, emotional state, and cognition are major dimensions of mental performance assessable in animals, with varying degrees of concordance with human mental functions. Influences of carbohydrates on these functions are not well studied, but some intriguing recent work suggests a need to advance this field of work. The behavior of 20 gorillas of various ages was observed across 5 zoos that switched animals to a diet lower in starch from w300 to w50 g/day; the revised diets also contained about three times more fiber than the previous diet. The extent of regurgitation/reingestion, a maladaptive behavior not observed in wild gorillas, was significantly less after this diet switch, and they also spent significantly more time eating (due to lesser caloric density of the new diet) (Less et al., 2014). Although this was far from a well-controlled study, it suggests that starch might exert an adverse behavioral effect in some contexts. Groups of 10 male Wistar rats given 10% sucrose or isocaloric maltodextrin to drink showed weight gain over controls drinking water, and in study maltodextrin caused greater weight gain and liquid calorie intake than did ingestion of the sucrose drink. Both carbohydrates decreased location ability of rats after 17 days on treatment compared with controls (Kendig et al., 2014). Groups of 16 male Swiss-Webster mice were fed 55% cornstarch, high-amylose corn starch, or a chemically modified high-amylose corn starch (12%e13% dietary RS) for 6 weeks. The RS-fed mice travelled less distance in an open field text, and the high-amylose-fed mice spent less time in open arms of a maze, but the mice fed the modified starch behaved similarly to mice fed cornstarch in the maze (Lyte et al., 2016). This suggests that some forms of RS may stimulate anxiety. These behavioral differences accompanied significant shifts in gut bacteria. Increased Actinobacteria due to high-amylose RS feeding compared with corn starch-fed mice was found. It will be important to determine if other fermentable dietary fibers exert similar behavioral effects. This field needs additional animal models and human studies. Gut/bacterial interactions may influence behavior possibly mediated by neuroendocrine factors produced by bacteria. The influences of dietary composition on gut microbial speciation may have profound significance for behavior.

3.2 Effects of Starches on Mental PerformancedHuman Studies Effects of carbohydrates on human cognition seem to be a needed but understudied area. The worldwide epidemic of T2D has stimulated research on adverse cognitive effects of this condition. Cognitive decline associated with T1D has also been studied. A recent meta-analysis of several hundred children with T1D showed that individuals with T1D scored worse than control subjects


without T1D on overall IQ, executive function, and motor speed, with worse effects the earlier onset of T1D and the poorer blood glucose control. In adults with T1D, the same cognitive impairments were seen as in children and memory was also impaired compared with control subjects without T1D. Seemingly, the longer the duration of T1D, the more cognition may be impaired, and improving blood glucose control as measured by hemoglobin A1C might be a key target (Tonoli et al., 2014), which suggests a possible role for resistant-starch food products in helping to lessen overall blood glucose responses to diet. Impaired glucose tolerance relates to cognitive decline with aging. Connections between adiposity, aging, and cognitive function have also involved sleep behavior. Sleep deprivation impairs cognitive function. One recent study hypothesized that hypoglycemia would exacerbate cognitive impairment associated with shorter sleep. In a randomized cross-over design, seven healthy men experienced total, moderate or no sleep deprivation, and the next morning had a hypoglycemic clamp by insulin infusion to achieve blood glucose concentration of 2.5 mmol/L for 30 min. Hypoglycemia and sleep deprivation impaired their reaction time and auditory-evoked potential, but hypoglycemia did not worsen these responses after total sleep deprivation (Jauch-Chara et al., 2010). This result indicates that blood glucose status does not mediate effects of sleep deprivation on mental function. The results also suggest reason to further study the role of availability and patterns of uptake of glucose in mental function, possibly affected by type and amount of dietary starch. Starch intake may be relevant to cognitive decline associated with aging, T2D and blood glucose control (Greenwood, 2003) in several ways. Highly palatable diets with greater fat and refined carbohydrate content are associated with obesity and its attendant greatly increased risk for T2D, which is associated with impaired cognition. Individuals with impaired glucose tolerance or lesser ability to rapidly clear glucose from the blood after a meal showed impaired verbal memory (Greenwood, 2003). Mean amplitude of glycemic excursions over 48 h of continuous glucose monitoring in 121 individuals with T2D was strongly correlated with cognitive function measured by the Mini Mental Status Exam (r ¼ 0.83) and a cognitive score based on executive function and attention (r ¼ 0.68) (Rizzo et al., 2010). Such studies suggest that starches with lesser ability to increase blood glucose (i.e., RSs) might prevent memory and other cognitive deficits. Food combinations that blunt postprandial blood glucose responses might have similar efficacy, such as increasing content of some dietary fibers in meals or avoiding meals high in carbohydrates in favor of more balanced mixtures of macronutrients. The body’s response to glucose involves secretion of intestinal glucagon-like protein-1 (GLP-1) which facilitates insulin action and glucose uptake. Neurons also produce GLP-1. GLP-1 receptor knockout mice show impaired memory and glucose intolerance, so this suggests a connection between glucose regulatory status and cognitive function (Burcelin, 2005). Dietary


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starch quality and content would be worthy of further investigation as to its influence on GLP-1 function. Postprandial effects of carbohydrates on some aspects of memory contrast with the findings of Greenwood (2003). Groups of 11 men or women, 60e89 years of age consumed 185 calorie drinks from safflower oil, whey protein (WP), or glucose or a noncaloric placebo. At 15 min after this breakfast, all caloric drinks improved cognitive performance on a paragraph recall (PR) test, and at 60 min on a delayed PR test. Only glucose improved connect-the-dot test score at 60 min after the meal (Kaplan et al., 2001). In young men (N ¼ 17) who ate 400 kcal meals of only carbohydrate, protein, or fat, memory test scores were greater after eating fat compared with carbohydrate (Fischer et al., 2001). Researchers compared rice flour starch, sucrose, and a nonstarch polysaccharide (NSP, Ambrotose complex) for effects on memory in groups of w25 adults, aged 45e60. Each group consumed 4 g of each carbohydrate and 100 mL water, and cognitive function tested before and 30 min after ingestion. Postdose decline in the Rey Auditory Verbal Learning Test (RAVLT) was prevented by NSP, postdose memory declined significantly after sucrose or starch intake, which both increased blood glucose at 30 min after ingestion, whereas NSP did not increase blood glucose (Best et al., 2015). A 12-week trial of the same NSP versus rice flour placebo ingested at 4 g twice-daily by groups of w50 adults aged 45e60 years showed improved RAVLT scores after ingesting NSP compared with rice flour starch (Best et al., 2010). In a randomized cross-over design, 47 young adults (mean age 23 years) ingested 5 g of oligofructose-enriched insulin or 5 g maltodextrin with a noncaffeinated beverage, cereal, and toast with jam. Two hours later, they performed cognitive tests. Inulin improved recognition memory and immediate and delayed recall (Smith et al., 2015). It is important to see if a placebo that did not increase blood glucose would have the same effect as NSP or inulin. In one study of 17 men of mean age 29 years, cognitive function test scores did not differ between an artificially sweetened placebo compared with 25 g glucose beverages (Ullrich et al., 2015). Because of the short duration of these studies, it is unlikely that the nondigestible carbohydrates (e.g., NSP) would be exerting their effects on the nervous system through their fermentation products. Research might well examine additional types of nondigestible carbohydrates such as RS for effects on cognition. Further work in this field may discover novel mechanisms of action, perhaps related to polysaccharide interactions with receptor proteins on intestinal cell surfaces that induce gutebrain interactions. Other aspects of mental performance and behavior connect with starch intake. Shorter sleep duration has been associated with obesity. European children (N ¼ 4300) were stratified according to amounts and types of carbohydrates (sugar, starch) eaten across daily meals and their sleep duration. The children in the highest quartile of starch intake at lunch and with shorter


sleep duration had an increased risk of higher body mass index (BMI). Those in the highest quartile of starch intake at breakfast also showed greatest risk for high BMI, whereas eating breakfast in general was associated with lower BMI (Hunsberger et al., 2015). Because of the link between obesity and overeating, the role of starches in hunger and fullness has also been studied, but starch quality does not seem to affect hunger responses much if at all in humans. Eight lean and eight obese women (mean age 46 years) consumed 400 kcal pancake breakfasts containing waxy maize starch, a chemically modified RS or either starch and 20 g WP. The combination of RS þ WP increased fat oxidation over 180 min after the meal and PYY at 180 min after the meal, compared with other treatments. WP significantly decreased hunger and increased satiety (Gentile et al., 2015). This study suggests that interactions between starches and other food components might be worth further study in developing antiobesity strategies.

3.3 Summary Because of the important of glucose regulation in cognitive function and the epidemic of glucose intolerance, dysregulation, and T2D, starch quality deserves more research in its effects on cognitive function, especially for the long term. A few studies suggest the possibility that substitution of highly digestible dietary starches with modest amounts of undigestible polysaccharides might modestly benefit cognition, especially some aspects of memory. Wellstandardized cognitive function tests are available, and collaborations between neuroscientists, psychologists, and nutritionists are essential for appropriate design of observational and interventional studies. Scientists must give more consideration to the nature of the placebo or control in human studies of carbohydrate effects on cognition. A “placebo” such as starch with obvious physiological effects to increase blood glucose makes difficult interpretation of cognitive effects of other treatments that do not have this physiological effect. More realistic dietary conditions in such experiments will facilitate their relevance.

