Advances in Food and Nutrition Research Volume 49

ADVANCES IN FOOD AND NUTRITION RESEARCH VOLUME 49 ADVISORY BOARD KEN BUCKLE University of New South Wales, Australia...

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ADVANCES IN

FOOD AND NUTRITION RESEARCH VOLUME 49

ADVISORY BOARD KEN BUCKLE University of New South Wales, Australia

MARY ELLEN CAMIRE University of Maine, USA

BRUCE CHASSY University of Illinois, USA

PATRICK FOX University College Cork, Republic of Ireland

DENNIS GORDON North Dakota State University, USA

ROBERT HUTKINS University of Nebraska, USA

RONALD JACKSON Quebec, Canada

DARYL B. LUND University of Wisconsin, USA

CONNIE WEAVER Purdue University, USA

RONALD WROLSTAD Oregon State University, USA

HOWARD ZHANG Ohio State University, USA

SERIES EDITORS GEORGE F. STEWART EMIL M. MRAK

(1948–1982) (1948–1987)

C. O. CHICHESTER

(1959–1988)

BERNARD S. SCHWEIGERT (1984–1988) JOHN E. KINSELLA (1989–1993) STEVE L. TAYLOR

(1995–

)

ADVANCES IN

FOOD AND NUTRITION RESEARCH VOLUME 49

Edited by

STEVE L. TAYLOR Department of Food Science and Technology University of Nebraska Lincoln, Nebraska USA

Elsevier Academic Press 525 B Street, Suite 1900, San Diego, California 92101-4495, USA 84 Theobald’s Road, London WC1X 8RR, UK

This book is printed on acid-free paper. Copyright ß 2005, Elsevier Inc. All Rights Reserved.

No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the Publisher. The appearance of the code at the bottom of the first page of a chapter in this book indicates the Publisher’s consent that copies of the chapter may be made for personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center, Inc. (www.copyright.com), for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to other kinds of copying, such as copying for general distribution, for advertising or promotional purposes, for creating new collective works, or for resale. Copy fees for pre-2005 chapters are as shown on the title pages. If no fee code appears on the title page, the copy fee is the same as for current chapters. 1043-4526/2005 $35.00 Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone: (þ44) 1865 843830, fax: (þ44) 1865 853333, E-mail: [email protected]. You may also complete your request on-line via the Elsevier homepage (http://elsevier.com), by selecting ‘‘Customer Support’’ and then ‘‘Obtaining Permissions.’’ For all information on all Academic Press publications visit our Web site at www.books.elsevier.com ISBN: 0-12-016449-3 PRINTED IN THE UNITED STATES OF AMERICA 05 06 07 08 9 8 7 6 5 4 3 2

1

CONTENTS

Contributors to Volume 49. . . . . . . . . . . . . . . . . . . . . . .

ix

Reinvention of the Food Guide Pyramid to Promote Health Paul A. Lachance and Michele C. Fisher I. II. III. IV. V. VI. VII. VIII. IX. X. XI. XII. XIII. XIV. XV.

Introduction . . . . . . . . . . . . . . . . . . . . . . . History of the Food Guides . . . . . . . . . . . . . . . Why Reinvent the Pyramid? . . . . . . . . . . . . . . . Alternate Solutions Pursued by the Consumer . . . . . Physical Activity as a Variable. . . . . . . . . . . . . . Impact of Cultural Diversity . . . . . . . . . . . . . . . Reinventing the Pyramid . . . . . . . . . . . . . . . . . The Calories for Nutrient to Assess Caloric Density . . Foods Providing the Most Nutraceuticals are Functional Foods . . . . . . . . . . . . . . . . . . . . Phytochemicals and Color-Coded Eating Plans . . . . . Addressing the Energy Intake Issues. . . . . . . . . . . Revamping the Cereal Grain–Based Food Group. . . . Communion with Other Pyramids . . . . . . . . . . . . How Should the Consumer Approach the Pyramid for Food Choice Guidance? . . . . . . . . . . . . . . . . . Practical Approach of Food Guides . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . .

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2 2 8 10 11 11 14 20

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28 29 32 33 34

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35 36 36

vi

CONTENTS

Plant Pigments: Properties, Analysis, Degradation Benoıˆt Schoefs I. Introduction . . . . . . . . . . . . . . . . . . . II. Spectroscopic, Molecular Structures, and Chemical Properties . . . . . . . . . . . . . . . III. Chemical Modifications Occurring During Food Treatments and Storage . . . . . . . . . . . . . IV. Methods of Analysis: An Overview . . . . . . . V. Pigment Identification and Quantification: the Problem of Standards . . . . . . . . . . . . . . VI. Extraction and Analysis: Case by Case . . . . . VII. Future Trends . . . . . . . . . . . . . . . . . . Acknowledgment . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . .

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42

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45

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53 59

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68 72 80 80 80

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. . . . .

Chitin, Chitosan, and Co-Products: Chemistry, Production, Applications, and Health Effects Fereidoon Shahidi and Reem Abuzaytoun I. II. III. IV.

Introduction . . . . . . . . . . . Chemistry. . . . . . . . . . . . . Applications of Chitin, Chitosan, Safety and Regulatory Status . . References . . . . . . . . . . . .

. . . . . . . . . . and their . . . . . . . . . .

. . . . . . . . . . . . Oligomers . . . . . . . . . . . .

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93 95 114 128 128

A Review of the Application of Sourdough Technology to Wheat Breads Charmaine I. Clarke and Elke K. Arendt I. II. III. IV.

Introduction . . . . . . . . . . . . . . . . . . . . . . . Microflora of Sourdough . . . . . . . . . . . . . . . . Positive Effects of Sourdough on Wheat Bread Quality. Understanding the Technological Functionality of Sourdough Application . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . .

138 139 142

. . . . .

146

CONTENTS

vii

V. Effect of Sourdough Incorporation on Bread Dough Structure . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

151 155 156

Detection of Insect Infestation in Stored Foods Somiahnadar Rajendran I. II. III. IV. V.

Introduction . . . . . . . . . . Insect Pests of Stored Foods . . Detection of Insects in Samples Detection in Storage Facilities . Conclusion . . . . . . . . . . . Acknowledgments . . . . . . . References . . . . . . . . . . .

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163 166 173 201 215 216 216

Compression and Compaction Characteristics of Selected Food Powders Gustavo V. Barbosa-Cańovas and Pablo Juliano I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . II. Modeling Compression and Compaction of Food Powders. III. Microstructural Approach for Compression and Compaction . . . . . . . . . . . . . . . . . . . . . . . IV. Compression and Compaction in Food Processing . . . . . V. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . .

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233 265

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285 288 300 301

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

309

. . . .

CONTRIBUTORS TO VOLUME 49

Numbers in parentheses indicate the page on which the authors’ contributions begin.

Reem Abuzaytoun, Department of Biology, Memorial University of Newfoundland, St. John’s, Canada (93) Elke K. Arendt, Department of Food and Nutritional Sciences, National University of Ireland, Cork, Ireland (137) Gustavo V. Barbosa-Cańovas, Biological Systems Engineering, Washington State University, Pullman, Washington, USA (233) Charmaine I. Clarke, Department of Food and Nutritional Sciences and National Food Biotechnology Centre, National University of Ireland, Cork, Ireland (137) Michele C. Fisher, Food and Nutrition Enterprises, LLC, Mullica Hill, New Jersey, USA (1) Pablo Juliano, Biological Systems Engineering, Washington State University, Pullman, Washington, USA (233) Paul A. Lachance, Department of Food Science, Rutgers, the State University of New Jersey, New Brunswick, New Jersey, USA (1) Somiahnadar Rajendran, Food Protectants and Infestation Control Department, Central Food Technological Research Institute, Mysore, India (163) Benoıˆt Schoefs, Dynamique vacuolaire et Re´ponses aux Stress de l’Environnement, Plante-Microbe-Environnement, Universite´ de Bourgogne a` Dijon, Dijon Cedex, France (41) Fereidoon Shahidi, Departments of Biochemistry and Biology, Memorial University of Newfoundland, St. John’s, Canada (93)

REINVENTION OF THE FOOD GUIDE PYRAMID TO PROMOTE HEALTH PAUL A. LACHANCE* AND MICHELE C. FISHER{ *Department of Food Science Rutgers, the State University of New Jersey New Brunswick, New Jersey, USA { Food and Nutrition Enterprises, LLC Mullica Hill, New Jersey, USA

I. II. III. IV. V. VI. VII. VIII.

IX. X. XI. XII. XIII.

XIV. XV.

Introduction History of the Food Guides Why Reinvent the Pyramid? Alternate Solutions Pursued by the Consumer Physical Activity as a Variable Impact of Cultural Diversity Reinventing the Pyramid The Calories for Nutrient to Assess Caloric Density A. Independence of ‘‘Organic’’ and Non-GMO Claims B. Further Rationales for Reinventing the Pyramid C. Health Claims Support the Reinvention of the Pyramid Foods Providing the Most Nutraceuticals Are Functional Foods A. Pyramid Must Recognize Bioactive Nutraceuticals Phytochemicals and Color-Coded Eating Plans Addressing the Energy Intake Issues Revamping the Cereal Grain–Based Food Group Communion with Other Pyramids A. Oldways Pyramids B. Willett and Stampfer Healthy Eating Pyramid How Should the Consumer Approach the Pyramid for Food Choice Guidance? Practical Approach of Food Guides References

