VITAMINS IN FOODSAnalysis, Bioavailability, and Stability

GEORGE F. M. BALL

Boca Raton London New York

A CRC title, part of the Taylor & Francis imprint, a member of the Taylor & Francis Group, the academic division of T&F Informa plc.

Published in 2006 by CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 2006 by Taylor & Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group No claim to original U.S. Government works Printed in the United States of America on acid-free paper 10 9 8 7 6 5 4 3 2 1 International Standard Book Number-10: 1-57444-804-8 (Hardcover) International Standard Book Number-13: 978-1-57444-804-7 (Hardcover) Library of Congress Card Number 2005049926 This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. A wide variety of references are listed. Reasonable efforts have been made to publish reliable data and information, but the author and the publisher cannot assume responsibility for the validity of all materials or for the consequences of their use. No part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www.copyright.com (http://www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC) 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe.

Library of Congress Cataloging-in-Publication DataBall, G.F.M. Vitamins in foods : analysis, bioavailability, and stability / by George F.M. Ball. p. cm. -- (Food science and technology ; 156) Includes bibliographical references and index. ISBN 1-57444-804-8 (alk. paper) 1. Food--Vitamin content. I. Title. II. Food science and technology (Taylor & Francis) ; 156. TX553.V5B358 2005 613.2'85--dc22

2005049926

Visit the Taylor & Francis Web site at http://www.taylorandfrancis.comTaylor & Francis Group is the Academic Division of Informa plc.

and the CRC Press Web site at http://www.crcpress.com

Dedication

This work is dedicated to my wife and dearest friend, Kazuko (Kako).

About the Author

George Ball has accumulated many years of commercial and research laboratory experience in pharmaceutical analysis, clinical analysis, biochemical analysis, and food analysis. He has contributed to original research publications relating to biochemistry (platelet function) and endocrinology (control of ovulation) and is the author of several books and book chapters on vitamins. He received the B.Sc. honors degree in agricultural sciences from the University of Nottingham, UK in 1975.

Preface

Optimal vitamin status is a prerequisite for good health, and governmentapproved food fortication strategies are deemed necessary to ensure adequate intake of certain vitamins. Knowledge about vitamin bioavailability from food is essential for the estimation of dietary requirements. Equally important is knowledge of a vitamins stability toward postharvest handling of food, processing, storage, and preparation for consumption. To acquire this knowledge, one must learn about vitamin chemistry and how the vitamin is absorbed and metabolized. Successful research into vitamin bioavailability and stability is entirely dependent on the development and validation of suitable analytical methods. Vitamin bioavailability from food is subject to many variables imposed by food constituents and the preparation of food. Great progress has been made over the past decade, largely attributable to innovative analytical methodology, but there are many inconsistencies and the continuation of a multipronged research effort from independent laboratories must be encouraged to achieve solid and vital data. I would like to acknowledge the expertise and diligence of Lynn Saliba at the British Library. George F. M. Ball

Contents

Part I

Properties of Vitamins3 4 4 5 6 6 7 9 9 10 11 11 12 13 14 14 15 16 17 17 17 18

Chapter 1. Nutritional Aspects of Vitamins 1.1 Denition and Classication of Vitamins . . . . . . . . . . . . . . . . . . . . . . . . 1.2 Nutritional Vitamin Deciency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3 Vitamin Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4 Vitamin Enhancement of Foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 Stability of Vitamins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5.1 Water Activity and Lipid Oxidation . . . . . . . . . . . . . . . . . . . . . . 1.5.2 First-Order Kinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5.3 Effects of Food Processing on Vitamin Retention. . . . . . . . . 1.5.3.1 Dehydration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5.3.2 Blanching . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5.3.3 Canning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5.3.4 Pasteurization and Ultra-High-Temperature Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5.3.5 Microwave Heating . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5.3.6 Hydrothermal Processes (Flaking, Pufng, and Extrusion) . . . . . . . . . . . . . . . 1.5.3.7 Freezing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5.3.8 Irradiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5.3.9 High Hydrostatic Pressure Treatment . . . . . . . . . . 1.5.3.10 Curing and Smoking . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5.4 Milling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5.5 Effects of Food Storage on Vitamin Retention . . . . . . . . . . . . 1.5.6 Effects of Domestic Cooking on Vitamin Retention . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Intestinal Absorption and Bioavailability of Vitamins: Introduction 2.1 General Principles of Solute Translocation . . . . . . . . . . . . . . . . . . . . . 2.2 Intestinal Absorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1 The Villus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.2 The Luminal Environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.3 Adaptive Regulation of Intestinal Nutrient Transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chapter 2.

23 24 24 25 25

Nonspecic Anatomical Adaptations to Changing Metabolic Requirements and Food Deprivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.3.2 Dietary Regulation of Intestinal Nutrient Carriers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.4 Digestion, Absorption, and Transport of Dietary Fat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.5 Transport of Glucose and Fructose: A Model for the Absorption of Some Water-Soluble Vitamins . . . . . . . . . 2.2.6 Effects of Dietary Fiber on Absorption of Nutrients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Bioavailability. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.1 General Concepts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.2 Methods for Estimating Vitamin Bioavailability in Human Subjects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.2.1 Plasma Response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.2.2 Urinary Excretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.2.3 Oral-Fecal Balance Studies and the Determination of Prececal Digestibility . . . . . . . . . . 2.3.2.4 Use of Stable Isotopes . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chapter 3. Vitamin A: Retinoids and the Provitamin A Carotenoids 3.1 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Chemical Structure, Biopotency, and Physicochemical Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.1 Structure and Biopotency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.1.1 Retinol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.1.2 Provitamin A Carotenoids . . . . . . . . . . . . . . . . . . . . . . . 3.2.2 Physicochemical Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.2.1 Appearance and Solubility . . . . . . . . . . . . . . . . . . . . . . . 3.2.2.2 Stability in Nonaqueous Solution . . . . . . . . . . . . . . . . 3.2.2.2.1 Retinoids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.2.2.2 Carotenoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 Vitamin A in Foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.1 Occurrence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.1.1 Vitamin A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.1.2 Provitamin A Carotenoids . . . . . . . . . . . . . . . . . . . . . . . 3.3.2 Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.2.2 Vitamin A in Milk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.2.3 Supplemental Vitamin A in Corn Flakes and Rice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2.2.3.1