4. DIETARY STARCHESdADVERSE EFFECTS 4.1 Animal Models Animal testing of dietary starches as food ingredients is prerequisite for such ingredients to be used in the human food supply. With advances in food chemistry, novel starch-derived ingredients may have interesting applications. Alpha-cyclodextrins (a-CD) are naturally occurring or enzymatically synthesized in vitro. They can help create delivery systems for lipid-soluble substances because they can form inclusion complexes with these molecules. Subchronic (90 days) general toxicity and genotoxicity tests of these compounds at up to 20% by weight of diet showed no adverse effects. A study


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feeding 0%, 1.5%, 5%, 10% or 20% a-CD versus 20% lactose control by weight of diet to groups of 25 pregnant Wistar rats from day 0 to 21 of gestation showed no adverse effects on pregnancy, and no fetoxicity or teratogenicity (Waalkens-Berendsen and Bar, 2004). Roasted wheat starch dextrins have rearranged glucoseeglucose bonds that decrease their digestibility, thus creating a type of RS, which as a fermentable dietary fiber may increase beneficial gut bacteria. A single dose of 2000 mg wheat dextrin/kg given by gavage to 50 female Sprague-Dawley rats showed no adverse signs over 14 days after dosing. A 90 days feeding of 0%, 1.25%, 2.5%, or 5% wheat dextrin by weight of diet also showed no adverse effects on histopathology or blood chemistry in groups of 20 male and 20 female Sprague-Dawley rats. Mutagenicity tests of the wheat dextrin by Ames assay or mouse lymphoma cell assay were negative (Wils et al., 2008). HBCD is used in sports beverages in Japan. Five male and five female SLC: Wistar rats were gavaged with 2000 mg HBCD/kg and showed no adverse effects over 14 days after dosing. HBCD was nonmutagenic in Ames assays (Choi et al., 2009). These studies support the safety of novel starch-derived food ingredients, but reports are lacking on chronic studies of such products.

4.2 Human Studies Several studies have examined the role of dietary starch in human health across the lifespan. In 2006, the EU set standards for total carbohydrate content of infant formulae of 9e14 g/100 kcal. Standards for added sugars in baby foods were set at w5 g/100 kcal, and total carbohydrate standards were set at 5e25 g/100 g, depending on the food type. In about 20 studies of carbohydrate intake of children ages 1e4 years, starch intake was slightly greater than 20% of energy and intake of sugars was slightly less than 30% of energy. Limits on sugar in baby foods might tend to limit the early preference for sweet taste that can establish a lifelong preference for sweetness, which might increase preference for an obesogenic diet. Decreasing the extent of early dental caries is also a reason for limiting early sugar intake. But little research exists on the effects of carbohydrate quality in complementary feeding and child development or long-term health effects. The brain requires glucose and a few studies showed benefits of glucose feeding on memory and attention in infants and young children. A diet of 40%e55% carbohydrate at a minimum was recommended for young children (Stephen et al., 2012). Dietary starches should reasonably constitute a significant portion of that total. No studies suggest adverse effects of dietary starches in early life. From principle components analysis of food intake of 388 rural South African adolescents (ages 11e15), greater intake of meat or of starch and folate were associated with greater BMI, but the association of starch and folate intake with BMI disappeared when adjusted for physical activity, socioeconomic status, and maternal education level (Pisa et al., 2015). This


suggests that dietary guidance for adolescents cautioning against overintake of meat and emphasizing healthier carbohydrate sources such as whole grains might be important to prevent greater BMI and its attendant presumptive longterm health risks (e.g., T2D). Data from the National Health and Nutrition Examination Survey (NHANES, 2009-10) of food recall data from 1200 adults, mean age of 41 years, showed that increased fruit and nonstarchy vegetable intake, excluding juices, was associated significantly with decreased BMI and waist circumference, but only in survey participants who did not exercise 150 min/ week or more (Cavallo et al., 2016). Because fruits and nonstarchy vegetables contain little to no starch, we may infer that the dietary substitutions made to increase fruit and vegetable intake would also tend to lessen overall starch intake. Thus, this study suggests that greater starch intake might adversely influence cardiovascular disease risk factors. However, many foods high in starch such as legumes and whole grains also contain dietary fibers, including RSs that would lessen cardiovascular disease risk. A recent review supported this idea, and especially noted the finding of the Nurse’s Health Study showing the least risk for T2D in women in the lowest tertile of glycemic index in their food intake and the highest tertile of cereal fiber intake (Maki and Phillips, 2015). A few recent human epidemiological studies associated high-starch intake with greater adiposity. In Sri Lanka, waist circumference, a risk factor for T2D, and carbohydrate intake across 100 women (ages 20e45) was significantly correlated (r ¼ 0.663). Diets in these women were predominantly rice-based, so starch intake was relatively great with total carbohydrate intake at w70% of daily energy (Rathnayake et al., 2014). In Northern China, 4154 participants, mean age of 54 years, were assessed for signs of metabolic syndrome and hyperlipidemia over 4.2 years, in association with intake of carbohydrates from starchy foods (rice, wheat, tubers). Those in the highest quartile of intake of carbohydrates from starchy foods had 50% greater risk of metabolic syndrome than those in the lowest quartile of intake of these foods (Feng et al., 2015). A threshold of increased risk for metabolic syndrome was estimated to be 220 g carbohydrate/day. A study of 132,479 participants (UK Biobank) assessed dietary intake by 24 h recall showing a significant positive association of BMI with increasing intake of starch or fat across all quintiles of intake. When adjusted for physical activity and total energy intake, starch intake was negatively associated with BMI across all quintiles of intake, and only fat intake remained positively associated with BMI across all quintiles of intake (Anderson et al., 2016). This study rebutted the current advice in the UK to limit sugar intake to decrease adiposity and emphasized a focus on fat and total energy intake rather than carbohydrates. Overall, physical activity seems to mitigate the adverse association of starch intake with increased BMI and risk for obesity-associated diseases. Another study considered the influence of high-starch intake on blood pressure in a combined group of 4680 men across several countries. This study showed that those at 2 standard deviations


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above the mean (w15% of energy or w120 g/day greater starch intake) had w1 mmHg lower diastolic and systolic blood pressure than the mean for this population sample (Brown et al., 2009). This study also refuted an assumed aspect of adverse effects of high-starch intake. An examination of adverse effects of starch needs to consider digestionRSs as well. Starchy foods are generally readily digestible and elevate blood glucose rapidly and greatly. This effect may be mitigated to some extent by the formation of RS from retrogradation after starch gelation. This is fairly well studied for potatoes. No matter the type of initial cooking, cooling potatoes increases RS and diminishes glycemic response to potatoes by w25% (Tian et al., 2016). It may be that this phenomenon blunts adverse effects of starch ingestion, but that remains to be seen. A review of RS safety studies showed that humans tolerated up to 60 g/day of RS without significant adverse effects (Goldring, 2004). Researchers observed some gastrointestinal symptoms such as bloating or flatulence. This dose is roughly the equivalent of 30 g dietary fiber per day. Animal studies have shown either increased or decreased incidence of colonic preneoplasia or neoplasia after RS feeding (Goldring, 2004). Short-term studies in humans with history of colon adenomas showed no adverse effects on colon cell proliferation of RS ingestion. More studies of RS and colon health are warranted. Current US FDA regulatory guidance includes a petition process for ingredient manufacturers to show scientific evidence that a novel product has beneficial physiological effect on human health consistent with dietary fibers. Those effects include but may not be limited to lowering blood glucose or cholesterol levels, lowering blood pressure, improved laxation and bowel function, increased mineral absorption in the intestinal tract, reduced energy intake, or promoting a feeling of fullness (US FDA, 2017).

4.3 Summary Studying adverse effects of starches, especially starch ingredients, continues to be needed in advancing food product development. Generally, food starches are well tolerated and pose little risk for adverse effects except those effects associated with long-term habits of overconsumption. But physical activity seems to mitigate increased risk for metabolic syndrome and obesity associated with diets high in starches.

5. CONCLUSIONS Glucose is a crucial fuel for muscles and the brain and dietary starches are the main providers of glucose in human diets. Replenishment and fortification of muscle glycogen stores is a concern mainly for extreme endurance athletes. Optimizing and individualizing starch ingestion to meet these special needs may reasonably involve design or discovery of novel food starches. There may be an additional opportunity to design starches that release glucose slowly and


adequately over two or more hours of physical activity, as a possibly more convenient substitute for the recommended intake of 20 or more grams of glucose every 20 min during a long activity. Adverse gastrointestinal symptoms might prove an important barrier to development of such products. Novel starches that fuel the brain optimally may be another research horizon. Much remains to be understood in the association of dietary and other habits with prevention of T2D, its attendant effects to diminish cognitive function and other chronic diseases of cognitive decline. Tighter regulation of blood glucose may be better for cognition. This suggests an opportunity for discovery and further development of diets and starches that provide glucose steadily, and without large spikes in the blood. Nutritional sciences clearly have a crucial role in cognition research in developing experimental diets and controls. These diets or foods need to simulate realistic human ingestive behavior. Recommendations for human dietary patterns that relatively mitigate development of cognitive decline must include recommendations for dietary starch content and type. There seems to be a certain symmetry in the findings that greater physical activity prevents adverse effects of eating a diet containing more starch, since greater physical activity and the need for dietary starch go hand in hand. Starch deserves more attention as a foundation of a healthy diet for physically active and cognitively sound humans.

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Index ‘Note: Page numbers followed by “f” indicate figures and “t” indicate tables.’