ADVANCES IN FOOD AND NUTRITION RESEARCH VOL 49 ISSN: 1043-4526

ß 2005 Elsevier Inc. All rights reserved

2

P. A. LACHANCE AND M. C. FISHER

I. INTRODUCTION Dietary guidelines and dietary food guides are tools used to educate the public about diet, nutrition, and health. The quality of an individual’s nutritional status is directly dependent on the quality of the input into nutrition, namely food, and applicable dietary supplements. Food is defined as any substance that is eaten to sustain psychological and physiological life, provide energy, and promote nutrition. It should be evident that dietary guidelines involve terms and goals pertaining to the intake of certain food components with the objective being to either strive to meet a certain set of dietary goals or strive to not exceed another set of goals (e.g., cholesterol intake). However, dietary food guides (e.g., pyramid) are food based but aimed at meeting and/or enhancing nutrition and therefore health. Consumers understand a dietary food guide because they eat food and not nutrients—nutraceuticals per se. Food is visible and nutrients are essentially invisible components of food. Food satisfies the palate and the psyche and provides satiation, whereas nutrients do not. For these reasons food education should be distinguished from nutrition education. It is a major flaw of the education scheme that food has not been differentiated from nutrition and health outcomes. The purpose of this chapter is to foster and make more consumer relevant the food-guide-pyramid approach to food selections and combinations aimed at enhancing the nutrition and health of the consumer.

II. HISTORY OF THE FOOD GUIDES Food guides are food and nutrition education tools that help consumers select and eat foods that provide them with adequate nutrition to maintain health. Health is defined as a continuing state of soundness and vigor of body and mind. Consumers select and purchase food. Although their ultimate goal is to obtain the nutrients from foods, many consumers do not know which nutrients, at what amounts, are found in foods so that they can ensure they meet their daily and overall nutritional needs. As a result they need a simple tool to help them choose the appropriate amounts and types of foods that will provide the nutrients needed for good health. More than 100 years ago, W. O. Atwater, the first director of the U.S. Department of Agriculture’s (USDA) Office of Experiment Stations, is believed to be the first person to use the scientific process to develop dietary guidance to improve health (Welsh et al., 1993). His food composition tables and dietary standards for the U.S. population were first published in 1894 (Atwater, 1894). He coordinated research on nutrient requirements, food composition, food consumption, and consumer economics that led to a

REINVENTION OF THE FOOD GUIDE PYRAMID

3

scientific connection between food composition, dietary intake, and health (USDA, 1993). Variety, proportionality, and moderation in healthful eating were promoted by Atwater in a Farmer’s Bulletin in 1902 (Welsh, 1994). In the bulletin, he states that ‘‘for the great majority of people in good health, the ordinary food materials . . . make a fitting diet, and the main question is how to use them in the kinds and proportions fitted to the actual needs of the body.’’ ‘‘Unless care is exercised in selecting food, a diet may result which is one-sided or badly balanced—that is, one in which either protein or fuel ingredients are provided in excess’’ (Atwater, 1902; Welsh et al., 1992). Atwater’s work formed the foundation for the development of an education tool, the food guide. In 1916, Hunt first grouped foods for purposes of ‘‘nutrition’’ education when she created the first USDA food guide (Ahlstro¨m and Ra¨sa¨nen, 1973). This USDA food guide had five food groups in which foods were placed based on their nutrient contents. The five groups included (1) vegetables and fruits, (2) milk, meat, eggs, fish, cheese, dry beans, peas, and peanuts, (3) cereals (breads and starches), (4) sugar, and (5) fat (Ahlstro¨m and Ra¨sa¨nen, 1973; Welsh, 1994) (Table I). This food guide also included the nutritional value of the foods consumed. Throughout the 1920s this food guide was very popular until the Great Depression created the need to consider economy in the planning of diets. Not only did consumers require guidance in planning nutritionally balanced diets, but they also needed help in doing so economically. As a result, food plans at four cost levels were developed by Stiebeling and Ward (1933). These plans were based on 12 major food groups that were to be used by consumers to assist them in making cost-effective weekly food purchases (Table I). The concept of nutrient density was used by these plans, which acknowledged that some foods could provide more nutrients at a lower cost. This concept of providing nutritious foods economically for a healthy diet remains a basic tenet of the USDA ‘‘nutrition’’ education program. The first Recommended Dietary Allowances (RDAs) were published in 1941 by the Food and Nutrition Board of the National Academy of Sciences (National Research Council, 1941). These were specific recommendations on intake levels for calories and nine essential nutrients that included protein, the minerals calcium and iron, and the vitamins A, D, thiamin, riboflavin, niacin, and ascorbic acid. Food rationing during World War II, along with these new RDAs, led to the development of the USDA’s National Wartime Nutrition Guide (Ahlstro¨m and Ra¨sa¨nen, 1973). This food guide was first published in 1943 and was known as the ‘‘Basic Seven.’’ It included the following food groups: (1) green and yellow vegetables, (2) oranges, tomatoes, and grapefruit, (3) potatoes and other vegetables and fruits, (4) milk and milk products, (5) meat, poultry, fish, eggs, and dried peas and beans,

4

TABLE I PRINCIPAL U.S. DEPARTMENT OF AGRICULTURE FOOD GUIDES FROM 1916 TO 1992a

Food guide Huntb (1916)

No. of groups Milk 5

Meats and other protein-rich foods

1 c milk þ 2 3 servings other

# of Servings Stiebelingc (1930s)

Meat

Breads

Vegetables and Fruits

Fats

Sugars

Cereals and other starchy foods

Vegetables and fruit

Fatty foods

Sugars

9

10

9

5

Milk, lean meat, poultry, fish

Dry beans, Eggs peas, and nuts

Flours, cereals

Leafy green yellow

Potatoes sweet potatoes

Other vegetables and fruits

Tomatoes Butter Other Sugars and citrus fats

# of Servings

2 c 9–10/wk

1/wk

As desired

11–12/wk

1

3

1

7 Basic Sevend (1940s) Foundation diet

Milk and milk products

Bread, flour, and cereal

Leafy green Potatoes, Citrus, tomato, yellow other fruit cabbage and vegetables salad greens

Butter; fortified margarine

# of Servings

2 c or more

Every day

1 or more

Some daily

12

1

Meat, poultry, fish eggs, peas, nuts, dried beans 1–2

2 or more

1 or more

—

—

—


Other

Protein-rich foods

Basic Foure (1956–1970s)

4

Meat group

Bread, cereal

Vegetable–fruit group

Foundation diet

2 c or more

2 or more

4 or more

5 Hassle-Freef (1979) Foundation diet

Milk–cheese group

4 or more (use dark green/yellow vegetables frequently, citrus daily) Vegetable–fruit group

2

Food Guide Pyramidg (1984þ) Total diet

a

6

Bread–cereal Meat, poultry, group fish and beans group 2 4

4 (include vitamin C source daily, dark green/yellow vegetable frequently)

Milk, yogurt, cheese

Meat, poultry, eggs, fish, dry beans, nuts

Breads, cereals, rice, pasta

Vegetable

2–3

2–3

6–11 3–5 whole grain dark green/deep yellow enriched starchy/legumes, other

Fats, sweets, alcohol group

(use depends on calorie needs)

Fruit

Fats, oils, sweets

2–4 citrus, other

Total fat 30% Sweets vary according to calories

From Welsh (1993). ‘‘Food for Young Children’’ (1916), ‘‘How to Select Foods’’ (1917), ‘‘A Week’s Food for an Average Family’’ (1921), ‘‘Good Proportions in the Diet’’ (1923). c ‘‘Planning for Good Nutrition’’ (1939) (published two previous food plans, 1933/1936). d ‘‘National Wartime Nutrition Guide’’ (1943), ‘‘National Food Guide’’ (1946). e ‘‘Essentials of An Adequate Diet’’ (1956), ‘‘Food for Fitness – A Daily Food Guide’’ (1958). f Food: ‘‘The Hassle-Free Guide to a Better Diet’’ (1979). g ‘‘Developing the Food Guidance Systems for ‘Better Eating for Better Health’’’ (1985). b