25 26 27 28 30 32 32 33 33 34 34 35 36

39 40 40 40 41 43 43 45 45 45 45 45 46 47 48 48 51 56

3.3.2.4 Provitamin A Carotenoids . . . . . . . . . . . . . . . . . . . . . . . 3.3.3 Vitamin A Equivalency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.4 Applicability of Analytical Techniques . . . . . . . . . . . . . . . . . . . 3.4 Intestinal Absorption, Metabolism, and Transport . . . . . . . . . . . . . 3.4.1 Absorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.2 Metabolic Events Within the Enterocyte . . . . . . . . . . . . . . . . . . 3.4.2.1 Esterication of Retinol . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.2.2 Conversion of Provitamin Carotenoids to Retinoids . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.3 Liver Uptake of Chylomicron Remnants and Storage of Vitamin A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.4 Plasma Transport of Retinol and Carotenoids . . . . . . . . . . . . 3.4.5 Tissue Uptake and Metabolism of Retinol . . . . . . . . . . . . . . . . 3.5 Bioavailability. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.2 In vivo Methods of Assessing b-Carotene Bioavailability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.2.1 Use of Radioisotopes in Cannulated Patients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.2.2 Animal Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.2.3 Serum, Plasma, or Chylomicron Responses not Involving Isotopic Tracers . . . . . . . . . . . . . . . . . . . 3.5.2.4 Methods Involving Stable Isotopes . . . . . . . . . . . . . . 3.5.3 In vitro Methods of Assessing b-Carotene Bioaccessibility and Bioavailability . . . . . . . . . . . . . . . . . . . . . . . 3.5.3.1 In vitro Digestion Methods to Assess b-Carotene Bioaccessibility . . . . . . . . . . . . . . . . . . . . . . . 3.5.3.2 In vitro Studies of b-Carotene Absorption Using Caco-2 Cells . . . . . . 3.5.4 Host-Related Factors Affecting the Bioavailability of b-Carotene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.5 Dietary Factors Affecting the Bioavailability of b-Carotene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.5.1 Location of Carotenoids in the Plant Source . . . . . 3.5.5.2 Food Matrix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.5.3 Dietary Protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.5.4 Dietary Fat and Energy . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.5.5 Dietary Fiber . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.5.6 Plant Sterols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.6 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6 b-Carotene Supplementation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6.1 Effect of Vegetable Consumption on Vitamin A Status in Populations at Risk of Vitamin A Deciency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

57 60 61 61 62 63 63 63 66 66 67 67 67 68 68 69 69 71 74 74 76 77 77 77 78 80 81 86 87 88 88

88

Effects of b-Carotene Supplementation on Breastmilk Carotenoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92 Chapter 4. Vitamin D 4.1 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 Chemical Structure, Biopotency, and Physicochemical Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.1 Structure and Biopotency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.2 Physicochemical Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.2.1 Appearance and Solubility . . . . . . . . . . . . . . . . . . . . . . 4.2.2.2 Stability in Nonaqueous Solution . . . . . . . . . . . . . . . 4.3 Vitamin D in Foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.1 Occurrence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.2 Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.3 Expression of Dietary Values . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.4 Applicability of Analytical Techniques . . . . . . . . . . . . . . . . . . 4.4 Intestinal Absorption, Transport, and Metabolism . . . . . . . . . . . . 4.5 Bioavailability. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chapter 5. Vitamin E 5.1 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2 Chemical Structure, Biopotency, and Physicochemical Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.1 Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.2 Biopotency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.3 Physicochemical Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.3.1 Appearance and Solubility . . . . . . . . . . . . . . . . . . . . . . 5.2.3.2 Stability in Nonaqueous Solution . . . . . . . . . . . . . . . 5.2.3.3 In Vitro Antioxidant Activity . . . . . . . . . . . . . . . . . . . . 5.3 Vitamin E in Foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.1 Occurrence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.2 Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.3 Expression of Dietary Values . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.4 Applicability of Analytical Techniques . . . . . . . . . . . . . . . . . . 5.4 Intestinal Absorption and Transport. . . . . . . . . . . . . . . . . . . . . . . . . . 5.4.1 Absorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4.2 Plasma Transport and Distribution . . . . . . . . . . . . . . . . . . . . . . 5.4.3 Preferential Secretion of 2R-a-Tocopherol Stereoisomers by the Liver . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4.4 Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5 Bioavailability. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5.1 Efciency of Vitamin E Absorption . . . . . . . . . . . . . . . . . . . . . .

3.6.2

107 108 108 108 108 109 110 110 112 113 113 114 115 116

119 120 120 121 122 122 122 122 123 123 126 128 128 128 129 129 129 130 130 130

Effects of Polyunsaturated Fats on Vitamin E Absorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5.3 Effects of Dietary Fiber on Vitamin E Absorption . . . . . . . 5.5.4 Effect of Plant Sterols on Vitamin E Bioavailability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.6 Vitamin E Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chapter 6. Vitamin K 6.1 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2 Chemical Structure, Biopotency, and Physicochemical Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2.1 Structure and Biopotency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2.2 Physicochemical Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2.2.1 Appearance and Solubility . . . . . . . . . . . . . . . . . . . . . . 6.2.2.2 Stability in Nonaqueous Solution . . . . . . . . . . . . . . . 6.3 Vitamin K in Foods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3.1 Occurrence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3.2 Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3.2.1 Effects of Hydrogenation . . . . . . . . . . . . . . . . . . . . . . . . 6.3.3 Applicability of Analytical Techniques . . . . . . . . . . . . . . . . . . 6.4 Intestinal Absorption and Transport. . . . . . . . . . . . . . . . . . . . . . . . . . 6.4.1 Absorption and Transport of Dietary Vitamin K . . . . . . . . 6.4.2 Bacterially Synthesized Menaquinones as a Possible Endogenous Source of Vitamin K . . . . . . . . . . . . . . . . . . . . . . . . 6.5 Bioavailability. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chapter 7. Thiamin (Vitamin B1) 7.1 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2 Chemical Structure, Biopotency, and Physicochemical Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2.1 Structure and Potency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2.2 Physicochemical Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2.2.1 Appearance and Solubility . . . . . . . . . . . . . . . . . . . . . . 7.2.2.2 Stability in Aqueous Solution . . . . . . . . . . . . . . . . . . . 7.3 Thiamin in Foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3.1 Occurrence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3.2 Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3.3 Applicability of Analytical Techniques . . . . . . . . . . . . . . . . . . 7.4 Intestinal Absorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.5 Bioavailability. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.5.1 Bioavailability of Thiamin in Foods . . . . . . . . . . . . . . . . . . . . . 7.5.2 Antithiamin Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5.5.2

131 131 132 132 132

137 138 138 139 139 139 139 139 141 142 142 143 143 143 145 146

149 149 149 150 150 150 151 151 152 154 154 155 155 156

7.5.2.1 Thiaminases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.5.2.2 Polyphenols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.5.3 Effects of Alcohol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.5.4 Effects of Dietary Fiber . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chapter 8. Flavins: Riboavin, FMN, and FAD (Vitamin B2) 8.1 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2 Chemical Structure, Biopotency, and Physicochemical Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2.1 Structure and Potency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2.2 Physicochemical Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2.2.1 Appearance and Solubility . . . . . . . . . . . . . . . . . . . . . . 8.2.2.2 Stability in Aqueous Solution . . . . . . . . . . . . . . . . . . . 8.3 Vitamin B2 in Foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3.1 Occurrence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3.2 Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3.3 Applicability of Analytical Techniques . . . . . . . . . . . . . . . . . . 8.4 Intestinal Absorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.4.1 Absorption of Dietary Vitamin B2 . . . . . . . . . . . . . . . . . . . . . . . 8.4.2 Absorption of Bacterially Synthesized Vitamin B2 in the Large Intestine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.5 Bioavailability. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