A AACC 76-21 method, 662e665 A/B1/B2 and B3 chains, 359, 360f Acetyl, 287e288 Acetylated starches, 365 Acetylation, 297e299 A-chains, 98e99 Acid hydrolysis, 291e293, 292f, 693, 694f A-crystalline, 356 ActiStar RM resistant starch, 837e839 ADP-Glu binding, 22e24, 23fe24f ADP-glucose pyrophosphorylase (ADP-Glc PPases) amino acid residues Arg, 45e46 Asp142, 45 Asp145, 45 Asp160, 45 Asp253, 46 Lys195, 44 Lys198, 44 Lys213, 44 pyridoxal-5-phosphate (PLP), 45 Tyr114, 44 Glu-1-P-binding site apparent affinity, 13e15, 14t LK44R/T54K, 15, 16f kinetic and regulatory properties, 7e8, 8t mechanism of activation, thioredoxin, 28e30 3PGA and Pi, 42e44, 43t physiological importance, 27e28 plant sources Arabidopsis thaliana, 41e42 barley, 30e36 Chlamydomonas reinhardtii, 30 endosperm, cereal grains, 36e37 kinetic constants, 30, 31te35t maize endosperm, 37e38 pea embryos, 37

rice endosperm and leaf, 40e41 tomato fruit, 38e39 wheat, 39e40 regulatory domain characterization, 47 structure-function relationships, 9e11, 10t Adsorbent agents, 734e735 Agricultural biotechnology, 180 Agrobacterium tumefaciens, 27, 45e46 Alcohol production process, 645 Ales, 637 Allosteric regulation, 26e27 Alpha-amylase, 636 Alpha-glucosidase, 637 Amaranth starch, 524 amylopectin, 526e527 amylose, 526 chemical modification, 530 digestibility, 530 food applications, 530e531 freezeethaw stability, 529e530 granule shape and size, 525e526, 525f minor constituents, 525 pasting properties, 527, 529f production, 525 retrogradation, 527e529 starch crystallinity, 526, 526f structure and functionality, 525e530 swelling power and solubility, 527 thermal properties, 527, 528t, 529f Amide, 287e288 Amine, 287e288 Ammonium sulfate, 17 a-Amylase hydrolysis, 785e786 Amyloglucosidase, 271 Amylomaltase-treated starches, 368, 368f Amylopectin, 97, 152, 356, 357te358t, 359e363, 360f, 360t, 362f, 382e385, 383f, 384t, 386t, 547, 595, 664t branch chains, 6 building blocks

873

874 Index Amylopectin (Continued ) Haworth conformation, 121 interblock chain length (IB-CL), 122e123 a-LDs, 122 Staudinger conformation, 121 C-chain distribution, 113 chain debranching, 194 chain disproportionation, 195 cluster model, 5, 5f cluster structure, 596 concentrations, 586e587 enzymes use, 108, 108f external and internal chain distribution b-limit dextrin (b-LD), 113 phosphorylase, 114e115 pullulanase, 115 sweet potato b-amylase and rabbit muscle phosphorylase, 113e114, 114f tangential flow filtration (TFF), 113e114 type 1 amylopectins, 116 type 2 amylopectins, 116 type 3 amylopectins, 116 type 4 amylopectins, 116 freezing pressure, 585 gelation, 604e605 granule matrix, 586e587 heterogeneous branched model, 595e596 hydroxypropyl starch paste, 589e590 lectin concanavalin A, 101 molecular aggregates, 100e101 molecular structures, 662, 664f molecular weight, 107 primary components, 771 retrogradation, 584, 604 short chain, 586e587 side chain elongation, 193e194 starch phosphate esters fungal glucoamylase, 124, 125f a-glucan, water dikinase (GWD), 123 location, 124 phosphoglucan, water dikinase (PWD), 123e124 unit chain length and distribution extra-long/super-long chains, 111e113 glucoamylase hydrolyses, 109e111 granule-bound starch synthase I (GBSS I), 111e113 HPAEC-PAD chromatograms, 111, 112f

H-1 protons, 109 molar ratios, 109, 110t unit chain profile, 109 units of clusters building block structure, 117, 118t chain distribution, 117e119 endo-acting enzymes, 117 isolation, 119e120, 120f Amylose, 5, 61e62, 97, 152, 357te358t, 359, 381e382, 547, 595, 664t amyl alcohols (Pentasol), 101 amylopectin retrogradation, 584 A-type granules, 585e586 B-type granules, 585e586 chain alignment, 588 fractionation method, 596 freezing pressure, 585 gelation, 604e605 hydroxypropyl starch paste, 589e590 molecular structures, 662, 664f primary components, 770 properties of, 105, 106t recombinant amylosucrase, 596 retrogradation, 584, 662, 665f starch amylose content blue value (BV), 102 chain length (CL), 102 iodine affinity (IA), 102e103 lectin concanavalin A, 104 lipid-amylose complexes (LAM), 103 structural analysis, 104e107, 106t V-type amylose aroma compounds, 605 cyclodextrins, 605 glucose residues, 605 structures, types, 607 VDMSO complexes, 606 Vglycerol complexes, 606 V-6I type complexes, 607e608 V-6II type complexes, 608 V-6III type complexes, 608e609 Viode complexes, 606 VKOH complexes, 606 V-8 type complexes, 609 Amylose-lipid complex (ALC), 160 Amylose-nanoparticles (AMNPs), 699e701, 703f Anhydrous glucose unit (AGU), 288 Annealing (ANN), 773e774, 830e831 crystallite surface stability, 234 gelatinization behavior, 235

Index with heat-moisture treatment (HMT), 233e234 high-amylose starches, 234 interblock chain lengths (IB-CL), 235 molecular rearrangements, 235 thermal properties, 234 wet-milling process, 234e235 Antisolvent process, 699e701, 700f Apparent amylose content (AAC), 379e382 Arabidopsis ADP-Glc PPase, 9e11, 10t Arabidopsis thaliana, 3e4, 41e42 Arabinoxylans (AXs), 340e341 Aroids amylopectin, 451e452 amylose, 451e452 crystallinity, 447 digestibility, 484 freeze-thaw stability, 481 granule shape and size, 439e440, 441te444t minor constituents, 429, 431te434t production, 428 retrogradation, 481 rheological properties, 482 swelling power and solubility, 459 thermal properties, 467, 467t viscosity and pasting, 474, 476te477t XRD, 447 Arracacha, granule shape and size, 440e444 Association of Official Analytical Chemists (AOAC), 255e256 ATP binding, 22

B Baked products, 567 amylopectin, 595e596 amylose, 596. See also V Amylose biscuits, 619e621, 620f and wafers warping, 611e612 cakes. See Cakes cookies, 618e619, 619f crust properties bread surface, 622e623, 622fe623f crust-to-crumb ratio, 623 “high” air humidity, 621 hydrothermal process, 621 oven volume vs. steam, 621, 622f steam injection, 621 food varieties, 610, 611t gelatinization temperature, 610 glucidic fraction, 595

875

granule size distribution, 611 hydrothermic transitions amylopectin gelation, 605 amylose gelation, 605 anhydrous potato, 603 gelatinization, 603e604 glass transition, 603 plasticizing effect, 603 retrogradation, 604 lipidic fraction, 597 mineral fraction, 597e598 nitrogen fraction, 597 nonglucidic fraction, 597 pan bread. See Pan bread pastries, 611 retrogradation, 610 staling process, 612 starch grain atomic force microscopy, 598 confocal laser scanning microscopy (CLSM), 598 crystalline and helical structures, 599 crystalline lattice, 600 crystalline powders, 599e600 degree of crystallinity, 602 differential scanning calorimetry (DSC), 602 dry starch granules, 598 hydration effect, 601 infrared spectroscopy, 602 potato starch granules, 598 resonance band intensities, 602 scanning electron microscopy (SEM), 598 scattering intensity, 602 synchrotron radiation, 598 ultrastructure, 598e599 X-ray micro-diffraction, 598 wheat starch, properties, 609 whole meal and white flour composition, 609, 609t yeast-leavened dough, 610 Bakery products, 568e569 Barley proteins, 30e36, 646 Bars, 571e572 B-chains, 98e99 B-crystalline, 356 Beer, 637 Beta-amylase, 636 Beverages emulsification/encapsulation, 575e578, 575fe577f

876 Index Biomedical and industry applications, 691, 731e732 Biosensors, 273e274 Biscuits, 619e621, 620f and wafers warping, 611e612 Black pepper oleoresins, 686, 687f BLAST analysis, 55 Bleaching, 293 Bottom-up process, 692 Branching enzyme (BE) amino acid residues, 71e73, 72t a-amylase family, 71, 72t assays, 64e65 branching enzyme-deficient mutants, 68e69 cDNA clones encoding, 69e70 a-1,4-glucanotransferase (D-enzyme), 75e77 isoamylase, 73e74 phosphorylase, 74e75 plant and algal branching enzymes, 65e67, 65t reserve tissue branching enzyme, 70e71 Branching enzyme-deficient mutants, 68e69 Bran starches, 165e166 Brewing applications, 634t amylopectin-synthesizing genes, 633e634 amylose chains, 633e634 barley grain, 649e650 biochemical parameters, 635 fermentable sugars maltose and maltotriose concentrations, 646 oligosaccharides, 646 sources, 642e643 sucrose, 647 fermentation, 634e635, 643e644, 644f flavor and aromatic characteristics, 635 fluorescence-assisted capillary electrophoresis, 633e634 human-controlled process, 634e635 mashing factors, 640e641 nonbarley beerlike beverages, 633 physical quality parameters, 635 processes, 635 rice, 651 Sorghum, 651 starch-degrading enzymes ales, 637 alpha-amylase, 636 alpha-glucosidase, 637 beer types, 637

beta-amylase, 636 dry beers, 637 lager beers, 637 limit dextrinase (LD), 635e636 nonbarley beer types. See Nonbarley beer types starch gelatinization, 648e649 starch synthesis genes, 650e651 substrateeenzyme system, 642 waxy starch, 650e651 whiskey and distilled products, 647e648 wild yeasts and specialty beers alcohol production process, 645 amino acids, 645e646 barley proteins, 646 carbohydrate source, 645 mashing process, 645e646 proteinases, 645e646 British gums, 224 Brookfield viscosity, 671e672, 672f BT1 protein, 37 Buckwheat starch, 531 amylopectin, 532 amylose, 532 digestibility, 534 food applications, 535e536 food processing, 534e535 freezeethaw stability, 534 future trend, 536 granule shape and size, 532 minor constituents, 531 pasting properties, 533 production, 531 retrogradation, 533 starch crystallinity, 532 structure and functionality, 532e534 swelling power and solubility, 533 thermal properties, 533 Building block backbone model, 98f, 99e100