Milk group

5

6


(6) bread, flour, and cereals, and (7) butter and fortified margarine (Welsh et al., 1993). Because certain foods were in limited supply during the war, this guide did not list a specific number of servings for each group but emphasized the selection of alternatives from other groups to meet nutritional needs. For example, if foods from group 2 were unavailable, then it was recommended that more foods from groups 1 and 3 be consumed to ensure nutritional adequacy. In 1946, after the war, this guide was revised to include the recommended number of servings per day for each food group. This format was then used for the next 10 years as a ‘‘nutrition’’ education tool (Ahlstro¨m and Ra¨sa¨nen, 1973). Problems associated with the Basic Seven were its complexity and lack of serving size information, so in 1956 a new food guide that streamlined the seven food groups to only four was issued by the USDA. It became known as the ‘‘Basic Four,’’ with only (1) milk, (2) meat, (3) vegetables and fruits, and (4) bread and cereals being the four food groups. For this new food guide the fat group was eliminated and the three vegetable and fruit groups were combined into one group (Ahlstro¨m and Ra¨sa¨nen, 1973). The minimum number of servings for each group was listed, as well as more accurate information on the appropriate serving sizes. The Basic Four was used to create a foundation diet that emphasized foods that provided protein, iron, calcium, vitamins A and C, thiamin, riboflavin, and niacin but provided little guidance on calories, fats, or sugars. It was assumed that individuals would meet their caloric and other nutrient needs by consuming more than the recommended amounts in the guide (Welsh et al., 1993). This emphasis on getting enough nutrients remained the focus of nutrition education for the next 20 years (Welsh et al., 1993). In this schematic the four food groups were presented as equal quadrants. Lachance and Fisher (1986) urged that the proportions recommended be presented akin to a ‘‘peace’’ symbol to emphasize the need for at least two-thirds of the food on the plate to be of plant origin, with the remaining being of animal and dairy origin. (Interestingly, the American Institute for Cancer Research introduced the concept as the ‘‘New American Plate’’ in April 2000.) The Basic Four food guide was modified in 1979 and became the ‘‘HassleFree Guide,’’ which added a fifth group, which included fats, sweets, and alcohol, to the Basic Four. Like the Basic Four, it recommended a foundation diet with daily servings from the milk, meat, fruit and vegetable, and grain groups, but it also separated low nutrient density foods from the other groups. The Hassle-Free Guide promoted the moderate consumption of fats, sugars, and alcohol and highlighted calories and dietary fiber (Welsh et al., 1993). The Dietary Guidelines for Americans have been published every 5 years since 1980 by the USDA and the Department of Health and Human Services


7

FIG. 1 U.S. Department of Agriculture Food Guide Pyramid. As revised in 1992. ‘‘Eat from the bottom up.’’

(HHS). The purpose of the Dietary Guidelines is to provide Americans 2 years and older with information on diet choices that will promote health and prevent disease (USDA, 2000). The Food Guide Pyramid is a graphic illustration of the Dietary Guidelines and was first issued in 1992 (Figure 1) (USDA, 1992). The current Food Guide Pyramid organizes foods according to a category (cereal, fruit, dairy, etc.) and nutrient content. For example, in the USDA Food Guide Pyramid, all fruits are grouped together and it is recommended that one select two to four servings from this group each day. Consumers are expected to select a recommended number of servings from each group to plan their diets. Internationally, food guides come in three major shapes: circular plate, oriental pagoda, and pyramid (Painter et al., 2002). The pyramid approach to food group guide combinations is used primarily in the United States, Puerto Rico, and the Philippines. In North America, Canada uses a rainbow concept, whereas Mexico and Central America use the most common and worldwide approach of a circular plate of food groupings. Whereas the emphasis of this chapter is to reconsider and reinvent the USDA Food Guide Pyramid, the recommendations for change must be science based. Yet the changes must not lose sight of the intended audience, which is John Q. Public, with essentially no education in science. Further, the recommendations made for the pyramid should be applicable as well to other food guide presentation approaches (circular, rainbow, etc.); however, this

8


chapter focuses on the pyramid approach as a food guide because it serves well as a model system.

III. WHY REINVENT THE PYRAMID? Why reinvent the Food Guide Pyramid? A major drawback of the various groupings of the pyramid is that they do not distinguish caloric differences or nutrient densities of the various foods within each grouping. Given the epidemic increase in obesity in America, one of the greatest challenges facing the Food Guide Pyramid is a plan for realizing nutritionally balanced diets that are low in energy. The USDA Center for Nutrition Policy and Promotion rightfully sees a need for the revision of the Food Guide Pyramid. The proposal of the USDA recognizes that the estimated energy requirements (EERs) of the dietary reference intakes (DRIs) to maintain weight is based on gender, age, height, weight, and activity level. The EER for men and women of reference body size decreases with age in years, but it increases for children up to the age of maturity. However, what plagues the United States is overweight and obesity in both children and adults. Some of the consumer issues of the Food Guide Pyramid elicited by the USDA pertain to perceptions of the difference between servings and portions, as well as serving size as related to the number of servings recommended. The fact is that few consumers use the current Food Guide Pyramid consciously, finding it to have too many details to follow. Another drawback to the current Food Guide Pyramid is that the actual practices of Americans as to choices made relative to the recommendations of the pyramid are poor. Most Americans do not ingest and thus meet the recommended food proportions of the Food Guide Pyramid. Americans consume too many servings of foods with added fats and sugars and do not eat enough fruits, vegetables, dairy products, lean meats, and foods made from whole grains (Kantor, 1998, 1999). Based on two estimations (Anonymous, 1994; USDA, 1998) of data on actual consumer practices visa`-vis the recommendations of the Food Guide Pyramid (Figure 2), one can graphically portray and contrast the food choices adult consumers have made. The ‘‘reality’’ pyramid reveals that the foods preferred and eaten are of the type best fitting the peak of the pyramid. For products with added sugars and discretionary fats, the estimated maximum that should be incorporated into the group at the peak is 27% of total food energy. However, ‘‘in reality’’ the average adult American chooses as much as 41% of their calories from this one food group (Anonymous, Consumer Reports on Health, 2000). The consequence is that their total food energy is substantially


9

FIG. 2 Actual consumption pyramid (based on 1996 CSFII data). The percentage of total energy consumption from discretionary fats, added sugars, and related food products is now 41% in contrast to the goal of ‘‘not to exceed 27%’’ (Anonymous, 2000).

derived from selecting poor nutrient dense food. It, thus, creates the question about whether the current pyramid as a food guide should be modified to increase the awareness of its intended capability to effectively lead to better food choice practices. The ‘‘reality’’ of this finding cries out for a missing indicator of nutrient density being made available to the consumer. Consumers have no other reference point about which foods, in what proportion, can sustain and enhance health. The Nutrition Facts panel on packaged foods of the Nutritional Labeling and Education Act of 1990 (NLEA) (Food and Drug Administration [FDA], 2003) serves the purpose of describing the calories per serving. It also provides information about what to avoid in terms of fat, cholesterol, and sodium. There has not been a marriage of the provisions of the NLEA, the Dietary Guidelines, and the Food Guide Pyramid. The E in NLEA has not really occurred. The NLEA is ‘‘regulated’’ by the FDA, which has sufficient regulatory crises to ever get around to be involved in education, but to its credit it has tried on at least two occasions, only to have to set education plans aside as a major regulatory based crisis erupted. The Dietary Guidelines are a joint HHS/USDA undertaking and the Food Guide Pyramid is a USDA education effort.

10


Meanwhile, and more importantly, the scientific evidence supporting health claims and functional foods promotes the concept that benefits can be derived from the proper choices of foods. This concept has reached a point at which one cannot deny the need to reappraise the pyramid and, thus, the health benefits that should accrue if one elects to follow a food guide and maximize the health benefits of the resultant food and meal combinations. For reasons that are not clear, consumers believe they are following a relative balance of the pyramid food groupings. They are confused because they observe that overweight and obesity are on the rise and they have been eating less visible fat, but they do not realize that in avoiding only fat (i.e., consuming a lower percentage of fat as a source of energy) (Chanmugam et al., 2003), they have increased their energy intake overall by up to 12–14% during the last 2 decades. It does not take long at that rate of excess over the need to become overweight and later obese, and for the phenomena to begin at younger and younger ages (Zizza et al., 2001).

IV. ALTERNATE SOLUTIONS PURSUED BY THE CONSUMER The consumer is seeking solutions to their dilemma of body weight control via a number of dieting solutions, in particular shifting the macronutrient (protein/carbohydrate) composition of their daily diet. Books describing weight loss plans, based on the proportions of dietary protein to carbohydrate, are so popular that several have been or are on the bestseller lists and have been translated into several languages. These favorite weight loss/ control plans advocate high-protein and low-carbohydrate dietary intake practices. Interestingly, the authors of two current popular weight loss books, Dr. Atkins’ New Diet Revolution (Atkins, 2002) and the South Beach Diet (Agatston, 2003) are physicians who are debated by other physicians and we have a contest that does not benefit the consumer. The client for these books and their plans are predominantly of the Baby Boomer and X generations. Otherwise, organized weight loss and physical activity businesses abound. It is important to note that children and seniors remain in limbo. The Atkins’ diet leaves the vegan in limbo, whereas the South Beach diet provides sought after vegetarian recipes. Since 1975 or so, adult Americans have consumed an additional 12–14 % more calories daily, which alone could help explain the epidemic rise in obesity. Over a span of about one generation, the increase in obesity has become a readily apparent association with increasing expenditures for eating away from home or purchasing food to take home (Lachance, 2000). The consumer concern for health becomes subservient to the need


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for convenience and rapid preparation and also influenced by the perception of the value in the amount of food for the cost.