156 157 159 160 160

165 165 165 167 167 167 168 168 169 171 171 171 172 173 173

Chapter 9. Niacin 9.1 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2 Chemical Structure, Biopotency, and Physicochemical Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2.1 Structure and Potency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2.2 Physicochemical Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2.2.1 Appearance, Solubility, and Other Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2.2.2 Stability in Aqueous Solution . . . . . . . . . . . . . . . . . . . 9.3 Niacin in Foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.3.1 Occurrence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.3.2 Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.3.3 Applicability of Analytical Techniques . . . . . . . . . . . . . . . . . . 9.4 Intestinal Absorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.5 Bioavailability. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.5.1 Niacin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.5.2 Tryptophan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

177 178 178 179 179 179 179 179 181 182 183 183 183 187 187

Chapter 10. Vitamin B6 10.1 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.2 Chemical Structure, Biopotency, and Physicochemical Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.2.1 Structure and Potency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.2.2 Physicochemical Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.2.2.1 Appearance and Solubility . . . . . . . . . . . . . . . . . . 10.2.2.2 Stability in Aqueous Solution . . . . . . . . . . . . . . . 10.3 Vitamin B6 in Foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.3.1 Occurrence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.3.2 Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.3.3 Applicability of Analytical Techniques . . . . . . . . . . . . . . . . 10.4 Intestinal Absorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.5 Bioavailability. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.5.1 Bioavailability of Vitamin B6 in Foods . . . . . . . . . . . . . . . . 10.5.2 Effects of Alcohol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.5.3 Effects of Dietary Fiber . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.5.4 Glycosylated Forms of Vitamin B6 . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chapter 11. Pantothenic Acid 11.1 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.2 Chemical Structure, Biopotency, and Physicochemical Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.2.1 Structure and Potency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.2.2 Physicochemical Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.2.2.1 Appearance and Solubility . . . . . . . . . . . . . . . . . . 11.2.2.2 Stability in Aqueous Solution . . . . . . . . . . . . . . . . 11.3 Pantothenic Acid in Foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.3.1 Occurrence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.3.2 Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.3.3 Applicability of Analytical Techniques . . . . . . . . . . . . . . . . 11.4 Intestinal Absorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.4.1 Digestion and Absorption of Dietary Pantothenic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.4.2 Absorption of Bacterially Synthesized Pantothenic Acid in the Large Intestine . . . . . . . . . . . . . . . 11.5 Bioavailability. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

189 190 190 191 191 192 193 193 194 197 198 199 199 201 201 202 205

211 211 211 212 212 213 213 213 213 216 216 216 217 217 218

Chapter 12. Biotin 12.1 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221 12.2 Chemical Structure, Biopotency, and Physicochemical Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221

Structure and Potency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Physicochemical Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.2.2.1 Appearance and Solubility . . . . . . . . . . . . . . . . . . 12.2.2.2 Stability in Aqueous Solution . . . . . . . . . . . . . . . 12.3 Biotin in Foods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.3.1 Occurrence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.3.2 Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.3.3 Applicability of Analytical Techniques . . . . . . . . . . . . . . . . 12.4 Intestinal Absorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.4.1 Digestion and Absorption of Dietary Biotin . . . . . . . . . . . 12.4.2 Absorption of Bacterially Synthesized Biotin in the Large Intestine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.5 Bioavailability. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

12.2.1 12.2.2

221 222 222 223 223 223 223 225 226 226 227 228 229

Chapter 13. Folate 13.1 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.2 Chemical Structure, Biopotency, and Physicochemical Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.2.1 Structure and Potency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.2.2 Physicochemical Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.2.2.1 Appearance, Solubility, and Ionic Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.2.2.2 Stability in Aqueous Solution . . . . . . . . . . . . . . . 13.3 Folate in Foods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.3.1 Occurrence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.3.2 Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.3.3 Applicability of Analytical Techniques . . . . . . . . . . . . . . . . 13.4 Absorption, Transport, and Metabolism . . . . . . . . . . . . . . . . . . . . . 13.4.1 Deconjugation of Polyglutamyl Folate . . . . . . . . . . . . . . . . 13.4.2 Absorption of Dietary Folate . . . . . . . . . . . . . . . . . . . . . . . . . . 13.4.3 Inuence of Folate-Binding Protein on the Absorption of Folate from Milk . . . . . . . . . . . . . . . . . . . . . . . 13.4.4 Adaptive Regulation of Folate Absorption . . . . . . . . . . . . 13.4.5 Salvage of Dietary 5-Methyl-5,6-DHF . . . . . . . . . . . . . . . . . 13.4.6 Absorption of Bacterially Synthesized Folate in the Large Intestine . . . . . . . . . . . . . . . . . . . . . . . . . 13.4.7 Plasma Transport and Intracellular Metabolism . . . . . . 13.4.8 Folate Homeostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.5 Bioavailability. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.5.2 Methods for Assessing Folate Bioavailability . . . . . . . . . 13.5.2.1 Plasma Response . . . . . . . . . . . . . . . . . . . . . . . . . . . .

231 232 232 233 233 234 236 236 238 244 245 245 246 247 249 249 250 251 251 252 252 252 252

13.5.2.2 Stable-Isotopic Methods . . . . . . . . . . . . . . . . . . . . . 13.5.2.3 Use of Ileostomy Subjects . . . . . . . . . . . . . . . . . . . 13.5.3 Inherent Bioavailability of Monoglutamyl and Polyglutamyl Folates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.5.4 Bioavailability of Naturally Occurring Folate in Fruits and Vegetables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.5.5 Bioavailability of Folate in Milk . . . . . . . . . . . . . . . . . . . . . . . 13.5.6 Effects of Soluble Food Components on Folate Bioavailability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.5.7 Effects of Dietary Fiber on Folate Bioavailability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.5.8 Bioavailability of Folate in Fortied Foods . . . . . . . . . . . . 13.5.9 Effects of Alcohol on Folate Status . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chapter 14. Vitamin B12 (Cobalamins) 14.1 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.2 Chemical Structure, Biopotency, and Physicochemical Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.2.1 Structure and Potency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.2.2 Physicochemical Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.2.2.1 Appearance and Solubility . . . . . . . . . . . . . . . . . . 14.2.2.2 Stability in Aqueous Solution . . . . . . . . . . . . . . . 14.3 Vitamin B12 in Foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.3.1 Occurrence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.3.2 Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.3.3 Applicability of Analytical Techniques . . . . . . . . . . . . . . . . 14.4 Absorption and Conservation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.4.1 Digestion and Absorption of Dietary Vitamin B12 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.4.2 Conservation of Vitamin B12 . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.5 Bioavailability. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.5.1 Efciency of Absorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.5.2 Bioavailability Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.5.2.1 Effects of Dietary Fiber . . . . . . . . . . . . . . . . . . . . . . 14.5.2.2 Effects of Alcohol . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.5.2.3 Effects of Smoking . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