C Cakes antiplasticizing effect, 615e616 m-differential scanning calorimetry heating curves, 616, 617f gel-forming systems, 614 Micro-ViscoAmyloGraph measurements, 616 pregelatinized starches, 617e618

Index protein-stabilized emulsion, 615 quality, 614 starch crystalline and amorphous structure ratios, 615, 616f sucrose limitation, 615e616 “three-ingredient” model systems, 616 types, 614, 615t wide-angle X-ray diffraction spectra, 616e617, 618f Canihua starch, 521 amylose, 522 granule shape and size, 522, 522f minor constituents, 522 pasting properties, 523e524, 524f production, 521 retrogradation, 524 starch crystallinity, 522, 523f structure and functionality, 522e524 thermal properties, 522e523, 523f Canned foods, 256 Capillary electrophoresis (CE), 270 Carbohydrates a-amylase hydrolysis, 785e786 disaccharides, 783 freeze-concentrated starch systems, 784 freezeethaw stability, 786 gelatinization temperature and enthalpy, 782e783 gel matrix structure, 785 guar gum, 786 linear nonstarch polysaccharides, 784e785 melting temperatures, 783e784 nonstarch polysaccharides, 784 plasticizing effect, 784 retrogradation, 783e784 starchesugar system, 782 starchewater ratio, 785e786 swelling capacity, 783 trisaccharides, 783 Carbon fixation, 3e4 Carbonyl (CO), 293 Carboxyl (COOH), 287e288, 293, 789e790 Cassava amylopectin, 449e450 amylose, 449e450 chemical modifications acetylation, 489 acetylation-cross-linking, 490 alkaline and acid treatment, 489 carboxymethylated starch, 490 complexing, 488

877

cross-linked starch, 488e489 hydroxypropylation, 489 oxidation, 489 oxidation-cross-linkingphosphorylation, 490 starch citrate, 490 starch succinate, 490 crystallinity, 446e447 digestibility, 483 freeze-thaw stability, 480e481 granule shape and size, 436e438, 436te437t, 438f, 439t minor constituents, 425te427t, 429 physical modifications annealing (ANN), 488 ball milling, 486e487 blending different native starches, 488 drum-drying, 486 electric field modification, 486 extrusion techniques, 486 heat-moisture treatment (HMT), 487e488 high-pressure-based treatments, 486 ɣ-irradiation, 486 microwave, 487 ultrasound, 487 production, 424e428, 425te427t retrogradation, 480e481 rheological properties, 481e482 swelling power and solubility, 453e457, 455te456t thermal properties, 460e465, 462te464t viscosity and pasting, 470e473, 471te472t, 474f XRD, 446e447 C-crystalline, 356 13 C-enriched plasma glucose, 814, 815f Cereal grains, 812 Cereals, 571e572 Chemical modification acetyl, 287e288 acetylation, 297e299 acid hydrolysis, 291e293, 292f amide, 287e288 amine, 287e288 anhydrous glucose unit (AGU), 288 applications, 284te285t, 308e316 health aspects, 308 industry trends, 316 nutritional value, 308 OSA-modified starches, 314e315 Triticum dicoccum, 314e315

878 Index Chemical modification (Continued ) Triticum durum, 314e315 in vitro digestibility, 314e315, 314t in vivo digestibility, 315 bleaching, 293 carbonyl (CO), 293 carboxyl (COOH), 293 cross-linking, 295 degree of substitution (DS), 288, 291t differential scanning calorimetry (DSC), 283e285 dimethyl sulfoxide (DMSO), 297e299, 298t distarch adipates, 295 distarch phosphates, 295 dual modification, 295e296 enzymatic degradation, 283e285 epichlorohydrin (EPI), 295 etherification, 294 freezeeThaw stability, 307e308, 309t gelatinization enthalpy, 304t granular size distribution, 288 hydroxypropylated starches, 287e288, 301e305 hydroxypropylation, 294, 297e299 microstructural and ultrastructural differences, 288 molar substitution (MS), 288, 291t morphological properties, 299e301 nanocrystal starches, 291e293 native starch, 285e287 octenyl succinic anhydride (OSA), 294 oxidation, 293 oxidized mucuna starch, 305e307 pasting properties, 305e307, 306t phosphoryl chloride (POCl3), 295 physical methods, 283e285 physicochemical properties, 288, 296e299 properties, 284te285t rate and efficiency, 288 refrigerated storage, 307e308, 309t rice starch, 402e405 acetylated rice starch, 403 acid-thinned rice starch, 403 cross-linked rice starch, 404e405 hydroxypropylated rice starch, 403e404 octenyl succinic anhydride modified starch, 403 phosphorylated rice starch, 404 sodium octenylsuccinate, 294 sodium trimetaphosphate (STMP), 295 source, 285e287

stabilization, 293e294 substitution, 293e294 swelling power, 296, 297t thermal characteristics, 301e305, 302te304t types, 283e285, 286te287t, 287e296 waxy maize starches (WMSs), 300e301 Chemical substitution, 200e201 Chinese chestnut starch, 459, 460t Chlamydomonas, 184 C. reinhardtii, 6e7, 30 13 C-labeled starch, 814 Clarity, 400 Classical breeding, 182e183 Coating technique, 752 13 CO2 breath test, 814 Cognitive function, 857 Coleus, granule shape and size, 440e444 Combined treatments, 365e366 Confections (candy), 572e573 Confocal laser scanning micrographs (CLSM), 165 Convective heat transfer coefficients, 582 Conversion techniques, 751e752 Cookies, 618e619, 619f Crop maturation, 170e171 Cross-linked starches, 364e365 Cross-linking, 295 Crystallinity, 713e716 Curcumin-loaded starch nanoparticles, 731e732

D Dairy products, 573e574 Debranching enzyme (DBE), 100, 194 Degraded starches, 364 Degree of substitution (DS), 288, 291t Deterioration mechanism components, 583 protein denaturation, 584 quality loss, 581e582 starch deterioration, 584e586 water crystallization, 582 water migration, 583e584 Dextrinization, 364 Dextrins, 364 Dietary fiber, 819e820 Dietary starches, 857 adverse effects animal models, 864e865 human studies, 865e867

Index mental performance animal studies, 861 human studies, 861e864 physical performance animal studies, 857e858 human studies, 858e860 Differential scanning calorimetry (DSC), 283e285, 389e390, 389f, 589 Digestibility, 401e402 Digestion and applications food-derived a-glucosidase inhibitors, 820 gastrointestinal hormones, 821 plasma glucose levels, 805 postprandial glycemia. See Postprandial glycemia postprandial hyperglycemia. See Postprandial hyperglycemia postprandial insulin and GIP response, 821 in vitro approaches, 813 in vivo approaches 13 C-labeled starch, 814 dual isotope method, 814e815, 816f hydrogen breath test, 813e814 ileostomy model, 815 intubation studies, 816 Digestion-resistant starches, 855 Dimethyl sulfoxide (DMSO), 100e101, 257, 297e299, 298t Disaccharides, 783 Distarch adipates, 295 Distarch phosphates, 295 Drug delivery system, 731e732 Drum drying, 363 Dry beers, 637 Dual isotope method, 814e815, 816f Dual modification, 295e296

E East Indian arrowroot, granule shape and size, 440e444 E. coli glycogen-branching enzyme, 194 Electronic tongues, 275 Electrospinning, 706 Electrospray, 706e707 Emulsion, 705 Endo-acting enzymes, 100, 117 Endogenous lipids, 167e168 Endosperm, cereal grains, 36e37 E-number E1422, 364e365

879

Enzymatic debranching/recrystallization, 701e705, 704f Enzymatic degradation, 283e285 Enzymatic hydrolysis, 597 Enzymatic methods, 265e267 Enzyme-treated starches, 364 Epichlorohydrin (EPI), 295 Etherification, 294 European food safety authority (EFSA), 206 Ewers’ polarimetric method, 258, 262e263 Expert Systems (ES), 275 Extrusion process, 683e684

F Fats, 548, 554 encapsulation, 681e682, 681t replacement, 574e575 Fermentable sugars maltose and maltotriose concentrations, 646 oligosaccharides, 646 sources, 642e643 sucrose, 647 Ferredoxin-thioredoxin system, 29 Films ASTM standard method, 748e749 barrier materials, 754 barrier properties hydrophilicity, 760 moisture barrier, 759e760 oxygen barrier, 757e758 permeability, 757 bio-based materials, 747e749 and synthetic materials, 749, 749f biopolymers, 748 chemico-physical properties, 747 coating technique, 752 conversion techniques, 751e752 film-forming mechanism, 755 food contact materials (FCMs), 750 food packaging, 753e754 free hydroxyl groups, 747, 754 full-scale processes, 752e753 gel formation, 755 heating and cooling cycle, 748 plasticizers, 751 plastic materials, global production, 750 solution casting, 751 synthetic polymers, 748 tensile properties chainechain interaction, 756e757 factors, 755e756