V. PHYSICAL ACTIVITY AS A VARIABLE The issue of the many facets of physical activity is not in the purview of this chapter. In terms of everyday physical aerobic activity, many factors other than food intake are overlooked. Security concerns bar reentry from stairwells of multistory buildings. Elevators are routinely used, even by college students, to escape a single flight of stairs. The computer age ties the individual to the screen, be it a game toy, e-mail, or serious research work. Living in air-conditioned environments lowers energy demand. What is eaten during say ‘‘Monday night football’’ is no help in a milieu of boisterous but limited opportunity for exercise. Of concern is the emulation by children. There has been a significant increase in the snacking of young adults (Zizza et al., 2001). Other lifestyle practices also are variables. The solution to obesity is multifaceted (Lachance, 1994), but expecting the Food Guide Pyramid to deal with energy output factors and the obvious energy inputs is naive. More effective would be providing the consumer with an indicator of the nutrient density of packaged and fast foods and thus help the consumer make more uncomplicated but sophisticated choices. We are proposing that an indicator of calorie density (cost) per 1% averaged daily value of 13 nutrients would help consumers realize when a food product is excessive in calories and/or unclassified as to location in the pyramid. The concept—calories for nutrient (CFN)—is presented later in this text and its application illustrated with example figures.

VI. IMPACT OF CULTURAL DIVERSITY The pyramid concept has been easily adapted to various cultures such as Arabic, Chinese, Cuban, Indian, Mexican, Native American, Russian, and Thai to name a few (USDA, 2003). The eating habits of Americans are diverse, which reflects the multicultural origins of Americans. Several key dietary pyramid patterns reflect the diversity of the U.S. consumer (Figures 3 through 6): vegetarian, Mediterranean, Latin American, and Asian. Unfortunately only 25% of adult Americans in 1996 (USDA CSFII) consumed the number of servings per day recommended by the USDA Food Guide Pyramid and the Dietary Guidelines on which the ‘‘official’’ pyramid (Figure 1) evolved (Katz, 1998). (The Food Guide Pyramid has not been revised since 1992 but is scheduled for a revision by 2005 or sooner.)

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FIG. 3 Vegetarian diet pyramid (Oldways, 1994). Most striking is the emphasis on whole grain and complete lack of cereal grain products that do not contain whole grain flour, such as pastas and breads.

FIG. 4 Mediterranean diet pyramid (Oldways, 1994). No specific mention is made of whole grain products. Legumes and nuts are given equal status with fruits and vegetables. Olive oil is specifically designated.


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FIG. 5 Latin American diet pyramid (Oldways, 1994). Equal status is given to grains, tubers, legumes, and nuts, along with fruits and vegetables.

FIG. 6 Asian diet pyramid (Oldways, 1994). Legumes, nuts, and seeds are given equal status with fruits and vegetables; included is tea as a beverage. Optional are alcoholic beverages on a daily basis.

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One of the major differences among the various pyramids regards the placement of legumes, nuts, and seeds. It is a more important category than simply their contribution to the protein content of the diet without a specific recommendation as to servings per day. Note that when legumes, nuts, and seeds of this food grouping are dissected out and placed into the foundation tier of the pyramid, one to two daily servings becomes specifically recommended. The USDA pyramid recommends six to nine servings per day of vegetables and fruits; however, only 24% of Americans consume five or more servings of fruits daily and only 50% consume three to five servings of vegetables per day (Katz, 1996). Federal agencies complicate the issues when they promote dietary interventions that emphasize deviating from the recommendations of the various food groupings. An example is the five-a-day program of the National Cancer Institute (NCI), which promotes the daily consumption of at least five servings per day of fruits and vegetables (NCI, 2003). It is an admirable goal but self-evidently not the official recommendation. Even with the five-a-day serving promotion, an increase in the daily eating of vegetables and fruits has been exceedingly poor ( 25). Requiring a CFN calculation to be displayed on the label of each product would aid consumers in ascertaining the caloric cost consequences of each food. See Table III for the CFN of some representative foods (for the nutrients in question). Food energy sources are no longer scarce in the diets of most Americans because if they were scarce, obesity would not be on the rise. Figures 14, 15, and 16 provide illustrations of the CFN concept in a given food as altered by the choice of the processed version chosen, for example of apples and boiled potatoes as compared to apple juice and french fries, or the choice of product because of changes in one ingredient (e.g., fat content of fluid milk). The CFN can be used to make choices between foods within a category or different types of foods or beverages or combinations of food such as a bagel with cream cheese (Figure 17). Representative CFNs for each food group of the reinvented pyramid are given in Figure 18. The CFNs rise accordingly as food groups move to the top tier. We suggest that the consumer will look for these low CFNs and understand that one serving per day of legumes, nuts, and seeds with a CFN of 20 in the foundation tier is diluted by the much lower CFNs of vegetables and fruits. A question can be how to differentiate between products that are truly whole grain (51% or greater whole-grain flour) and traditional refined cereal

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FIG. 14 Effect of milk choice on caloric density (calories for nutrient [CFN]). Higher fat products have a higher caloric density.

FIG. 15 Effect of apple choice on caloric density (calories for nutrient [CFN]). The CFN of apple products reflects loss in nutrients via processing.

FIG. 16 Effect of potato choice on caloric density (calories for nutrient [CFN]). The CFN of potato products reflects added fat and some loss of nutrients via preparation.


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FIG. 17 Effect of spread choice on caloric density (calories for nutrient [CFN]) for bagel. The CFN of adding one ounce of cream cheese or one tablespoon of butter.

FIG. 18 Average caloric density (calories for nutrient [CFN]) for each food group of the pyramid. The CFN for each major food group of the pyramid reflects the high content of nutrients, in particular for the vegetable and fruit groups. Foods with high caloric value but poor nutrient content need to be used sparingly.

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grain products such as white bread, cornmeal, and rice, in comparison with those products containing some fraction of cereal grain ingredients but that are ‘‘fun’’ foods more in line with desserts. These ‘‘tasty’’ cereal grain products (croissants, donuts, danishes and pastries, cookies and cakes, etc.), which have a cost of 50 calories or more per average 1% of the daily RDA must be booted up to the fun/dessert category at the top of the pyramid. This means that the cereal grain tier has the whole-grain and the basic cereal food products such as enriched bread, but that those foods that are not 51% whole grain and have a high CFN content because of added sugars, fat, or both (such as apple pie) also automatically qualify for the peak tier of the pyramid. Beverages such as water, milk, and tea or an occasional glass of wine may be sources of caloric energy but with other health benefits because of their role as functional foods. Carbonated beverages and drinks with low juice content (e.g., token 10% or less) are recreational and belong in the peak of the pyramid. A. INDEPENDENCE OF ‘‘ORGANIC’’ AND NON-GMO CLAIMS

As consumer acceptance of the ‘‘organic food’’ increases, it is repeating the phenomena of shifting business away from Mom-and-Pop health food stores to specialty corporate market chains (e.g., Wild Oats Natural Marketplace). Classic supermarkets also are purveyors of the standardized (meeting criteria established by the USDA) organic products including an increasing array of combined ‘‘organic’’ and non–genetically modified organism (non-GMO) fresh and snack foods. In terms of the Food Guide Pyramid, no changes are needed because these factors are independent variables offered by the purveyors and the purchase decision chosen by the consumer. B. FURTHER RATIONALES FOR REINVENTING THE PYRAMID

Equally important justifications for the modification of the foundation of the Food Guide Pyramid are the data of the consistent coherent tradition of emerging scientific activity pertaining to (1) the scientific agreement that underlies the approval of positive health claims and (2) nutraceutical content. Our attempt to justify the various food groups by the ‘‘glycemic index’’ and ‘‘dietary fiber’’ content of a set of representative foods fails because the data are too limited. In general, the hypothesis was that foods in the ‘‘funfood’’ peak would be low in fiber and higher in glycemic index, whereas the foundation foods of vegetables, legumes, nuts, seeds, and fruits would be sources of dietary fiber and have lower glycemic indices. The available


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glycemic index data (Monro, 2003; Foster-Powell, 2002) are too limited to use (see Table III for evidence of gaps). What does emerge is the need to reemphasize the importance of plant foods and the concurrent density of bioactive phytochemicals (nutraceuticals), which are in addition to the density of nutrients in vegetables, legumes, nuts, seeds, and fruits, as well as whole grains in the food choices that are based on the reinvented pyramid. The findings of the scientific literature (Albert et al., 2002; Cohen et al., 2000; Finley, 2003; Hebert et al., 1998; Jian et al., 1999; Kris-Etherton et al., 1999; Michaud et al., 1999; Tsai et al., 2004; Verhoven et al., 1996; Zhang et al., 1992) relative to realizing health benefits transcend the ‘‘nutrient–nutritive value’’ rationale and food group classification of the USDA pyramid. C. HEALTH CLAIMS SUPPORT THE REINVENTION OF THE PYRAMID

As mandated by the 1990 NLEA, health claims in food labeling can be petitioned from the FDA. The intent is to educate the public about recognized diet–disease interrelationships. Several relationships with significant scientific agreement have been identified and several have been issued. Many of the official health claims have a direct association with one or more of the plant food groups (see Table II). There are now 15 or more health claims, which are based on ‘‘significant scientific agreement.’’ In the interim, an FDA proposal was issued permitting the placement of a health claim on the label but identifying one of four categories (A, B, C, or D) of scientific rigor substantiating the health claim. Purveyors do not desire to have a ‘‘qualified’’ product claim in a ‘‘B, C, D’’ format to be misconstrued by the consumer as an overall product rating and the proposal is unlikely to survive. The following discussion is offered as evidence of the need (based on the scientific agreement of the health claim) to relocate the Food Guide Pyramid base, or foundation tier, from cereal grains to vegetables, legumes, seeds, nuts, and fruits (citrus and berries). Some health claims point to particular body systems (e.g., bone) or biomarkers (e.g., blood cholesterol level); however, the irrefutable observation can be made that the greater the number of different health claims that are positively associated to a particular food grouping, the stronger the emphasis should be to advocate choosing and consuming foods from particular food grouping(s) (see Table II). Thus, one could argue that the emphasis on the health-claim benefits of fruits and vegetables in ‘‘reducing the risk of cancer’’ or the role of leafy vegetables as a source of food folate and thus neural tube defect prevention and thus the