253 255 256 257 258 260 262 263 264 264

275 276 276 277 277 278 278 278 279 281 281 282 283 283 283 284 284 284 285 285

Chapter 15. Vitamin C 15.1 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289 15.2 Chemical Structure, Biopotency, and Physicochemical Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290 15.2.1 Structure and Potency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290

Physicochemical Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.2.2.1 Solubility and Other Properties . . . . . . . . . . . . . 15.2.2.2 Stability in Aqueous Solution . . . . . . . . . . . . . . . 15.3 Vitamin C in Foods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.3.1 Occurrence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.3.2 Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.3.3 Applicability of Analytical Techniques . . . . . . . . . . . . . . . . 15.4 Intestinal Absorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.4.1 General Principles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.4.2 Transport Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.4.2.1 Ascorbic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.4.2.2 Dehydroascorbic Acid . . . . . . . . . . . . . . . . . . . . . . . 15.4.3 Efciency of Ascorbate Absorption in Humans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.5 Bioavailability. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.5.1 Bioavailability of Vitamin C in Foods . . . . . . . . . . . . . . . . . 15.5.2 Effects of Dietary Fiber . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.5.3 Effects of Alcohol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

15.2.2

291 291 292 292 292 294 299 300 300 301 301 302 302 303 303 304 305 305

Part II

Analysis of Vitamins311 312 312 313 314 314 314 314 316 316 318 320

Chapter 16. Analytical Considerations 16.1 Bioassays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16.2 In Vitro Analytical Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16.3 Analytical Approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16.4 Preparation of Sample Extracts for Analysis . . . . . . . . . . . . . . . . . 16.4.1 Extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16.4.2 Cleanup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16.5 Method Evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16.5.1 Measurement Value and Uncertainty . . . . . . . . . . . . . . . . . 16.5.2 Quality Assurance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16.5.3 Food Reference Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16.5.4 Method Validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chapter 17. 17.1 17.2 17.3 17.4 Extraction Techniques for the Water-Soluble Vitamins Vitamin B1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Vitamin B2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Niacin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Vitamin B6 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

321 322 323 326

17.5 Pantothenic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17.6 Biotin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17.7 Folate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17.8 Vitamin B12 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17.9 Vitamin C. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chapter 18. Microbiological Methods for the Determination of the B-Group Vitamins 18.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18.2 General Principles. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18.2.1 Turbidimetric Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18.2.2 Methods Based on the Measurement of Metabolic Carbon Dioxide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18.3 Conventional Turbidimetric Method Using Test Tubes . . . . . . . 18.3.1 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18.3.2 Laboratory Facilities and Cleaning of Glassware . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18.3.3 Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18.3.4 General Assay Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18.3.4.1 Maintenance of Stock Cultures . . . . . . . . . . . . . . 18.3.4.2 Preparation of the Inoculum Culture . . . . . . . . 18.3.4.3 Preparation of the Assay (Basal) Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18.3.4.4 Extraction of the Vitamin from the Test Material . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18.3.4.5 Setting Up the Assay . . . . . . . . . . . . . . . . . . . . . . . . 18.3.4.6 Quantication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18.3.5 Partial Automation of the Assay Procedure . . . . . . . . . . . 18.4 Turbidimetric Method Using Microtiter Plates . . . . . . . . . . . . . . . 18.5 Assays of Individual B-Group Vitamins . . . . . . . . . . . . . . . . . . . . . 18.5.1 Vitamin B1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18.5.2 Vitamin B2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18.5.3 Niacin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18.5.3.1 Determination of Total Niacin . . . . . . . . . . . . . . . 18.5.3.2 Determination of Bound Nicotinic Acid . . . . . 18.5.3.3 Determination of Added Nicotinic Acid . . . . 18.5.4 Vitamin B6 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18.5.5 Pantothenic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18.5.6 Biotin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18.5.7 Folate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18.5.8 Vitamin B12 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

328 328 329 331 332 333

339 339 339 341 342 342 343 344 344 345 346 347 348 348 349 350 350 351 351 352 354 354 355 356 356 359 360 360 361 363

Chapter 19. 19.1 19.2 19.3

19.4

19.5

19.6

19.7

19.8

Physicochemical Analytical Techniques (Excluding HPLC) AOAC Titrimetric Method for Vitamin C. . . . . . . . . . . . . . . . . . . . Direct Spectrophotometric Determination of Vitamin C. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Colorimetric Methods for Niacin and Vitamin C. . . . . . . . . . . . . nig 19.3.1 Determination of Niacin by the Ko Reaction (AOAC Method) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.3.2 Colorimetric Methods for Vitamin C . . . . . . . . . . . . . . . . . . Fluorometric Methods for Thiamin, Riboavin, Vitamin B6, and Vitamin C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.4.1 Thiamin (AOAC Method) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.4.2 Riboavin (AOAC Method) . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.4.3 Vitamin B6 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.4.4 Vitamin C (AOAC Method) . . . . . . . . . . . . . . . . . . . . . . . . . . . Enzymatic Methods for Nicotinic Acid and Ascorbic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.5.1 Nicotinic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.5.2 Ascorbic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Continuous-Flow Analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.6.1 Segmented-Flow Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.6.2 Flow-Injection Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.6.3 Applications to Food Analysis . . . . . . . . . . . . . . . . . . . . . . . . 19.6.3.1 Fat-Soluble Vitamins . . . . . . . . . . . . . . . . . . . . . . . . . 19.6.3.2 Thiamin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.6.3.3 Riboavin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.6.3.4 Thiamin and Riboavin Simultaneously . . . . 19.6.3.5 Niacin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.6.3.6 Vitamin C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gas Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.7.1 Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.7.2 Column Technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.7.3 Detectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.7.4 Derivatization Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.7.5 Quantication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.7.6 Applications to Food Analysis . . . . . . . . . . . . . . . . . . . . . . . . 19.7.6.1 Vitamin E . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.7.6.2 Thiamin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.7.6.3 Niacin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.7.6.4 Vitamin B6 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.7.6.5 Pantothenic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . Supercritical Fluid Chromatography . . . . . . . . . . . . . . . . . . . . . . . . 19.8.1 Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.8.2 Instrumentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

369 372 373 373 374 375 375 375 376 377 378 378 379 380 380 380 381 381 381 382 382 382 383 385 385 385 386 386 387 387 387 388 388 388 389 390 390 391