880 Index Films (Continued ) glass transition temperature, 756e757 high-amylose maize, 756 low-density polyethylene (LDPE), 755e756 nonplasticized films, 755e756 solution-cast amylopectin films, 757 textile industry, 747 thermoplast, 748 thermoplastic starch (TPS), 751 waxy maize starch, 754e755 Flavors, 561e562 Flow injection analysis (FIA) amyloglucosidase, 271 hydrolysis products, starch, 270e271, 271f microwaves, 272 starch-hydrolyzing enzymes, 270e271 Fluorescence-assisted capillary electrophoresis, 633e634 Fluorescent agent, 733e734 Fluorimetric method, 265 Fluorophore-assisted carbohydrate electrophoresis (FACE), 109e111 Food components aroma and flavor compounds, 790 carbohydrates. See Carbohydrates carboxyl acids, 789e790 chemical composition and morphology, 769 lipids processing conditions, 775 starchelipid complex formation, 776e777 starchelipid complex structure, 775e776 starchelipid functionality, 777e778 phase transitions annealing, 773e774 gelatinization, 772e773 retrogradation, 774 physical properties, 772 polyols, 789e790 polyphenols, 789e790 primary components, 770e771 proteins starcheprotein aggregation/segregation, 779e781 starcheprotein functionality, 781e782 starcheprotein structure, 779 starchy endosperm cell, 778e779 salts electrostatic repulsion forces, 787

gelatinization enthalpy, 788 sodium chlorideestarch systems, 787e788 starch pressurized systems, 788 thermal and physicochemical properties, 786e787 secondary components, 771e772 starch granules, 770 surfactants, 788e789 thermal and physicochemical properties, 775 Food contact materials (FCMs), 750 Food ingredient, 375e377 Food packaging, 753e754 Forward genetics, 182e183 Fourier transform infrared spectroscopy (FTIR), 262, 716e717 Free fatty acids, 378 Freeze-concentrated starch systems, 784 Freeze-induced denaturation, 584 Freezeethaw stability, 307e308, 309t, 400e401 Friabilin proteins, 335 Frozen foods deterioration mechanism, 583. See also Deterioration mechanism detrimental effects, 581 food quality chemical modification, 588e590 genetical modification, 586e587 physical modification, 587e588 texture stabilizer and regulator, 586 polysaccharide stabilizers, 581 Fruits, 561e562

G Gamma irradiation, 698e699 Gamma irradiationeassisted nanoprecipitation method, 699e701 Gas chromatography (GC), 269 Gastric emptying rate, 809 Gastrointestinal hormones, 810e811 Gelatinization (GT), 166e167, 304t, 354, 354f, 385, 389e397 Gelling, 169 Gel-permeation chromatography (GPC), 101 Gene technology classical breeding, 182e183 forward genetics, 182e183 mutagenesis, 182e183 phytoglycogen, 182e183

Index plant bioreactors, 182 plant models and starch crop sequences, 184 precise genome editing (PGE), 184e185 targeted starch bioengineering, 183e184 targeting-induced local lesions (TILLING) protocols, 183 Genetic modification, 407 Ghost, 353 Giant taro, granule shape and size, 440e444 Glass noodles, 367 a-1,4-Glucanotransferase (D-enzyme), 75e77 Glucan-water dikinase (GWD), 378e379 Glucidic fraction, 595 Glucoamylase hydrolyses, 109e111 Glucoamylases, 100 a-D-Glucopyranosyl, 595 Glucose bioavailability, 855e856 Glucose-dependent insulinotropic polypeptide (GIP), 811 Glucose transporter (GLUT2), 809 a-Glucosidase inhibitors prediabetes, 817 T2DM, 816e817 Glutenin macropolymers, 584 Glycation end products (AGEs), 857 Glycemic index (GI), 827 Food and Agriculture Organisation (FAO), 817 high-GI products, 818 high-postprandial glycemia, 818e819 low-GI products, 818 low-postprandial glycemia, 819 World Health Organisation (WHO), 817 Granular-bound starch synthase I (GBSSI), 159 Granular cold-water-swelling starch (GCWS) methods, 226e227 single starch, 227 waxy maize starch, 227 Granular size distribution, 288 Granule-bound starch synthase (GBSS), 6e7, 48 amylose, 61e62 Arabidopsis leaves, 63 C. reinhardtii, 63 GBSSI, 61

GBSSIa, 62e63 GBSSIb, 62e63 GBSSII, 61 maltooligosaccharides, 63 pea embryo and potato tuber GBSSIs, 61e62 Granule-bound starch synthase I (GBSSI), 111e113, 382 Ground soaked rice, 376 Guar gum, 786

H Haworth conformation, 121 Heating dry starch, 236e237 Heat-moisture treatment (HMT), 367, 830e831 A-type crystallinity, 229 B-type crystallinity, 229 C-type crystallinity, 229 granule swelling and amylose leaching, 230 lauric acid, 232 moisture contents, 228e229 processing temperatures, 228e229 resistant (RS) starch, 230e232 slowly digestible starch (SDS), 230e232 treatment times, 228e229 unmodified thickeners, 233 Heterotetrameric endosperm enzyme, 38 High amylose and amylose-only starch, 195e196 chain branch point formation, 195 High-fat foodstuffs, 256 High-molecular amylose, 359 High-performance anion exchange chromatography equipped with pulsed amperometric detector (HPAEC-PAD), 109e111, 267e268, 385 High-performance liquid chromatography (HPLC), 267e268 High-performance size-exclusion chromatography (HPSEC), 104 High-pressure homogenization, 242e243, 693e695, 695f High-speed jet homogenizer, 242e243 Holm method, 266 Homology-dependent gene silencing approaches, 183e184 Hoya carnosa, 42e44

881

882 Index Hydrocyclone, 356 Hydrogen breath test, 813e814 Hydrophilic interaction chromatography, 267 Hydrophobic moiety, 153e154 Hydroxypropylated starch (HP-starch), 287e288, 294, 297e299, 301e305, 365, 589e590

I Ileostomy model, 815 Indian chestnut starch, 430e435 amylopectin and amylose, 453, 454t Inhibited starches, 364e365, 367 Inorganic phosphate (Pi), 7e8 Insoluble starch-iodine complex, 260e261 Intermediate components, 159 Intermediate material (IM), 97, 126e128 Intestinal starch digestion, 808e809 Intestinal transit time, 810 In vitro digestibility, 314e315, 314t In vivo digestibility, 315 Isoamylase, 73e74

L Lager beers, 637 Leaf starch, 3e4 Lectin concanavalin A, 101, 104 Limit dextrinase (LD), 635e636 D-Limonene water interfacial tension and surface tension, 668e669, 670t Linear amylose molecules, 662, 664t Linear nonstarch polysaccharides, 784e785 Lipid-amylose complexes (LAM), 103 Lipidic fraction, 597 Lipids, 160, 548 processing conditions, 775 starchelipid complex formation, 776e777 starchelipid complex structure, 775e776 starchelipid functionality, 777e778 Long-grain rice, 374e375 Low amylose GBSS suppression, 196e197 and waxy-type starches, 196 Luff-Schoorl starch, 260e261 Lysophosphatidyl choline (LPC), 335e336, 378 Lysophosphatidylethanolamine (LPE), 378 Lysophospholipids (LPLs), 378

M Magnetic separation techniques, 735 Maize endosperm, 37e38 Maize kernels, 151 Maltooligosaccharides, 63 Maltose (Type B), 600 Maltotriose (Type A), 600 Martin process, 327 Meat products, 570e571 Metabolomes, 190 Methacrylic anhydride starch, 699e701 Methyl thiazolyl tetrazolium (MTT) assay, 723e725 Microencapsulation AACC 76-21 method, 662e665 carbohydrate source, 661 chemical modifications CAPSUL solution, 668e669, 669f cross-linking, 667 degree of substitution (DS), 667e668 hydrophilic lipophilic balance (HLB) number, 667e668 hydrophobic treatment, 667 octenyl succinic anhydride (OSA) starch, treatment process, 667t, 668 regulatory limitations, sodium starch octenyl succinate, 667e668, 667t severe pH and salinity processes, 666e667 stabilization, 667 surface/interfacial tensions, 668e669, 670t energy storage biopolymer, 661 extrusion process, 683e684 fat encapsulation, 681e682, 681t gelatinization, 662 intermolecular interactions, 662e665 linear amylose molecules, 662, 664t octenyl succinic anhydride (OSA) starch, 661 physical modifications, 669e670 plating application black pepper oleoresins, 686, 687f N-ZORBIT 2144, 684, 685f N-ZORBIT M, 686, 686f water-soluble and oil-soluble materials, 685e686 Rapid Visco Analyzer RAV-4, 662e665 retrogradation, 662 spray-congealing process, 682e683, 682f spray-drying microencapsulation, 661. See also Spray-drying microencapsulation

Index starch hydrolysis, 665e666 starch sources, 662, 663t vitamin encapsulation oil-soluble vitamins, 678e680 surface oil, 680, 681f vitamin E oil, 678e680, 680f Micronization, 239 Microwaves, 236 Milled rice kernels, 151 Milling, 239e240 Mimetics, 574e575 Mineral fraction, 597e598 Minor constituents, 547e549 Modified starches, 551e552 Moisture, 548e549 Moisture loss, 583 Moisture-resistant materials, 760 Molar substitution (MS), 288, 291t Molecular farming, 180 Molecular variation process, 582 Mono-acyl lysophospholipids, 597 Multiangle laser light scattering with differential refractive index (MALLSRI) detection, 382 Multifunctional antimicrobial film, 732e733 Myofibrillar proteins, 584