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role of soluble fiber in disease prevention gives priority to the foundational position of vegetables, legumes, seeds, nuts, and fruits great merit. In contrast, a health claim for the ‘‘fun foods,’’ as well as sugars and oils at the peak of the pyramid, has not been forthcoming and the likelihood that a heath claim would emerge for foods within this category is practically nil. Of the 12 functions of food, 9 are psychosocial. It makes psychological, social, and hedonistic commonsense that foods with poor nutrient density should reside in the peak of the pyramid and that most foods chosen as a finishing touch to a meal invariably have a high caloric density. Of intermediate recognition in the third tier are dairy products as a major source of calcium-rich and vitamin D–enriched foods and ‘‘the reduced risk of osteoporosis.’’ The meat group, also on the third tier, offers high-quality sources of protein and other nutrients. However, note that the health-claim benefits of soy and soy products are properly placed as ‘‘legumes’’ in the foundation tier of the reinvented pyramid.

IX. FOODS PROVIDING THE MOST NUTRACEUTICALS ARE FUNCTIONAL FOODS Fruits and vegetables per se are recognized as being associated with a reduced risk of cardiovascular disease (CVD) and cancer. In fact, practically all the ‘‘approved’’ health claims can be associated with the base of the reinvented pyramid and the number of qualifying food diminishes as one moves up the pyramid. For example, vegetables, legumes, nuts, seeds, and fruits are all sources of dietary fiber and thus ‘‘a reduced risk of cancer and a reduced risk of coronary heart disease’’ (FDA, 1998; Rolls et al., 2004). In fact, if one searches for the food combinations with the most benefit in thwarting both cancer and CVD, then it becomes evident that the composition of the vegetables, legumes, seeds, nuts, and fruits delivers certain health attributes (Dragsted et al., 2004). These include high potassium, low sodium (unless added), an array of antioxidants including the vitamin E of tree nuts, a naturally occurring selection of monounsaturated and polyunsaturated lipids, and a number of vitamins, such as folates, and minerals such as magnesium, manganese, zinc, and others. The array of nutrient-dense foods including legume (especially soy and peanut) proteins coupled with whole-grain products makes protein complementation fully satisfactory for the vegan. The synergy in the function of these nutrients with the bioactive properties of phytochemicals such as the non-vitamin precursor carotenoids and the flavonoids, to name a few, confirms the powerhouse of the reinvented pyramid by making the base of the pyramid a source and density of health-promoting factors rather than a source and density of macronutrient calories.


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A. PYRAMID MUST RECOGNIZE BIOACTIVE NUTRACEUTICALS

The results of emerging science substantially support a strong association between certain foods and their phytochemicals or biomarkers and the thwarting of actual chronic disease. An estimated 40% of all human cancers are believed to be related to diet (World Cancer Research Fund, 1997). Finley (2003) proposed that the ‘‘antioxidant responsive element’’ may explain the protective effects of cruciferous vegetables on cancer and observes that ‘‘the relationship of diet to cancer is not necessarily because of the inclusion of carcinogens in our diet, but may be a consequence of the exclusion of anti-carcinogens from our diets.’’ One can make the analogous arguments relative to the exclusion of dietary hypocholesterolemic compounds and the risk of CVD. This is a new and vital rationale for the role of diet in chronic disease prevention. For the consumer, this premise moves away from foods ‘‘to avoid’’ to a positive message of which foods ‘‘to emphasize.’’ Therefore, it is imperative to make appropriate changes in the pyramid array and therefore evolve an improved guidance as to (1) what foods and food groups (categories) to emphasize on a daily/frequency basis and (2) to reeducate the public about the major rationales in the pyramid and the consequential health benefits. Whereas all foods are invariably sources of energy, not all foods are substantial sources of nutrients and complementary bioactive nutraceutical compounds that thwart the pathogenesis of chronic diseases. The phytochemicals that have been found to be beneficial can be categorized into several chemical composition groupings (Table IV) (Guhr and Lachance, 1997). These are fiber; antioxidants; allylic sulfides; isothiocyanates; indoles; terpenes; flavonoids; phytoestrogens; and saponins. The flavonoids have many subgroupings such as catechins (e.g., in tea) and proanthocyanidins (e.g., in purple grapes). If one desires to arrange these phytochemicals by food sources, then the categories are simpler and recognizable. These are five: dark green and yellow vegetables, legumes (including soy bean products and peanuts) and tree nuts, citrus and berries, the cruciferous vegetables such as broccoli, cabbage, cauliflower, kale, turnip etc., and the sulfur-rich vegetables garlic, onion, leek, and chive. Both black and green tea may be the beverage of choice.

X. PHYTOCHEMICALS AND COLOR-CODED EATING PLANS Two sets of writers (one on the East Coast and one on the West Coast) have published books about healthy eating plans based on favoring certain combinations of food plants based on their natural colors (Heber and

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TABLE IV A LIST OF SOME PHYTOCHEMICAL GROUPS, THEIR SOURCES AND ORGANOLEPTIC/AESTHETIC OR NUTRITIVE PROPERTIES

Phytochemical group 1.

Phenols

2. Indoles

Some phytochemicals in the phytochemical group Simple phenols, phenolic acids, hydroxy cinnamic acid derivatives, flavonoids Indole-3-carbinol, indole-3acetonitrile, ltryptophan

3. Isothiocyanates Phenethyl isothiocyanate, benzyl isothiocyanate, sulforaphane

4. Allylic sulfur compounds

5. Monoterpenes 6. Monoterpene like 7. Carotenoids

8. Antioxidant vitamins 9. Antioxidant mineral

Diallyl sulfide, diallyl disulfide, S-allyl cysteine, allyl propyl disulfide, ajoene d-limonene, d-carvone Perryl alcohol a-carotene, bcarotene, acryptoxanthin, b-cryptoxanthin, lutein, lycopene, zeaxanthin Vitamin C, vitamin E Selenium

Sources Almost all fresh fruits and vegetables, cereal grains, tea (black and green), nuts Cruciferous vegetables (including brussel sprouts, kale, cabbage, broccoli, cauliflower, spinach, watercress, turnip, radish) Cruciferous vegetables (including brussel sprouts, kale, cabbage, broccoli, cauliflower, spinach, watercress, turnip, radish) Allium vegetables (including garlic, onion, leek, shallot, chive, scallion) Citrus oils, vegetable oils, spice oils Cherries Most red to yellow fruits and vegetables

Organoleptic/aesthetic or nutritive properties Flavor, color, or aroma

Pungent flavor

Pungent flavor

Flavor

Flavor, aroma Flavor Color

Fruits and vegetables, Nutrient whole cereal grains Garlic, whole cereal Nutrient grains


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Bowerman, 2002; Joseph et al., 2002). Both diets provide an eating plan of several days that illustrates how best to combine foods of different color categories, thus reflecting the different phytochemicals that impart both color and medicinal properties within color category. Starting with the premise that a number of foods have been used for their medicinal properties, the authors organize into four (Joseph et al., 2002) or seven (Heber and Bowerman, 2002) major color-coded categories of fruits and vegetables and then demonstrate the proper assemblage into model recipes. The Heber and Bowerman colors are as follows: red, in which one finds strawberries and raspberries and tomatoes and red bell peppers; orange-yellow, in which one finds oranges, mangoes, grapefruit, and the vegetables carrots, sweet potatoes, and winter squash; the green fruit such as kiwi and avocado; the green vegetables such as kale, broccoli, and spinach; and the blue-purple fruit such as blueberries, concord grapes, and dried plums and the vegetables purple cabbage and eggplant. The color system for planning diets does permit a recipe or meal-planning method to indirectly access most phytochemical protective compounds. One cannot expect the consumer to be aware of the chemical classification of phytochemicals wherein color is not a predominant or concurrent attribute. The limiting goal has to be convincing the consumer of the importance of eating a variety of fruits and vegetables, seeds, nuts, or legumes each day. In other words, the foundation foods are vegetables, legumes, nuts, seeds, and fruits, colored or not. The consumer further needs to realize that if a fruit or vegetable is not part of the breakfast habit, the probability of obtaining five to seven servings of fruits or vegetables in a given day is practically nil. Needless to say, if the consumer does not have a breakfast habit, other changes in diet choices will be needed. Innumerable scientific studies (Bidlack et al., 1998, 2000; Ho et al., 1992, 1993; Lachance, 1997) have been published on various aspects of these nutraceutical phytochemicals. These studies range from epidemiological studies, clinical trials, and basic in vitro and in vivo cell line and animal and related mechanistic investigations. The end points of these trials have been aimed at thwarting the pathogenesis of major chronic diseases such as CVD, cancer, and diabetes. The most coherent, consistent beneficial results have been those in which the conditions were preventative or ameliorated by distinct foods or the combinations of whole foods. It is again clear, therefore, that the base of the pyramid must be allocated to the promotion of the daily intake of vegetables, legumes, seeds, nuts, and fruits to emphasize the importance of these foods for their health benefits.