19.8.3 Columns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.8.4 Applications to Food Analysis . . . . . . . . . . . . . . . . . . . . . . . . 19.9 Capillary Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.9.1 Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.9.2 Capillary Zone Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . 19.9.3 Micellar Electrokinetic Capillary Chromatography . . . 19.9.4 Operational Aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.9.5 Applications to Food Analysis . . . . . . . . . . . . . . . . . . . . . . . . 19.9.5.1 Thiamin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.9.5.2 Riboavin, FMN, and FAD . . . . . . . . . . . . . . . . . . 19.9.5.3 Niacin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.9.5.4 Vitamin C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chapter 20. Determination of the Fat-Soluble Vitamins by HPLC 20.1 Nature of the Sample. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.2 Extraction Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.2.1 Alkaline Hydrolysis (Saponication) . . . . . . . . . . . . . . . . . . 20.2.1.1 Vitamin A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.2.1.2 Carotenoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.2.1.3 Vitamin D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.2.1.4 Vitamin E . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.2.2 Alcoholysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.2.3 Enzymatic Hydrolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.2.4 Direct Solvent Extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.2.4.1 Vitamin A and Carotene . . . . . . . . . . . . . . . . . . . . . 20.2.4.2 Carotenoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.2.4.3 Vitamin D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.2.4.4 Vitamin E . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.2.4.5 Vitamin K . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.2.5 Matrix Solid-Phase Dispersion . . . . . . . . . . . . . . . . . . . . . . . . 20.2.6 Supercritical Fluid Extraction . . . . . . . . . . . . . . . . . . . . . . . . . . 20.2.6.1 Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.2.6.2 Instrumentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.2.6.3 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.3 Cleanup Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.3.1 Precipitation of Sterols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.3.2 Open-Column Chromatography . . . . . . . . . . . . . . . . . . . . . . 20.3.2.1 Magnesia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.3.2.2 Alumina . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.3.2.3 Silica Gel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.3.3 Solid-Phase Extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.3.3.1 General Considerations . . . . . . . . . . . . . . . . . . . . . . 20.3.3.2 Application in Vitamin D Determinations . .

393 394 394 394 396 396 397 399 399 399 406 408 409

419 419 419 421 422 422 423 424 424 425 426 427 427 428 428 429 430 430 430 431 435 436 436 436 436 437 437 437 438

20.4

HPLC Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.4.1 Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.4.2 Explanations of Chromatographic Terms . . . . . . . . . . . . . 20.4.2.1 Retention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.4.2.2 Separation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.4.2.3 Resolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.4.2.4 Efciency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.4.3 The Column . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.4.4 Chromatographic Modes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.4.4.1 Normal-Phase Chromatography . . . . . . . . . . . . . 20.4.4.1.1 Adsorption Chromatography. . . . . . . . . . . . . . . . . 20.4.4.1.2 Polar Bonded-Phase Chromatography. . . . . . . . . . . . . . . . . 20.4.4.2 Reversed-Phase Chromatography . . . . . . . . . . . 20.4.4.3 Two-Dimensional HPLC . . . . . . . . . . . . . . . . . . . . . 20.4.5 Detection Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.4.5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.4.5.2 Absorbance Detection . . . . . . . . . . . . . . . . . . . . . . . 20.4.5.3 Fluorescence Detection . . . . . . . . . . . . . . . . . . . . . . 20.4.5.4 Electrochemical Detection . . . . . . . . . . . . . . . . . . . 20.4.5.5 Mass Spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . 20.5 Applications of HPLC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.5.1 Vitamin A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.5.1.1 Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.5.1.2 Quantication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.5.1.3 Normal-Phase Separations . . . . . . . . . . . . . . . . . . 20.5.1.4 Reversed-Phase Separations . . . . . . . . . . . . . . . . . 20.5.2 Provitamin A Carotenoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.5.2.1 Sources of Variation in the Methodology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.5.2.2 Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.5.2.3 Potential Problems with the Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.5.2.4 Normal-Phase Separations . . . . . . . . . . . . . . . . . . 20.5.2.5 Reversed-Phase Separations . . . . . . . . . . . . . . . . . 20.5.2.5.1 C18-Bonded Phases . . . . . . . . . . . . . . 20.5.2.5.2 C30-Bonded Phases . . . . . . . . . . . . . . 20.5.3 Vitamin D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.5.3.1 Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.5.3.2 Quantication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.5.3.3 Cleanup Procedures . . . . . . . . . . . . . . . . . . . . . . . . . 20.5.3.4 Normal-Phase Separations . . . . . . . . . . . . . . . . . . 20.5.3.5 Reversed-Phase Separations . . . . . . . . . . . . . . . . .

439 439 440 440 440 441 442 442 444 444 444 447 448 452 453 453 454 455 456 457 457 457 457 463 465 466 469 469 469 472 474 475 475 486 489 489 489 499 500 500

Vitamin E . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.5.4.1 Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.5.4.2 Quantication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.5.4.3 Normal-Phase Separations . . . . . . . . . . . . . . . . . . 20.5.4.4 Reversed-Phase Separations . . . . . . . . . . . . . . . . . 20.5.5 Vitamin K . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.5.5.1 Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.5.5.2 Normal-Phase Separations . . . . . . . . . . . . . . . . . . 20.5.5.3 Reversed-Phase Separations . . . . . . . . . . . . . . . . . 20.5.6 Simultaneous Determination of Two or Three Vitamins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.5.6.1 Normal-Phase Separations . . . . . . . . . . . . . . . . . . 20.5.6.2 Reversed-Phase Separations . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Determination of the Water-Soluble Vitamins by HPLC 21.1 HPLC Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.1.1 The Column . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.1.2 Chromatographic Modes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.1.2.1 Ion Exchange Chromatography . . . . . . . . . . . . . 21.1.2.2 Ion Exclusion Chromatography . . . . . . . . . . . . . 21.1.2.3 Reversed-Phase Chromatography . . . . . . . . . . . 21.1.2.4 Reversed-Phase Ion-Pair Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.1.3 Derivatization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.2 Applications of HPLC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.2.1 Thiamin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.2.1.1 Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.2.1.2 Methodology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.2.2 Vitamin B2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.2.2.1 Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.2.2.2 Methodology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.2.3 Niacin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.2.3.1 Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.2.3.2 Methodology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.2.4 Vitamin B6 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.2.4.1 General Considerations . . . . . . . . . . . . . . . . . . . . . . 21.2.4.2 Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.2.4.3 Methodology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.2.5 Pantothenic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.2.5.1 Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.2.5.2 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chapter 21.

20.5.4

505 506 510 511 526 527 528 540 541 546 549 562 567

585 585 585 585 587 588 589 591 592 592 592 594 598 598 600 612 612 612 624 624 625 626 638 638 639

Biotin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.2.6.1 Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.2.6.2 Application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.2.7 Folate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.2.7.1 General Considerations . . . . . . . . . . . . . . . . . . . . . . 21.2.7.2 Cleanup Procedures . . . . . . . . . . . . . . . . . . . . . . . . . 21.2.7.3 Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.2.7.4 Methodology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.2.8 Vitamin B12 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.2.9 Vitamin C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.2.9.1 Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.2.9.2 Methodology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.2.10 Multiple Vitamin Analyses . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.2.10.1 Thiamin and Riboavin . . . . . . . . . . . . . . . . . . . . 21.2.10.2 Riboavin and Pyridoxine . . . . . . . . . . . . . . . . . 21.2.10.3 Nicotinamide and Pyridoxine . . . . . . . . . . . . . . 21.2.10.4 Three or More Vitamins . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chapter 22. 22.1 22.2 Biospecic Methods for Some of the B-Group Vitamins Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Immunoassays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22.2.1 The Immunological Reaction . . . . . . . . . . . . . . . . . . . . . . . . . . 22.2.2 Radioimmunoassay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22.2.2.1 Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22.2.2.2 Determination of Pantothenic Acid . . . . . . . . . 22.2.3 Enzyme-Linked Immunosorbent Assay . . . . . . . . . . . . . . . 22.2.3.1 Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22.2.3.2 Determination of Pantothenic Acid . . . . . . . . . 22.2.3.3 Determination of Vitamin B6 . . . . . . . . . . . . . . . . Protein-Binding Assays. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22.3.1 Radiolabeled Protein-Binding Assays . . . . . . . . . . . . . . . . . 22.3.1.1 Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22.3.1.2 Determination of Biotin . . . . . . . . . . . . . . . . . . . . . . 22.3.1.3 Determination of Folate . . . . . . . . . . . . . . . . . . . . . 22.3.1.4 Determination of Vitamin B12 . . . . . . . . . . . . . . . 22.3.2 Enzyme-Labeled Protein-Binding Assays . . . . . . . . . . . . . 22.3.2.1 General Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . 22.3.2.2 Determination of Biotin . . . . . . . . . . . . . . . . . . . . . . 22.3.2.3 Determination of Folate . . . . . . . . . . . . . . . . . . . . . 22.3.2.4 Determination of Vitamin B12 . . . . . . . . . . . . . . . Biomolecular Interaction Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . 22.4.1 Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