N Nanocomposites, 727e728, 729te730t Nanoprecipitation, 699e701, 702fe703f amylose-nanoparticles (AMNPs), 699e701, 703f antisolvent process, 699e701, 700f gamma irradiationeassisted nanoprecipitation method, 699e701 methacrylic anhydride starch, 699e701 relative crystallinity (RC), 699e701 Near-infrared reflectance analysis (NIRA), 261 Near-infrared spectroscopy, 269e270 Nitrogen, 548, 597 Nonbarley beer types brewing process, 638f, 639e640 malting process, 638e639, 638f pseudograins, 638 Sorghum, 638 Nonglucidic fraction, 597 Nonstarch polysaccharides, 784 Nonthermal treatments freeze-drying, 244 freezing and thawing, 244 high-pressure treatment, 240

883

high-pressure homogenizers, 242e243 high-speed jet homogenizer, 242e243 ultrahigh pressure (UHP) treatment, 241e242 milling, 239e240 pulsed electric field (PEF) technology, 243 ultrasonic treatment, 237e239 Nuclear magnetic resonance studies, 586e587 Nutritional value, 308 N-ZORBIT 2144, 684, 685f N-ZORBIT M, 686, 686f

O Octenyl succinic anhydride (OSA) starch, 661 regulatory limitations, 667e668, 667t treatment process, 667t, 668 Oral insulin delivery, 732e733 Oryza sativa L., 373 OSA-modified starches, 314e315 Osmotic pressure treatment (OPT), 237 Oxidation, 364 Oxygen permeability (OP), 757e758

P Pachyrhizus, 440e444 Pan bread amylopectin retrogradation, 613, 614f bacterial a-amylases, 613 calorimetry tests, 613 fermentation, 612 forced convection and microwave ovens, 612 gelatinization temperature, 612 gluten proteins, 612 heating rate, 613e614 hydrothermal processing, 613 macroscopic properties, 613 Pasta, 571e572 Pasting property, 167e169, 168f Pastries, 611 Pea embryos, 37 Pea starch, 601 Pentosans, 340e341 Pericarp, 165e166 Pet products, 570 Phosphate monoesters, 160, 378e379 3-Phospho-glycerate (3PGA), 7e8 Phospholipids, 160 Phosphorus, 378e379, 548 Phosphorylase, 74e75

884 Index Phosphorylase stimulation assay, 64e65 Phosphorylation, 180e181 engineering, 197 indirect engineering, 197e198 starch, in plant, 197 Photosynthesis, 3e4 Physical modification chemical changes, 244 nonthermal treatments, 237e244 overview, 223e224 rice starch, 405e407 extrusion, 406 gamma-irradiated rice starch, 406e407 hydrothermal treatment, 405e406 sonicated rice starch, 406 thermal treatments, 224e237 Phytoglycogen, 159, 182e183 Pickering emulsion, 728e731 Plant a-1,4-glucan-synthesizing enzymes ADP-Glu binding, 22e24, 23fe24f ADP-glucose pyrophosphorylase (ADP-Glc PPases) Glu-1-P-binding site, 13e15 kinetic and regulatory properties, 7e8 mechanism of activation, thioredoxin, 28e30 physiological importance, 27e28 structure-function relationships, 9e11 allosteric regulation, 26e27 ATP binding, 22 catalysis Asp145 and Asp280, 24e25 K198, 24e25 R33, 24e25 Rffh and GlmU, 24e25 potato tuber ADP-Glc PPase activity catalytic and regulatory subunits, 12, 13t crystal structure, 17e19 homotetramer catalytic subunit structure, 19e21 small and large subunits, 11e13 amino acid sequence similarity, 11 Arg33 and Lys43, 11e12 LargeK44R/T54K, 12 Lys44 and Thr54, 11e12 phosphate (Pi), 13 phylogenetic analysis, 16 sequence comparison, 11e12, 12t Plant starch synthesis leaf starch, 3e4 storage tissues, 4e5, 5f

Platinum redox electrode, 274 Polarimetry, 262e263. See also Ewers’ polarimetric method Polyelectrolyte complex formation, 705e706 Polyols, 789e790 Polyphenols, 789e790 Polysaccharides, 97e98 Postprandial glucose response, 808 Postprandial glycemia cereal grains, 812 food composition, 812e813 food matrix, 812 gastric emptying rate, 809 gastrointestinal hormones, 810e811 glucose transporter (GLUT2), 809 intestinal starch digestion, 808e809 intestinal transit time, 810 physicochemical characteristics, 811 postprandial glucose response, 808 sodium-glucose cotransporter (SGLT-1), 809 starch characteristics, 811e812 starch-derived glucose, 808 Postprandial hyperglycemia dietary fiber, 819e820 a-glucosidase inhibitors prediabetes, 817 T2DM, 816e817 glycemic index (GI) concept Food and Agriculture Organisation (FAO), 817 high-GI products, 818 high-postprandial glycemia, 818e819 low-GI products, 818 low-postprandial glycemia, 819 World Health Organisation (WHO), 817 persons risk healthy persons, 808 prediabetes, 807e808 type 2 diabetes, 806e807 postprandial plasma glucose regulation, 806 Potato starch, 601 acetylated starches, 365 amylopectin, 357te358t, 359e363, 360f, 360t, 362f amylose, 357te358t, 359 B-crystalline, 356 C-crystalline, 356 combined treatments, 365e366

Index composition, 355, 356t cross-linked starches, 364e365 degraded starches, 364 developments, 366e368 amylomaltase-treated starches, 368, 368f inhibited starches, 367 small granular potato starch, 367 food industry derivatives, 363e366 functionality, 363e366 gelatinization water, 354, 354f granular and molecular structure, 356e361 granules, 356e359, 357te358t hydroxypropylated starch, 365 market, 355 overview, 353e354 particle size distribution, 357te358t physical processing, 363 production, 355, 356t retrogradation, 354 semi-cross-linked behavior, 354 starch octenyl succinates, 366 Potato tuber ADP-Glc PPase activity catalytic and regulatory subunits, 12, 13t crystal structure, 17e19, 18f homotetramer catalytic subunit structure Cys12, 19 sulfate 1, 20e21 sulfate 2, 21 sulfate 3, 21 sulfate-binding region and amino acid residues, 19e20, 20f Potentiometry (nonenzymatic electrochemical detection), 273 Precise genome editing (PGE), 184e185, 206 Pregelatinized starch, 587e588 applications, 226 double-drum equipment, 225e226 extrusion, 225 hot-drum pregelatinized starch products, 225e226 procedure, 225 Product development, starch amylopectin, 547 amylose, 547 bakery products, 568e569 bars, 571e572 beverages emulsification/encapsulation, 575e578, 575fe577f cereals, 571e572

885

characteristics, 546 components, 546e549 confections (candy), 572e573 dairy products, 573e574 dressings, 567e568 factors, 558e562, 558t fat replacement, 574e575 fats, 548 flavors, 561e562 food applications, 549e551 food ingredients, 561e562 fruits, 561e562 functional properties, 563e578 baked, 567 fried, 567 frozen, 564e565 functional differences, 566 impinged air processed, 567 instant products, 565e566 microwaved, 567 puffed, 566 snack foods, 566e567 thermal processing, 563e564 gravies, 567e568 lipids, 548 meat products, 570e571 mimetics, 574e575 minor constituents, 547e549 modified starches, 551e552 moisture, 548e549 native/common starches, 549e551, 550f nitrogen, 548 other condiments, 567e568 packaging, 559 pasta, 571e572 pet products, 570 phosphorus, 548 properties, 546 proteins, 548, 562 salts, 561 sauces, 567e568 shear, 558e559 source, 546 spices, 561e562 starch selection methods, 552e555 desired function, 552 fats, 554 food system (pH), 553 gums, 554 postprocessing, 554 processing method, 552e553

886 Index Product development, starch (Continued ) salts, 554 shear, 553 storage, 554e555, 555te557t water-soluble materials, 553e554 storage, 559 structure, 546 substitution, 574e575 sweeteners, 560 temperature, 558 trace elements, 548 water, 548e549, 559e560 Propylene oxide, 366 Proteins, 160e161 denaturation, 584 macropolymers, 583e584 starcheprotein aggregation/segregation, 779e781 starcheprotein functionality, 781e782 starcheprotein structure, 779 starchy endosperm cell, 778e779 Pseudocereal starches amaranth starch, 524 amylopectin, 526e527 amylose, 526 chemical modification, 530 digestibility, 530 food applications, 530e531 freezeethaw stability, 529e530 granule shape and size, 525e526, 525f minor constituents, 525 pasting properties, 527, 529f production, 525 retrogradation, 527e529 starch crystallinity, 526, 526f structure and functionality, 525e530 swelling power and solubility, 527 thermal properties, 527, 528t, 529f buckwheat starch, 531 amylopectin, 532 amylose, 532 digestibility, 534 food applications, 535e536 food processing, 534e535 freezeethaw stability, 534 future trend, 536 granule shape and size, 532 minor constituents, 531 pasting properties, 533 production, 531 retrogradation, 533 starch crystallinity, 532

structure and functionality, 532e534 swelling power and solubility, 533 thermal properties, 533 canihua starch, 521 amylose, 522 granule shape and size, 522, 522f minor constituents, 522 pasting properties, 523e524, 524f production, 521 retrogradation, 524 starch crystallinity, 522, 523f structure and functionality, 522e524 thermal properties, 522e523, 523f quinoa starch, 509e510, 510t amylopectin, 513e514, 514t amylose, 512e513, 513t digestibility, 519e520 food applications, 520e521 food processing, 520 freezeethaw stability, 519 granule shape and size, 511 minor constituents, 511 pasting properties, 517e518, 517f, 518t production, 510 retrogradation, 518e519 seeds, 509e510 starch crystallinity, 511e512, 511f structure and functionality, 511e520 swelling power and solubility, 514e515 thermal properties, 515e517, 515f, 516t Pulsed electric field (PEF) technology, 243 Pyridoxal-5-phosphate (PLP), 45 Pyrodextrins, 224