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XI. ADDRESSING THE ENERGY INTAKE ISSUES Can one expect the Food Guide Pyramid to assist in helping consumers with their estimated energy requirements (EERs)? Without separate guidance on the cost in calories needed to meet the RDAs and access to the health benefits of the nutraceuticals of functional foods, the Food Guide Pyramid has limitations. The irony is that nutritionists conducting nutrient assessments know that the most highly variable diet analysis data are that of energy intake, yet it is the only ‘‘nutrient’’ that consumers can measure for themselves as often as they wish by stepping onto a scale, preferably in the nude and facing a full-length mirror. The key question the consumer needs to answer is ‘‘did this amount of calories cause a change, up or down, in body weight?’’ Without a change in choices made, serving size, and activity levels, the chances of a change in body weight occurring are low. Poorly recognized is the role of ‘‘where’’ the choice of foods is made and the impact of serving size. There is increasing evidence that consumers making purchases away from home (not where they are eaten) ‘‘disinhibits’’ their health concerns and practices in favor of the economic or emotional value of the food eaten. The correlation of rate of increase in obesity with the rate of spending for food purchased away from home is startling (Lachance, 2000). This observation brings in question the limitation of the CFN concept if it were to appear only on packaged foods. A solution would be the requirement that the first-level packaging of each fast-food item should be required to include the imprinting of total calories for the particular serving size purchased and the CFN. On cups and beverage containers, three sets of the CFN could be easily imprinted in advance for carbonated beverage, milkshake, and Slurpee drinks. The proposed reinvented pyramid is a simpler presentation for addressing the energy intake concerns faced by consumers. It brings up the issue of whether the presentation of food groups should return to the plate configuration rather than the pyramid. In initial studies the USDA claims that the pyramid was favored over the plate; however, the official plate displayed equal proportions of four major food groups. Partitioning the plate into a ‘‘peace ‘‘ symbol presentation with emphasis on two-thirds plant foods to one-third animal foods is okay, but ‘‘hungry’’ portions can still defeat the control of calories. At a steakhouse, the proportions would obviously be reversed. The pyramid does add a segment at the peak for discretionary fats and added sugars. We again propose that any food with a CFN higher than 50, such as donuts and various desserts or snacks, should be parked in the peak tier of the pyramid. National survey data (National Health and Nutrition Examination Survey) has revealed that this ‘‘peak’’ segment is toppling


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with the provisioning of about 40% of the energy intake because so many food choices with a CFN of 50 or greater make up this upper tier of the pyramid. The reinvented Food Guide Pyramid as proposed makes the low CFN foods the primary (e.g., foundation of the pyramid) source of nutrients and nutraceuticals at low energy cost and, depending on the choice of preparation selected (steamed vs deep fried), highly likely to be effective in the control of body weight. One can couple the advantage of products of high health benefit and low CFN, with the third dimension being the adoption of routine physical activity levels such as walking stairs to walking miles indoors (as on a treadmill) or outdoors. There may also be a spiritual dimension because, in theory, regular church-goers have a lower rate of obesity than that observed in the general population.

XII. REVAMPING THE CEREAL GRAIN–BASED FOOD GROUP Bread is the staff of life, but donuts are not. People rarely eat one-half of a croissant or a danish. The vending machine dispenser has a package of four Oreo cookies, not two medium cookies. There is no serving container for sale at the cinema that provides only one cup of popcorn, but that is the official USDA/FDA serving size! On the other hand, cultures that eat tortillas rather than bread, eat several tortillas. A Chinese meal is understood to include rice, unless other versions of the food (e.g., pork fried rice) are requested. In Italian restaurants, a pasta side-dish is automatic, with the choice being what style of pasta is preferred. Croutons may or may not be included in the salad, provided as part of the meal or ordered a` la carte. Whole-grain flour is defined as having the compositional ingredients of bran, germ, and endosperm in the flour, as is present in the native state. For health-claim purposes, the food must include 51% of whole-grain flour by weight. In addition to dietary fiber, whole-grain flour has considerable antioxidant properties, as well as an array of B vitamins and key minerals that are over and above providing dietary fiber. There is no question that whole grains exert health benefits, but the American intake is about 20–30% of the cereal grain category. There is a long history of cardiovascular health benefits associated with the routine consumption of whole-grain products (Jacobs et al., 1998, 1999; Liu et al., 1999). Evidence of benefits of whole grains in diabetes has emerged (Liu et al., 2000). The foundation of the classic USDA pyramid is based on cereal grain foods serving as energy sources, but energy sources are no longer limiting

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in the dietary. Only whole grains and the products thereof have shown significant health benefits. Because the whole-grain cereal products exert the scientifically derived health benefits, these foods must become the second tier of the pyramid. Interestingly, the foods of these bottom two tiers are able to provide for the complete sustenance of the vegan. This fact demonstrates the essentiality of the bottom two tiers of a revised pyramid of food guidance. It should be noted that those cereal grain products that have a CFN of 50 or more should not be accounted for in this tier but moved to the peak of the pyramid as foods to use sparingly.

XIII. COMMUNION WITH OTHER PYRAMIDS A. OLDWAYS PYRAMIDS

As pointed out by Ferro-Luzzi and Sette in 1989, the Mediterranean diet pyramid is based on an amalgamation of the diverse dietary patterns of several countries. The concept of the Mediterranean diet is said to have originated from the Seven Countries Study initiated by Ancel Keys in the 1950s (Hu, 2003). There are 15 countries that border on the Mediterranean. One can best describe the diet in the Mediterranean countries as having ‘‘a high availability of protective food items’’ (DeCarli and La Vecchia, 1991), with an emphasis on fruits, vegetables, vegetable fats, high-soluble dietary fiber, and a consumption of alcoholic beverages. It is a diet in which legumes and nuts are joined with vegetables and fruits as one tier. Potatoes are coupled with cereal grains. Oldways Preservation and Trust Exchange, a nonprofit company, has created diet pyramids that represent traditional cultural eating patterns associated with good health (Escobar, 1997; Oldways, 2003). The ‘‘healthy eating pyramids’’ developed by Oldways Preservation and Trust Exchange include Mediterranean, Asian, Latin American, and vegetarian pyramids. A comparison of the Vegetarian Pyramid (Figure 3), the Mediterranean Pyramid (Figure 4), the Asian Pyramid (Figure 5), and the Latin American Pyramid (Figure 6) reveals that all have in common the coupling into one tier of fruits, vegetables, legumes, and nuts (seeds are included in the Asian Pyramid). The Mediterranean Pyramid recommends a high fat intake, but it is substantially from olive oil, which is singled out, and no other oils are mentioned. The Latin American Pyramid also incorporates grains and tubers in the same foundation tier. The Vegetarian Pyramid separates seeds and nuts and emphasizes whole grains (essentially and naively excluding any mention of refined grains or products thereof ).


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B. WILLETT AND STAMPFER HEALTHY EATING PYRAMID

Willett and Stampfer (2003) have proposed a ‘‘rebuilding’’ of the 1992 USDA Food Guide Pyramid, which they call the Healthy Eating Pyramid. First, they make a case for distinguishing between healthy and unhealthy types of fats and carbohydrates. Fruits and vegetables are recommended, but dairy product consumption is limited. The authors state that the emphasis on percentage of calories from fat in the diet does not necessarily relate to the incidences of coronary heart disease (CHD). They point to the high incidence of CHD in Finland in contrast to Crete and the even higher incidence in Japan albeit only 8–10% calories are from fat. Obviously the type of fat makes a difference and their pyramid builds its base on plant oils and whole-grain foods. ‘‘Refined’’ grain products as well as potatoes, pasta, and sweets are to be used sparingly. Vegetables and fruits are promoted in the second tier, with emphasis ‘‘in abundance’’ for vegetables. Nuts and legumes are promoted as a separate third tier. The bottom three tiers fulfill the needs of the vegan. The Healthy Eating Pyramid is not a mirror image of the Mediterranean Pyramid. It is strongly based on a philosophy of the redistribution of fats and types of fats. In the animal food tiers, eggs, poultry, and fish are okay up to two servings. The dairy products tier suggests only one to two servings per day and suggests the alternative of a calcium supplement. No mention is made of the much needed complementation of vitamin D. Alcohol in moderation with meals is okay, as well as a multivitamin preparation. No mention is made of supplement ready-to-eat (RTE) cereals such as TotalÕ cereal.