21.2.6

645 645 646 646 646 648 650 653 676 677 677 682 701 701 715 715 716 720

22.3

22.4

735 735 735 737 737 737 738 738 740 741 741 741 741 743 743 745 747 747 747 747 748 749 749

Biosensor-Based Immunoassay for Supplemental Biotin and Folate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 750 22.4.3 Biosensor-Based Protein-Binding Assay for Supplemental and Endogenous Vitamin B12 . . . . . . . . . . 751 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 752 Chapter 23. Summarized Appraisal of Analytical Techniques 23.1 Microbiological Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.2 High-Performance Liquid Chromatography . . . . . . . . . . . . . . . . . 23.2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.2.2 Fat-Soluble Vitamins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.2.2.1 Vitamin A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.2.2.2 Carotenoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.2.2.3 Vitamin D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.2.2.4 Vitamin E . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.2.2.5 Vitamin K . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.2.3 Water-Soluble Vitamins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.2.3.1 Thiamin and Flavins . . . . . . . . . . . . . . . . . . . . . . . . . 23.2.3.2 Niacin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.2.3.3 Vitamin B6 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.2.3.4 Pantothenic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.2.3.5 Biotin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.2.3.6 Folate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.2.3.7 Vitamin C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.3 Supercritical Fluid Chromatography . . . . . . . . . . . . . . . . . . . . . . . . 23.4 Capillary Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.5 Flow-Injection Analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.6 Biospecic Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.7 Evaluation of Vitamin Bioavailability From Food Analysis Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.7.1 Fat-Soluble Vitamins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.7.2 Water-Soluble Vitamins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.7.2.1 Thiamin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.7.2.2 Vitamin B2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.7.2.3 Niacin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.7.2.4 Vitamin B6 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.7.2.5 Pantothenic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.7.2.6 Biotin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.7.2.7 Folate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.7.2.8 Vitamin B12 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.7.2.9 Vitamin C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

22.4.2

757 758 758 759 759 759 760 760 760 761 761 762 762 763 763 763 763 764 764 765 765 767 767 767 767 768 768 769 769 769 769 770 770 770 777

Part I

Properties of Vitamins

1Nutritional Aspects of Vitamins1.1 Denition and Classication of Vitamins

Vitamins are a group of organic compounds that are, in very small amounts, essential for the normal functioning of the human body. They have widely varying chemical and physiological functions and are broadly distributed in natural food sources. Thirteen vitamins are recognized in human nutrition and these may be conveniently classied into two groups according to their solubility. The fat-soluble vitamins are represented by vitamins A, D, E, and K; also included are the 50 or so carotenoids that possess varying degrees of vitamin A activity. The water-soluble vitamins comprise vitamin C and the members of the vitamin B group, namely thiamin (vitamin B1), riboavin (vitamin B2), niacin, vitamin B6, pantothenic acid, folate, and vitamin B12. This simple classication reects to some extent the bioavailability of the vitamins, as the solubility affects their mode of intestinal absorption and their uptake by tissues. The solubility properties also relate to the distribution of vitamins in the various food groups, and have a direct bearing on the analytical methods employed. For many of the vitamins, biological activity is attributed to a number of structurally related compounds known as vitamers. The vitamers pertaining to a particular vitamin display, in most cases, similar qualitative biological properties to one another, but, because of subtle differences in their chemical structures, exhibit varying degrees of potency. Provitamins are vitamin precursors, that is, naturally occurring substances which are not vitamins themselves, but which can be converted into vitamins by normal body metabolism. Most of the vitamins are absolutely essential in the human diet because the body tissues cannot synthesize them. Two notable exceptions are vitamin D and niacin. Cutaneous synthesis of vitamin D depends on adequate exposure of the skin to sunlight, and the synthesis of niacin depends on a sufcient intake of its amino acid precursor, tryptophan, bound within protein. Plants have the ability to synthesize vitamins, except for vitamin B12, and serve as primary sources of these dietary essentials.3

4

Nutritional Aspects of Vitamins

1.2

Nutritional Vitamin Deciency

Several B-group vitamins serve as coenzymes for enzymes that function in the catabolism of foodstuffs to produce energy for the organism. A typical coenzyme consists of a protein (apoenzyme) to which the vitamin is attached. The vitamin portion of the coenzyme is usually responsible for the attachment of the enzyme to the substrate. If the specic vitamin is not available to form the coenzyme, the sequence of chemical changes in the metabolic process cannot proceed and the product whose change is blocked accumulates in the tissues: alternatively, metabolism is diverted to another pathway. For some B-group vitamins, clinical deciency results in a biochemical defect, which is manifested as a disease with characteristic symptoms. Other vitamins have less dramatic deciency symptoms in humans, but their deciency in certain animal species may give rise to distinctive signs. Some human individuals can benet from vitamin supplements, indicating that they may have been subclinically vitamin decient to begin with. The causes of nutritional vitamin deciency are any one or combination of the following: inadequate ingestion, poor absorption, inadequate utilization, increased requirement, increased excretion, and increased catabolism. The capacity of the body to store vitamins is another factor: humans can store thiamin for only about 2 weeks, whereas vitamin B12 can be stored in the liver for several years.

1.3

Vitamin Requirements

Metabolic processes must respond to the immediate needs of the body and therefore vitamin requirements are subject to continuous variation between certain limits. The Food and Nutrition Board of the Institute of Medicine in the U.S. denes a requirement as the lowest continuing intake level of a nutrient that, for a specic indicator of adequacy, will maintain a dened level of nutriture in an individual. A recommended dietary allowance (RDA) of a nutrient is the average daily dietary intake level that is sufcient to meet the requirement of nearly all (97 98%) apparently healthy individuals in a particular life stage and gender group. The RDA is derived from an estimated average requirement (EAR), which is an estimate of the intake at which the risk of inadequacy to an individual is 50%. RDAs have been published for vitamins A, D, E, and K, thiamin, riboavin, niacin, vitamin B6, folate, vitamin B12, and vitamin C. In the case of pantothenic acid and biotin, there is insufcient evidence to calculate an EAR, and a reference adequate intake (AI) is

Vitamins in Foods: Analysis, Bioavailability, and Stability

5

provided instead of an RDA. The AI is a value based on experimentally derived intake levels or approximations of observed mean nutrient intakes by a group (or groups) of apparently healthy people [1].