Q Qualitative analysis, 259e262 Quantitative analysis, 260e261

R Raman spectroscopy, 716e717 Random-coil conformation, 153e154 Rapidly digestible starch (RDS), 401e402, 855e856 Rapid Visco Analyzer RAV-4, 186e187, 662e665 Reactive extrusion, 696e698 Recrystallization, 583e584 Redox regulation, 29e30

Index Refrigerated storage, 307e308, 309t Relative crystallinity (RC), 699e701 Reserve tissue branching enzyme, 70e71 Resistant starch (RS) ActiStar RM resistant starch, 837e839 classification, 830 cooking time and particle size, 845 dietary fiber content, 827e828 food matrixes, 840e845 functional properties, 839 gluten-free products, 841, 847 high-amylose resistant starch, 839 hydrothermal cooking, 844 maize flour, 844e845 molecular and physicochemical characteristics, 828e829 nutraceutical starch, 827 postprandial insulin and glucose responses, 840e841 production, 828 products, 840 raw ingredients, 845e846 ready-to-eat breakfast cereal ring, 840e841 retrogradation process, 844 RS3 (Novelose 330), 840 RS4-type ingredients, 839 starch fraction digestion, 841, 842te843t starch hydrolysis, 827e828 starch modifications, nutritional purpose chemical modifications, 836e837, 838t enzymatic modifications, 831e836, 835t granular structure, 830 molecular arrangement, 830 physical modifications, 830e831, 832te833t thermal stability, 846 uncooked cereal starches, 829e830 wheat flour, 844e845 Resistant starch-rich ingredient (RSRI), 620e621 Retrogradation, 152, 154e155, 354, 397e402 Reverse genetics, 183e184 Rice, 651 amylopectin, 382e385, 383f, 384t, 386t amylose, 381e382 apparent amylose content (AAC), 379e382 chemical modification, 402e405 acetylated rice starch, 403 acid-thinned rice starch, 403

887

cross-linked rice starch, 404e405 hydroxypropylated rice starch, 403e404 octenyl succinic anhydride modified starch, 403 phosphorylated rice starch, 404 clarity, 400 constitutes of, 377e379 differential scanning calorimetry (DSC), 389e390, 389f digestibility, 401e402 endosperm, 40e41 food ingredient, 375e377 food processing applications, 402e407 free fatty acids, 378 freezeethaw stability, 400e401 functionality, 379e388 future perspectives, 407e408 gelatinization (GT), 385, 389e397 genetic modification, 407 glucan-water dikinase (GWD), 378e379 granule-bound starch synthase I (GBSSI), 382 granule shape and size, 379, 380f ground soaked rice, 376 high-performance anion exchange chromatography equipped with pulsed amperometric detector (HPAECPAD), 385 indica and japonica, 374e375 kernel and broken rice, 375 long-grain rice, 374e375 lysophosphatidyl choline (LPC), 378 lysophosphatidylethanolamine (LPE), 378 lysophospholipids (LPLs), 378 market size, 375 minor constituents, 378e379 multiangle laser light scattering with differential refractive index (MALLSRI) detection, 382 noodle production, 375e376 Oryza sativa L., 373 pasting properties, 392e396, 393fe395f phosphate-monoester, 378e379 phosphorus, 378e379 physical modification, 405e407 extrusion, 406 gamma-irradiated rice starch, 406e407 hydrothermal treatment, 405e406 sonicated rice starch, 406 processing quality, 375 production, 373e374, 373t rapidly digestible starch (RDS), 401e402

888 Index Rice (Continued ) retrogradation, 397e402 rheological properties, 395f, 396e397 short-grain rice, 374e375 starch crystallinity, 379e381 structure, 379e397 swelling power, 387e388, 387t, 388f traditional method, 375e376 water solubility, 387e388, 387t, 388f waxy rice flour, 375 RS3 (Novelose 330), 840 RS4-type ingredients, 839

S Salts, 561 electrostatic repulsion forces, 787 gelatinization enthalpy, 788 sodium chlorideestarch systems, 787e788 starch pressurized systems, 788 thermal and physicochemical properties, 786e787 Sauces, 567e568 Self-assembly, 707 Semi-cross-linked behavior, 354 Shear, 558e559 Short-grain rice, 374e375 Slowly digestible starch (SDS), 855e856 cooking time and particle size, 845 food matrixes, 840e845 gluten-free products, 847 glycemic index (GI), 827 health beneficial effects, 827e828 hydrothermal cooking, 844 maize flour, 844e845 molecular and physicochemical characteristics, 828e829 nutraceutical starch, 827 production, 828 raw ingredients, 845e846 retrogradation process, 844 starch modifications, nutritional purpose chemical modifications, 836e837, 838t enzymatic modifications, 831e836, 835t granular structure, 830 molecular arrangement, 830 physical modifications, 830e831, 832te833t thermal stability, 846 uncooked cereal starches, 829e830

Small-angle X-ray scattering (SAXS), 241e242 Small granular potato starch, 367 Sodium-glucose cotransporter (SGLT-1), 809 Sodium starch octenyl succinate (SSOS), 661 Soluble starch synthase I (SSI), 48e51 Solvent emulsificationediffusion technique, 732e733 Sonic spray ionization (SSI), 274 Sorghum, 651 Sorptionedesorption isotherms, 601 Spices, 561e562 Spray-congealing process, 682e683, 682f Spray-drying microencapsulation, 661 advantages, 670, 670t Brookfield viscosity, 671e672, 672f conditions, 673, 673t denser/glassy matrix, 676e677 droplet size, 671e672, 671f dynamic spray-drying process, 674, 675f economy and flavor sensory profile, 677e678 emulsion formulations, 670e671, 671t film-forming property, 677 flavor microencapsulation, 673e674, 674t high-load flavor systems, 678 high-pressure homogenization, 670e671 low-molecular weight carbohydrates, 676e677 nondestructive solvent, 673e674 onefold orange flavor, 677e678, 679f oxidation level, 677, 678f retention, 676, 676t, 677f scanning electron microcopy (SEM) images, 674e676, 675f vitamin encapsulation oil-soluble vitamins, 678e680 surface oil, 680, 681f vitamin E oil, 678e680, 680f water evaporation and matrix material deposition, 674e676 SS. See Starch synthases (SS) Stabilization, 293e294 Stable thickeners, 365e366 Starch bioengineering additional minor components, starch functionality, 202 analytical technologies, molecular structure molecular analysis, 186 physical and rheological analysis, 186e187

Index demands and opportunities, 179e180 extending functionality, existing starches, 203 food systems, 201e202 stable processing and storage stable starch, 202e203 gene technology classical breeding, 182e183 forward genetics, 182e183 mutagenesis, 182e183 phytoglycogen, 182e183 plant bioreactors, 182 plant models and starch crop sequences, 184 precise genome editing (PGE), 184e185 targeted starch bioengineering, 183e184 targeting-induced local lesions (TILLING) protocols, 183 high-amylose and resistant starch, 203 importance, 179e180 metabolic reactions amylopectin, 193e195 cereals and potato, 187e189, 189f granule size, 198e199 high amylose, 195e196 increased starch yield, engineering starch degradation, 192 initial step, starch biosynthesis, 191e192 low amylose, 196e197 maintaining starch structure, 192 metabolomes, 190 molecular size, 198 pleiotropic effects, 187e189 scanning electron microscopy (SEM) images, 187e189, 188f starch biosynthesis, regulation, 187e190, 188f starch phosphorylation, 197e198 thermorobust crops, 192e193 yield penalty, 190e191 physical and chemical properties chain length and amylose, 200 chemical substitution, 200e201 complexity of, 201 engineering solubility, 201 structural components, 199e200, 199f predictive models, 205 starch biosynthesis, complexity, 204 starch granule assembly, 205

889

Starch components A-chains, 98e99 amylopectin, 97 amylose, 97 B-chains, 98e99 building block backbone model, 98f, 99e100 debranching enzymes, 100 endo-acting enzymes, 100 glucoamylases, 100 glucosyl chain, 98e99, 98f intermediate material (IM), 97 internal chains, 99 long and short chains, 99 proteins, 97e98 waxy, 97 Starch crop improvement, 180 Starch crystallinity, 379e381 Starch deterioration, 584e586 Starch fractionation, 100e101 Starch granule, 325e326 amylopectin, 180e181, 181f average branch chain length distributions, 155e156, 156f bimodal distribution, 155e156 B-type polymorph, 156e159 extra-long branch chains, 159 gelatinization properties, starches, 155e156, 157te158t amylose, 153f, 180e181, 181f complete hydrolysis, 152e153 double-helical conformation, 154e155 hydrophobic moiety, 153e154 polyiodide ions, 153e154 random-coil conformation, 153e154 retrogradation, 154e155 assembly, 205 biosynthesis, 151 density of, 151 intermediate components, 159 internal structures of blocklets, 163e164 bran starches, 165e166 elongated granules, 165 pericarp, 165e166 relative locations, amylose and amylopectin, 162e163 structural variations, hilum to periphery, 164 lipids, 160 morphology of, 161e162, 162fe163f phosphate monoesters, 160