XIV. HOW SHOULD THE CONSUMER APPROACH THE PYRAMID FOR FOOD CHOICE GUIDANCE? The consumer wants to eat ‘‘healthy.’’ There is no way to explain the plethora of print and some electronic media that promotes a proper diet. Dieting and cookbooks pervade the nonfiction bestseller lists and other indicators of concern. They are trying to understand why certain foods are acceptable in one culture and not in another, for example, white rice. Is eating pasta really all that bad when there are so many Italian restaurants? Some foods have a historical documentation of sparing diseases and death, such as potatoes. But why are high-CFN ‘‘potato chips’’ on the diet plate? The consumer has been eating less red meat than in the days after World War II. Fish is very expensive and the controversy of overfishing and

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mercury/dioxin contamination makes the consumer hesitate. Consumers drink less fluid milk than decades ago and they wonder whether that is smart in view of the numerous osteoporosis stories found everywhere. They are slowly switching from drinking considerable amounts of carbonated beverages to bottled waters. Tea remains the beverage of choice but not to the young. Are we really being honest with ourselves when we are told in various ways that there are three meals a day and two snacks? How important is it to eat a healthy breakfast? Are the home fries and the eggs really all that bad? (This combination, in terms of protein quality, is superior to eggs alone.) Unequivocally, for a guaranteed amount of the most nutrients and the benefits of protective compounds in the foods, we should plan on plant foods dominating at least two-thirds of the plate (the lower two tiers of the pyramid) and most of that should be first vegetables, second fruits (would you believe tomatoes?), and third a handful of nuts or a serving of a legume (would you believe peas), and all three complemented by a salad of mixed greens, carrot pieces, and maybe a few olives. Whole-grain products also complement with nutrients and nutraceuticals. The entree can be most anything from mussels to eggplant parmigian to chicken breast to hamburger or steak, half of which, if eaten at a restaurant, you can take home for another meal. XV. PRACTICAL APPROACH OF FOOD GUIDES A consumer friendly and instructional food guide not only should be a list of food groupings, but should also convey the proportions recommended from the various food groups. Whereas health claims for foods within a grouping assist the user in identifying the relative importance of certain foods, the balance of the dietary depends on the combinations of food servings made from within a food guide segment and in combination with foods from other groupings. The reinvented pyramid is a solid guide, based on the science underpinning of health claims and a high density of phytochemicals having protective health benefits. Coupled with an index of physiological costs in calories per nutrient, the consumer can learn to make healthy choices whether at home or on the road. REFERENCES Agatston, A. 2003. ‘‘The South Beach Diet: The Delicious, Doctor-Designed, Fool Proof Plan for Fast and Healthy Weight Loss’’. Rodale Press, Emmaus, PA. Ahlstro¨m, A. and Ra¨sa¨nen, L. 1973. Review of food grouping systems in nutrition education. J. of Nutr. Educ. 5, 13–17.


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Albert, C.M., Gaziano, M., Willett, W.C., and Manson, J.E. 2002. Nut consumption and decreased risk of sudden cardiac death in the Physicians’ Health Study. Arch. Intern. Med. 162, 1382–1387. Anonymous 1994. Market Research of America. Anonymous, Consumer Reports on Health (March 2000). 12(3). Anonymous 2000. Food Pyramid: Ideal vs. Real. Atkins, R.C. 2002. ‘‘Dr. Atkins’ New Diet Revolution’’. Harper Collins, New York. Atwater, W.O. 1894. Foods: Nutritive Value and Cost. Farmers’ Bulletin no. 23, p. 357. Washington, DC. Atwater, W.O. 1902. Principles of Nutrition and Nutritive Value of Food. Farmers’ Bulletin no. 142, p. 48. Washington, DC. Bidlack, W.R., Omaye, S.T., Meskin, M.S., and Jahner, D. 1998. ‘‘Phytochemicals: A New Paradigm’’. Technomic Publishing Co., Lancaster, PA. Bidlack, W.R., Omaye, S.T., Meskin, M.S., and Topham, D.K.W. 2000. ‘‘Phytochemicals as Bioactive Agents’’. Technomic Publishing Co., Lancaster, PA. Chanmugam, P., Guthrie, J.F., Cecilio, S., Morton, J.F., Basiotis, P., and Anand, R. 2003. Did fat intake in the United States really decline between 1989–1991 and 1994–1996? J. Am. Diet Assoc. 103, 867–872. Cohen, J.H., Kristal, A.R., and Stanford, J.L. 2000. Fruit and vegetable intakes and prostate cancer risk. J. Natl. Cancer Inst. 92, 61–68. DeCarli, A. and La Vecchia, C. 1991. Diet and cancer In ‘‘The Mediterranean Diets in Health and Disease’’ (G. A. Spiller, ed.), pp. 287–303. Van Nostrand Reinhold, New York. Dragsted, L.M., Pedersen, A., Hermetter, A., Basu, S., Hansen, M., Haren, G.R., Kall, M., Dragsted, L.O., Pedersen, A., Hermetter, A., Basu, S., Hansen, M., Haren, G.R., Kall, M., Breinholt, V., Castenmiller, J.J.M., Stagsted, J., Jakobsen, J., Skibsted, L., Rasmussen, S.E., Loft, S., and Sandstro¨m, B. 2004. The 6-a-day study: Effects of fruit and vegetables on markers of oxidative stress and antioxidative defense in healthy nonsmokers. Am. J. Clin. Nutr. 79, 1060–1072. Escobar, A. 1997. Are all food pyramids created equal? Nutrition Insights Center for Nutrition Policy and Promotion 12, 75–77. Ferro-Luzzi, A. and Sette, S. 1989. The Mediterranean diet: An attempt to define its present and past composition. Eur. J. Clin. Nutr. 43(Suppl. 2), 13–29. Finley, J.W. 2003. The antioxidant responsive element (ARE) may explain the protective effects of cruciferous vegetables on cancer. Nutr. Rev. 61, 250–258. Food and Drug Administration 1998. Staking a claim to good health. FDA Consumer Magazine Washington, DC. Nov/Dec. Food and Drug Administration 2003. Nutrition labeling. http://vm.cfsan.fda.gov.label.html. Foster-Powell, K. 2002. International table of glycemic index and glycemic load values. Am. J. Clin. Nutr. 76, 5–56. Guhr, G. and Lachance, P.A. 1997. Role of phytochemicals in chronic disease prevention In ‘‘Nutraceuticals: Designer Foods III Garlic, Soy and Licorice’’ (P.A. Lachance, ed.), pp. 311–364. Food & Nutrition Press, Trumbull, CT. Heber, D. and Bowerman, S. 2002. ‘‘What Color Is Your Diet?’’. Regan Books, New York. Hebert, J.R., Hurley, T.G., Olendski, B.C., Teas, J., Ma, Y., and Ha, J.S. 1998. Nutritional and socioeconomic factors in relation to prostate cancer: A cross-national study. J. Natl. Cancer Inst. 90, 1637–1647. Ho, C.-T., Lee, C.Y., and Huang, M.-T. 1992. ‘‘Phenolic Compounds in Food and their Effects on Health I and II’’. p. 338/p. 402. American Chemical Society. 506 and 507. Washington, DC. Hu, F.B. 2003. The Mediterranean diet and mortality—olive oil and beyond. N. Engl. J. Med. 348, 2595–2596.

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Huang, M.-T., Tosihhiko, O., Ho, C.-T., and Rosen, R.T. 1993. ‘‘Food Phytochemicals for Cancer Prevention’’. Vols 1 and 2 p. 427/p. 450. American Chemical Society. 546 and 547. Washington, DC. Jacobs, D.R., Jr., Meyer, K.A., Kushi, L.H., and Folsom, A.R. 1998. Whole-grain intake may reduce the risk of ischemic heart disease in postmenopausal women: The Iowa Women’s Study. Am. J. Clin. Nutr. 68, 248–257. Jacobs, D.R., Jr., Meyer, K.A., Kushi, L.H., and Folsom, A.R. 1999. Is whole grain intake associated with reduced total and cause-specific death rates in older women? The Iowa Women’s Study. Am. J. Public Health. 89, 322–329. Jian, M.G., Hislop, G.T., Howe, P., and Ghadirian, P. 1999. Plant foods, antioxidants and prostate cancer risk: Findings from case-control studies in Canada. Nutr. Cancer. 34, 173–184. Joseph, J.J., Nadeau, D.A., and Underwood, A. 2002. ‘‘The Color Code. A Revolutionary Eating Plan for Optimum Health’’. Hyperion Books, New York. Kantor, L. S. 1998. A Dietary Assessment of the U.S. Food Supply: Comparing per Capita Food Consumption with Food Guide Pyramid Serving Recommendations. U.S. Dept. of Agriculture. Agricultural Economic Report no. 772. Kantor, L. S. 1999. A comparison of the U.S. food supply with the Food Guide Pyramid recommendations In ‘‘America’s Eating Habits: Changes and Consequences’’ (E. Frazao, ed.), pp. 71–95. Agriculture Information Bulletin no. 750. Washington, DC. Katz, F. 1998. USDA surveys show what Americans eat. Food Technol. 52, 50–54. Kris-Etherton, P.M., Yu-Poth, S., Sabate, J., Ratcliffe, H.E., Zhao, G., and Etherton, T.D. 1999. Nuts and their bioactive constituents: Effects on serum lipids and other factors that affect disease risk. Am. J. Clin. Nutr. 70(Suppl), 504–511. Lachance, P.A. 1994. Scientific status summary—human obesity. Instit. Food Technol. 48(2), 127–138. Lachance, P.A. 1997. ‘‘Nutraceuticals: Designer Foods: Garlic, Soy, Licorice’’. Food and Nutrition Press, Trumbull, CT. Lachance, P.A. 2000. Food fortification with vitamin and mineral nutraceuticals In ‘‘Essentials of Functional Foods’’ (M.K. Schmidl and T.P. Labuza, eds), pp. 293–302. Aspen Publishers, Inc., Gaithersburg, MD. Lachance, P.A. and Fisher, M.C. 1986. Educational and technological innovations required to enhance the selection of desirable nutrients. Clin. Nutr. 5, 257–267. Liu, S., Munson, J.E., Stampler, M.J., Hu, F.B., Giovannucci, E., Rimm, E., Manson, J.E., Hennekens, C.H., and Willett, W.C. 1999. Whole-grain consumption and risk of coronary heart disease: Results from the Nurses’ Health Study. Am. J. Clin. Nutr. 70, 412–419. Liu, S., Stampfler, M.J., Hu, F.B., Giovannucci, E., Colditz, G.A., Hennekens, C.H., and Willett, W.C. 2000. A prospective study of whole-grain intake and risk of type 2 diabetes mellitus in US women. Am. J. Public Health. 90, 1409–1415. Michaud, D.S., Spiegelman, D., Clinton, S.K., Rimm, E.B., Willet, W.C., and Giovannucci, E.L. 1999. Fruit and vegetable intake and incidence of bladder cancer in male prospective cohort. J. Natl. Cancer Inst. 91, 605–613. Miller, H.E., Rigelhof, F., Marquart, L., Prakash, A., and Kanter, M. 2000. Antioxidant content of whole grain breakfast cereals, fruits and vegetables. J. Am. Coll. Nutr. 19, 312S–319S. Monro, J. 2003. Redefining the glycemic index for dietary management of postprandial glycemia. J. Nutrition 133(12), 4256–4258, 2003 Dec. National Cancer Institute 2003. National Cancer Institute’s 5 A Day for Better Health Program web site. http://www.5aday.gov. National Research Council, Committee on Food and Nutrition 1941. ‘‘A yardstick for good nutrition—recommended dietary allowances’’, p.2. Washington, DC. Oldways Preservation and Exchange Trust 2003. Healthy Diet Pyramids. www.oldwayspt.org.