1.4

Vitamin Enhancement of Foods

The terms restoration, fortication, enrichment, standardization, and nutrication have been used to describe various ways of enhancing the vitamin content of foods [2]. Restoration involves the replacement, in full or in part, of vitamin losses incurred during processing. The addition of vitamins A and D to skimmed milk powder, and of vitamin D to evaporated milk, are examples of nonlegislative vitamin restoration in the U.K. Other examples are the replacement of B-group vitamins in our to compensate for the losses incurred in the milling of cereals to low extraction rates, and the addition of vitamin C to instant potato. Fortication refers to the addition of vitamins to foods that are suitable carriers for a particular vitamin, but which do not necessarily contain that vitamin naturally. It is especially carried out to fulll the role of a food in the diet. Thus margarine is fortied with vitamin A in many countries to replace the vitamin A that is lost from the diet when margarine is substituted for butter. Vitamin D is added to margarine at higher levels than found in butter as a public health measure, as the extra is considered necessary for the population as a whole. In the U.S., fortication of cereals and grains with folic acid began in 1996 and, since January 1998, all cereal grain products are fortied with 140 mg of folic acid/100 g. In the U.K., fortication of foods with folic acid is still voluntary. Enrichment refers to the addition of vitamins above the initial natural levels to make a product more marketable. Standardization refers to additions designed to compensate for natural uctuations in vitamin content. For instance, milk and butter are subject to seasonal variations in their vitamin A and D contents, and hence these vitamins are added to some dairy products to maintain constant levels. Nutrication means the addition of vitamins to formulated or fabricated foods marketed as meal replacers. Vitamins are also added to perform specic processing functions. For instance, b-carotene (the principal source of dietary vitamin A) is added to products such as pasta, margarine, cakes, and processed cheeses to impart color. Vitamin E and vitamin C (in the form of ascorbyl palmitate) can be used as antioxidants to stabilize pure oils and fats. Ascorbic acid is used for a variety of purposes in food processing, such as a reducing agent involved in the formation of the cured meat color in the curing of bacon and ham.

6

Nutritional Aspects of Vitamins

1.5

Stability of Vitamins

New food product development aims to retain as much of the naturally occurring vitamin content as possible, to protect added vitamins, and to minimize the appearance of undesirable breakdown products. Factors that play a role in the degradation of vitamins during processing and storage include temperature, air or oxygen, light, moisture content, water activity, pH, degradative enzymes, and metal trace elements, particularly iron and copper. Ryley et al. [3] discussed the use of kinetic data and mathematical models for predicting vitamin retention in food processing.

1.5.1

Water Activity and Lipid Oxidation

The water activity of a food is one of the primary factors determining the rate of food deterioration by various biochemical reactions. Raw foods with a high content of active water, such as leafy vegetables and meat, deteriorate in only a few days, whereas dry seeds, containing only structural water, can be stored for years under proper conditions. Water activity measures the availability of the water present in the system: it is more closely related to the physical and chemical properties of food than is total moisture content. Within the heterogeneous food matrix, the reactivity of each constituent is inuenced by its afnity for surrounding water molecules and the competing inuences of neighboring hydrophilic or hydrophobic chemical groups. Changes in the environment, such as heat, light, pressure, pH, additives, and modication of particle size, may alter the molecular state of water, and thereby inuence constituent reactivities and functional properties. The total water-binding energies of constituent chemical groups are reected in the equilibrium water vapor pressure or absolute humidity of the food material. At constant temperature, the vapor pressure may be expressed as equilibrium relative humidity (ERH) or the related analogous water activity. Water activity (Aw) can be dened as the ratio of the equilibrium vapor pressure exerted by the food material ( p) to the vapor pressure of pure water ( p0) at the same temperature: Aw p EHR p0 100 (1:1)

Small changes in temperature, or other factors which inuence Aw, may induce profound changes in the quality and stability of a food product. Therefore, Aw measurements provide a simple and convenient

Vitamins in Foods: Analysis, Bioavailability, and Stability

7

means of evaluating water-binding activity. The nonlinear, generally sigmoidal relationship between Aw and total moisture content, expressed as a moisture sorption isotherm, is a fundamental characteristic of a food product [4]. A low water activity (Aw , 0.6) does not permit microbial or enzymatic activity. It is generally accepted that growth of bacteria and most yeasts virtually ceases at Aw , 0.9, while molds do not grow at Aw , 0.7 [5]. The presence of water has a profound effect on lipid oxidation h as discussed by Labuza [6]. This has relevance to vitamin A and carotenoids, which undergo coupled oxidation in the presence of lipids. The kinetics of lipid oxidation are best studied in model systems using a swellable solid matrix, since the variables can be controlled. At Aw 0 (as in a dehydrated food), lipid oxidation is rapid, but the rate of oxidation decreases as the water activity increases up to Aw 0.5, where a leveling-off occurs. The protective effect of water within this range of water activity may be due to two mechanisms working together. First, hydrogen bonding takes place between water and lipid hydroperoxides produced during the propagation stage of lipid peroxidation. This bonding prevents the metal-catalyzed decomposition of the hydroperoxides into peroxyl and alkoxyl radicals that intensify propagation of the chain reaction. Second, hydration of metal catalysts makes them less effective through changes in their coordination sphere. In the intermediate moisture range of Aw 0.55 0.85, the solvent and mobilization properties of water become dominant, and lipid oxidation increases with increasing water activity. The catalysts present are more easily mobilized and swelling of the solid matrix exposes new catalytic sites. This detrimental effect of water has been conrmed in actual intermediate moisture foods [7]. As might be expected from the effect of water on lipid oxidation, the loss of all-trans-retinol suspended on microcrystalline cellulose in the dark, in air, increased with increasing water activity in the range of intermediate-moisture foods (Aw 0.4 0.75) [8]. In contrast, increasing the water activity from 0 to 0.73 decreased the rate of b-carotene degradation in the dark [9]. A protective effect of water against oxidation of carotenoids in the intermediate-moisture region was also observed by Ramakrishnan and Francis [10]. This difference in behavior between retinol and b-carotene can possibly be attributed to the poorer solubility of b-carotene in water.