890 Index Starch granule (Continued ) phospholipids, 160 phosphorylation, 180e181 phytoglycogen, 159 proteins, 160e161 semicrystalline structure, 151 starch retrogradation, 169e170 starch synthases (SS), 58e59 storage tissues, 4e5 Starch granule-associated proteins (SGAPs), 338 Starch measurement, 255 analysis methods, 259t classical methods, 259e261 modern methods, 261e273 recommended methods, 259, 260t regulations, 255e256 sample preparation, 256e258, 257f Starch nanocrystals (SNCs), 693 Starch nanoparticles (SNPs) acid hydrolysis, 693, 694f applications adsorbent agents, 734e735 biomedical and industry applications, 691, 731e732 curcumin-loaded starch nanoparticles, 731e732 delivery system, 731e732 fluorescent agent, 733e734 magnetic separation techniques, 735 multifunctional antimicrobial film, 732e733 nanocomposites, 727e728, 729te730t oral insulin delivery, 732e733 pickering emulsion, 728e731 solvent emulsificationediffusion technique, 732e733 superparamagnetic iron oxide nanoparticle (SPION), 735 bottom-up process, 692 crystallinity, 713e716 digestibility properties, 721, 722f electrospinning, 706 electrospray, 706e707 emulsion, 705 enzymatic debranching and recrystallization, 701e705, 704f Fourier transform infrared spectroscopy (FTIR), 716e717 gamma irradiation, 698e699 high-pressure homogenization, 693e695, 695f

molecular weight distributions, 713, 714fe715f morphological structure and size distribution, 707, 708te712t nanoprecipitation, 699e701, 702fe703f amylose-nanoparticles (AMNPs), 699e701, 703f antisolvent process, 699e701, 700f gamma irradiationeassisted nanoprecipitation method, 699e701 methacrylic anhydride starch, 699e701 relative crystallinity (RC), 699e701 polyelectrolyte complex formation, 705e706 preparation, 691e692, 692f properties, 691 protein interaction, 726, 727f Raman spectroscopy, 716e717 reactive extrusion, 696e698 rheological properties, 719e721, 720f self-assembly, 707 steam jet cooking, 699 surface functionalization chemical modification, 725 physical modification, 726 swelling properties, 721e723 thermal properties, 717e718 top-down process, 692 toxicological experiments, 723e725, 724f ultrasonication, 696, 697fe698f Starch octenyl succinates, 366 Starch phosphate esters fungal glucoamylase, 124, 125f a-glucan, water dikinase (GWD), 123e124 location, 124 phosphoglucan, water dikinase (PWD), 123e124 Starch properties gelatinization, 166e167 gelling, 169 pasting, 167e169, 168f syneresis, 169e170 Starch residue fermentation and products, 855e856 Starch retrogradation, 169e170 Starchesugar system, 782 Starch synthase II (SSII), 48 C. reinhardtii, 53 intermediate material, 51 maize du1 gene, 51e52 SSIIa activity, 52e53

Index Starch synthase III (SSIII), 48, 54e55 Starch synthase IV (SSIV), 48 A. thaliana, 56 BLAST analysis, 55 primer, 57 SSIVa/SSIVb, 56e57 Western blots, 56 Starch synthases (SS), 47 branching enzyme, 64e77 characterization, 48, 49f double mutants, 57e58 granule-bound starch synthase (GBSS) amylose, 61e62 Arabidopsis leaves, 63 C. reinhardtii, 63 GBSSI, 61 GBSSII, 61 GBSSIa, 62e63 GBSSIb, 62e63 maltooligosaccharides, 63 pea embryo and potato tuber GBSSIs, 61e62 soluble starch synthase I (SSI), 48e51 starch granule, 58e59 starch synthase II (SSII) C. reinhardtii, 53 intermediate material, 51 maize du1 gene, 51e52 SSIIa activity, 52e53 starch synthase III (SSIII), 54e55 starch synthase IV (SSIV) A. thaliana, 56 BLAST analysis, 55 primer, 57 SSIVa/SSIVb, 56e57 Western blots, 56 waxy protein structural gene, 60e61 Starch synthase V (SSV), 48 Starch synthesis, enzyme reactions, 6e7 Staudinger conformation, 121 Steam jet cooking, 699 Storage tissues, 4e5, 5f Substitution, 293e294 Sucrose, 3e4 Superparamagnetic iron oxide nanoparticle (SPION), 735 Super soft wheat flour, 328 Surfactants, 788e789 Sweet potato amylopectin, 450e451, 451t amylose, 450e451, 451t crystallinity, 447

891

digestibility, 483e484 granule shape and size, 438 minor constituents, 429, 430t production, 428 rheological properties, 482 swelling power and solubility, 457e459, 458t thermal properties, 466e467, 466t viscosity and pasting, 474, 475t XRD, 447 Swelling power, 387e388, 387t, 388f Swollen starch granules, 169 Syneresis, 169e170

T Targeted starch bioengineering, 183e184 Targeting-induced local lesions (TILLING) protocols, 183 Thermal treatments annealing, 233e236 granular cold-water-swelling starch (GCWS) methods, 226e227 single starch, 227 waxy maize starch, 227 heating dry starch, 236e237 heat-moisture treatment (HMT), 228e233 osmotic pressure treatment (OPT), 237 pregelatinized starch, 224e226 Thermoplastic starch (TPS), 751 Thermorobust crops, 192e193 Thioredoxin, 28e30 Tomato fruit, 38e39 Top-down process, 692 Traditional Chinese medicine starches XRD, 448, 448t Trisaccharides, 783 Triticum dicoccum, 314e315 Triticum durum, 314e315 Tubers and leguminous starches, 597 Tuber starches African yam bean, 422 amylose and amylopectin, 448e453 arracacha, 422 biotechnological methods, 493e494 buffalo gourd, 424 cassava, 422 chemical modifications, cassava acetylation, 489 acetylation-cross-linking, 490 alkaline and acid treatment, 489

892 Index Tuber starches (Continued ) carboxymethylated starch, 490 complexing, 488 cross-linked starch, 488e489 hydroxypropylation, 489 oxidation, 489 oxidation-cross-linkingphosphorylation, 490 starch citrate, 490 starch succinate, 490 Chinese water chestnut, 422 coleus, 423 digestibility, 483e484 east Indian arrowroot, 423 elephant foot yam, 422 food applications, 484e485 functionality, 435e484 future trends, 494e495 giant taro, 423 granule shape and size, 435e445 kudzu, 423 lotus root, 423 minor constituents, 428e435 modifications Amorphophallus, 492 Colocasia and Xanthosoma starches, 492 Peruvian carrot, 493 sweet potato, 490e491 modifying tropical starches, 485e494 Oca, 423 Peruvian carrot, 422 physical modifications, cassava annealing (ANN), 488 ball milling, 486e487 blending different native starches, 488 drum-drying, 486 electric field modification, 486 extrusion techniques, 486 heat-moisture treatment (HMT), 487e488 high-pressure-based treatments, 486 ɣ-irradiation, 486 microwave, 487 ultrasound, 487 production, 424e428 Queensland arrowroot, 423 retrogradation and freezeethaw stability, 480e481 rheological properties, 481e483 shoti, 423

starch-rich traditional Chinese medicinal plants, 424 structure, 435e484 swamp taro, 423 sweet potato, 422 swelling power and solubility, 453e459 tannia, 422 taro, 422 thermal properties, 460e470 tropical belt, 421 tropical root crops, 421e424 viscosity and pasting, 470e479 winged bean, 424 XRD and starch crystallinity, 445e448 yams, 422

U UDP-Glc, 58e59 Ultrahigh pressure (UHP) treatment, 241e242 Ultrasonication, 696, 697fe698f Ultrasonic treatment, 237e239 Unit chain profile, 109 Unmodified thickeners, 233

V Visible spectroscopy (colorimetry), 263e265 Vitamin encapsulation oil-soluble vitamins, 678e680 surface oil, 680, 681f vitamin E oil, 678e680, 680f

W Water crystallization, 582 Water migration, 583e584 Water solubility, 387e388, 387t, 388f Water vapor permeability (WVP), 759 Waxy, 97 Waxy maize starch, 754e755 Waxy protein structural gene, 60e61 Waxy rice amylopectin, 359 Western blots, 56 Wet-milling process, 234e235 Wheat, 39e40, 325e326, 601 functionality, food industry, 344e346 granular and molecular structure amylopectin (AP), 330e331 amylose (AM), 330e331

Index gelatinization temperatures, 333, 333t gelation, 330 large and small granules, 332 size and shape, 331, 331t hydrolysis dextrose equivalent (DE), 341e342 enzymatic hydrolysis, 342 hydrolysates, 344 saccharification, 343 starch depolymerization, 342 minor components amylose-lipid complex formation, 337, 337f arabinoxylans (AXs), 340e341 friabilin proteins, 335 lipids, 335e336 lysophospholipids, 336 pentosans, 340e341 polar lipids, 338e339 puroindolin isoforms, 338 starch granule-associated proteins (SGAPs), 338 production A-starch, 328 A-type and B-type granules, 326 B-starch, 326e327 decanters, 327 defiberd milk, 327 hydrophobicity, 326e327 laboratory-milled flour, 329e330 Martin process, 327e328 potato starches, 326e327 process overview, 328, 329f sodium chloride (NaCl), 329e330 sodium hydroxide (NaOH), 329e330 starch milk, 328

super soft wheat flour, 328 water, 326e327 wheat kernels, 326 Whiskey, 647e648 Wild yeasts alcohol production process, 645 amino acids, 645e646 barley proteins, 646 carbohydrate source, 645 mashing process, 645e646 proteinases, 645e646

X

Xerosicyos danguyi, 42e44

Y Yams amylopectin, 452e453, 452t amylose, 452e453, 452t crystallinity, 447 digestibility, 484 freeze-thaw stability, 481 granule shape and size, 440, 445t minor constituents, 430, 435t production, 428 retrogradation, 481 rheological properties, 483 swelling power and solubility, 459 thermal properties, 467e470 viscosity and pasting, 474e479, 478t XRD, 447 Yellow dextrins, 364 Yield penalty, 190e191

893


Starch in Food: Structure, Function and Applications

Starch in Food: Structure, Function and Applications

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