1.5.2

First-Order Kinetics

The analytical approach in calculating and predicting food quality deterioration involves a kinetic/mathematical model. The model may

8

Nutritional Aspects of Vitamins

include equations of mass and energy balance, thermodynamics, transport and chemical properties, and coefcients. These equations have been simplied for specic conditions. Saguy and Karel [11] produced a status report on the modeling of quality deterioration during food processing and storage. Most reactions involving the deterioration of a nutrient t a rst-order mathematical expression: dC kC dt (1:2)

where C is the nutrient concentration at a given reaction time (t), and k is the degradation reaction rate constant. When Equation (1.1) is integrated, and log (C/C0) versus t is plotted, the slope of the straight line obtained denes the rate constant (k), where C0 is the initial concentration of nutrient. As an example, consider the effect of microwave processing on watersoluble vitamins, as carried out by Okmen and Bayindirli [12]. Vitamin solutions were heated at constant temperatures of 60, 70, 80 and 908C in the microwave oven. Aliquots (1 ml) were taken from the heat-treated samples after 20, 40, 60 and 80 min intervals and analyzed for vitamin concentration. The rst-order reaction rate constants at the four different temperatures were calculated from the slopes of the log (C/C0) versus t plots shown in Figure 1.1. Other reaction orders besides rst are possible. A zero-order reaction occurs if the loss of nutrient is so small in the time period studied that the value of C does not change signicantly. Therefore, the right-hand side of Equation (1.2) is a constant [13]. Ascorbic acid oxidation in liquid infant formula under limited presence of dissolved oxygen takes place by a second-order reaction [14].0 0.02 log (C/Co) 0.04 0.06 0.08 0.1 0 20 40 60 Time (min) 80

60 C 70 C 80 C 90 C

FIGURE 1.1 A typical graph of log (C/C0) vs. time for vitamin C. (From Okmen, Z.A. and Bayindirli, A.L., Int. J. Food Prop., 2 (3), 255, 1999. Copyright 1999. With permission.)

Vitamins in Foods: Analysis, Bioavailability, and Stability 1.5.3 Effects of Food Processing on Vitamin Retention

9

Commercial food processing preserves food quality and extends shelf life by destruction of food-spoilage microorganisms and certain endogenous enzymes, which could otherwise promote spoilage and/or reduce nutritive value. Since all processed foods have to be stored until they are consumed, proper food packaging is essential to maintain preservation. Time and temperature during processing and storage are closely controlled in good manufacturing practice. Distribution controls, however, are less stringent, except for very perishable products. The main factors contributing to vitamin losses are oxidation (air exposure), heat (temperature and time), catalytic effect of metals, pH, action of enzymes, moisture, irradiation (light or ionizing radiation), and various combinations of these factors. Some vitamins are sensitive to processing and storage, while others are more or less stable. The water-soluble vitamins are susceptible to leaching losses during commercial washing and blanching, and domestic cooking. Vitamin C is very susceptible to chemical oxidation during processing, storage, and cooking. Thiamin is heat-sensitive in neutral and alkaline foods, and is unstable in air. Riboavin is notoriously susceptible to decomposition by light. Niacin and vitamin B6 are stable under a variety of processing conditions. Vitamins A and E are destroyed under conditions that accelerate the oxidation of unsaturated fats, such as access of air, heat, light, trace metal ions, and storage time. Vitamin K is stable to heat, but extremely sensitive to both uorescent light and sunlight. Vitamin D is little affected by processing and storage. Some losses of certain vitamins during food processing are inevitable. However, one should consider the relative importance of the loss of a specic vitamin from a particular commodity. For example, vitamin C loss from milk during pasteurization and refrigerated storage is relatively unimportant, as milk is an insignicant source of this vitamin in the daily diet compared to other foods, such as citrus fruits and juices. Another point to consider is that natural variations in the vitamin content of a raw food material may affect the content of that vitamin in the nal product more than the processing itself. Retention studies of vitamins to assess the effects of food processing on the nutritive value of foods are of great importance to food technologists and consumers. In this section, the broad effects of various processing techniques on vitamin retention are discussed. The effects of processing on specic vitamins are discussed in the relevant chapters. 1.5.3.1 Dehydration

Removal of the biologically active water from foods through dehydration stops the growth of microorganisms, whilst also reducing the rate of

10

Nutritional Aspects of Vitamins

enzyme activity and chemical reactions. Rancidity of the lipid constituents of dehydrated foods is reduced if the protective structural water is left intact. Fruits, vegetables, juices, meats, sh, milk, and eggs are among the foodstuffs commonly subjected to dehydration processes. Appropriate drying techniques are used in the processing of dehydrated foods. The sun-drying of fruit, sh, meat, and grain is still of importance in certain parts of the world. In the tunnel-drying of fruits and vegetables, the produce is spread onto trays or a conveyer and passed into a high-velocity air stream in the temperature range of 60 938C. Spray-drying is a highly efcient process for drying milk, eggs, and coffee. In spray-drying, liquids are dispersed in ne droplets and sprayed into an upward-owing stream of hot air. Materials that can be made into a paste, such as mashed potatoes and tomato puree, can be dried by spreading thinly on steam-heated revolving drums. As the product is in direct contact with the hot drums, vitamin losses would be expected to be greater than those resulting from tunnel driers and spray driers. In commercial freeze-drying, the frozen food is placed into a chamber, which is evacuated and then heated. Because of the low pressure, ice does not melt, and the water vapor passes directly from a solid to a vapor phase (sublimation). The freeze-drying process is applied mainly to meats and results in the least change in the physical characteristics of the product. The result is a dry, porous product, though there will always be some small amount of residual water. Compression of freeze-dried foods gives improved stability in storage by reducing the surface area exposed to oxygen and moisture. Except for sun-drying, the actual process of dehydration does not cause major losses of vitamins. Retention of ascorbic acid is better in rapid drying at high temperatures than in slower drying at lower temperatures. Drying methods that expose the food to air result in losses of vitamin A, b-carotene, and vitamin C due to oxidation. Freezedrying, which is carried out in the absence of oxygen, does not cause loss of vitamin C. 1.5.3.2 Blanching

The blanching of vegetables and fruits entails subjecting the fresh produce to temperatures in the range of 75 958C for 1 10 min prior to canning, freezing, or dehydration. Blanching serves several purposes: as a cleaning process; to reduce the volume of bulky vegetables by wilting; to expel air from the plant tissues, thereby reducing the potential for oxidative changes; and to inactivate endogenous enzymes that would otherwise cause quality deterioration. Blanching with hot water is still the most common system, despite the signicant loss of water-soluble vitamins through leaching. This is because of the relatively low capital and

Vitamins in Foods: Analysis, Bioavailability, and Stability

11

running costs compared with more efcient procedures, such as steam or microwave blanching. The immediate scalding of plant tissues is desirable to minimize the oxidation of ascorbic acid by ascorbic acid oxidase. If the inactivating temperature of about 858C is not immediately reached, the breakdown of cell structure will allow contact between active enzyme and substrate. Thus inefcient blanching will incur some loss of vitamin C by oxidation, as well as by leaching [15]. Unlike thiamin, ascorbic acid is essentially stable to the heating conditions during blanching. When peas were blanched at 82 888C for 3 min, losses of thiamin, niacin, vitamin B6, and vitamin C were 4, 20, 18, and 17%, respectively [16]. 1.5.3.3 Canning The heat treatment of canned foods will ideally sterilize the food, that is, destroy all viable microorganisms present. Some microorganisms and their spores are extremely resistant to heat, and very stringent heat treatments would be re