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Painter, J., Rah, J.-H., and Lee, Y.-K. 2002. Comparison of international food guide pictorial representations. J. Am. Diet. Assoc. 102, 483–489. Rolls, B.J., Ello-Martin, J.A., and Tohill, B.C. 2004. What can intervention studies tell us about the relationship between fruits and vegetable consumption and weight management? Nut. Revs. 62, 1–17. Roos, E.B., Hirvonen, T., Mikkila, V., Karnoven, S., and Rimpela, M. 2001. Household educational level as a determinant of consumption of raw vegetables among male and female adolescents. Prev. Med. 33, 282–291. Slavin, J.L., Martini, M.C., Jacobs, D.R., and Marquart, L. 1999. Possible mechanisms for the protectiveness of whole grains. Am. J. Clin. Nutr. 70, 459S. Stiebeling, H.K. and Ward, M. 1933. Diets at four levels of nutrition content and cost. USDA Circular no. 296. Washington, DC. Thompson, L.U. 1994. Antioxidants and hormone-mediated health benefits of whole-grains. Crit. Rev. Food Sci. Nutr. 34, 473. Tsai, C.-J., Leizmann, M.F., Hu, F.B., Willett, W.C., and Giovannucci, E.L. 2004. Frequent nut consumption and decreased risk of cholecystectomy in women. Am. J. Clin. Nutr. 80, 76–81. U.S. Dept. of Agriculture. 1998. Continuing Survey of Food Intakes by Individuals and 1994–1996 Diet and Health Knowledge Survey and related materials [CD-ROM]. U.S. Dept. of Agriculture. 2000. Backgrounder—2000 Dietary Guidelines for Americans, 5th Ed. http://www.usda.gov.cnpp/Pubs/DG2000/Backgr.PDF. U.S. Dept. of Agriculture. 2003. Ethnic/Cultural and Special Food Guide Pyramids. http://www. nal.usda.gov/fnic/etext/000023.html. Verhoven, D.T., Goldbohm, R.A., vanPopel, G., Verhagen, H., and van der Brandt, P.A. 1996. Epidemiological studies on Brassica vegetables and cancer risk. Cancer Epidemiol. Biomarkers Prev. 5, 733–740. Welsh, S. 1994. Atwater to present: Evolution of nutrition education. J. Nutr. 124, 1799S–1807S. Welsh, S., Davis, C., and Shaw, A. 1992. A brief history of food guides in the United States. Nutr. Today. Nov/Dec, 6–11. Welsh, S., Davis, C., and Shaw, A. 1993. USDA’s Food Guide—Background and Development. U.S. Dept. of Agriculture. Human Nutrition Information Service. Miscellaneous Publication no. 1514. Willett, W.C. and Stampfer, M.J. 2003. Rebuilding the food pyramid. Sci. Am. 288, 64–71. World Cancer Research Fund and AICR Food 1997. Nutrition and the Prevalence of Cancer: A Global Perspective. Washington, DC. Zhang, Y., Talalay, P., Cho, C., and Posner, G. 1992. A major inducer of anticarcinogenic protective enzymes from broccoli: Isolation and elucidation of structure. Proc. Natl. Acad. Sci. 89, 2399–2403. Zizza, C., Siega-Riz, A.M., and Popkin, B.M. 2001. Significant increase in young adults’ snacking between 1977–78 and 1984–1996: Represents a cause for concern! Prev. Med. 32, 303–310.

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PLANT PIGMENTS: PROPERTIES, ANALYSIS, DEGRADATION BENOIˆT SCHOEFS Dynamique vacuolaire et Re´ponses aux Stress de l’Environnement Plante-Microbe-Environnement, Universite´ de Bourgogne a` Dijon Dijon Cedex, France

I. Introduction II. Spectroscopic, Molecular Structures, and Chemical Properties A. Common Properties B. Carotenoids C. Tetrapyrroles D. Plant Polyphenolic Compounds E. Alkaloids III. Chemical Modifications Occurring during Food Treatments and Storage A. Degradation due to Enzymatic Activities B. Degradation by Heat C. Degradation due to Acidification D. Degradation due to Oxygen Exposure E. Degradation due to Light F. Detection of Adulterants and Control of Quality IV. Methods of Analysis: An Overview A. General Cases B. Spectroscopic Methods C. Chromatographic Separation D. Mass Spectrometry V. Pigment Identification and Quantification: The Problem of Standards A. Noninvasive Methods B. Evaluation of the Color and Its Perception C. Use of Chlorophyll Fluorescence to Estimate the Freshness or the Maturity of Plants VI. Extraction and Analysis: Case by Case A. Pigments from Intact Fruits and Vegetables B. Anthocyanins in Seeds C. Water-Soluble Carotenoids from Flower Tissues D. Pigments from Juices and Drinks E. Pigments from Oil F. Carotenoids from Raw and Cooked Pasta



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G. Pigments from Vegetable Purees H. Carotenoids from Cheese I. Pigments from Textile J. Pigments in Pharmaceutical Products: The Pigment–Cyclodextrin Complexes K. Pigments from Animal Tissues VII. Future Trends Acknowledgment References

I. INTRODUCTION What do children choosing sweets in a shop and their adults selecting fruits in a grocery have in common? Both use the appearance and decide to buy the most colorful items. This very daily example shows that the colors of foods have a strong influence on our perception of acceptability and palatability (Blendford, 1995; Hendry and Houghton, 1996; Newsome, 1990). ‘‘Plant pigment’’ is a generic expression used to designate a large number of colored molecules synthesized by photosynthetic organisms. On the basis of their chemical structure, they can be classified into four families: tetrapyrroles (e.g., chlorophyll), carotenoids (e.g., b-carotene), polyphenolic compounds (e.g., anthocyanins), and alkaloids (e.g., betalains). An example of the chemical structure of a member of each family of compounds is presented in Figure 1. The number of molecules belonging to each family is quite large, so several volumes would be necessary to describe their particular properties. The description of the physicochemical properties of pigments has been the topic of comprehensive reviews and books (Britton, 1988; Britton et al., 1995; Gross, 1991; Schoefs, 2002). Plant pigments have positive roles in human health (Franceschi et al., 1994; Gerster, 1993; Groten et al., 2000; Kumpulainen and Salonen, 1996; Mayne et al., 1994; Shim et al., 2003). Although animal tissues contain plant pigments, these tissues are not able to synthesize them. Therefore, the pigments must be obtained from food (Baker, 1992; Castenmiller et al., 1999; Depee et al., 1998). Once assimilated, pigments enter the biochemical pathways along which they may be eventually modified (Parkinson and Brown, 1981). For instance, b-carotene is split into two parts, one of these serving as a precursor of vitamin A (Goswani and Barua, 2003), an important molecule for vision, skin protection, and cell growth. Several studies have established the minimum daily intake of these valuable pigment molecules. Because consumption of fresh foods has decreased and that of processed foods has increased, food colors have become an important aspect of the pigment food formulation process. Unfortunately, during food

PLANT PIGMENT ANALYSIS

43

FIG. 1 Example of the chemical structure of a member of each family of pigments synthesized by plants.

processing, the natural pigment content may be altered or destroyed. A very demonstrative example is the color change that occurs during the processing of mangoes or pineapple flesh. In the fresh fruit, the main carotenoid is the diepoxy-carotenoid violaxanthin (yellow), which during processing is transformed to the faint-yellow auroxanthin, a furanoid carotenoid (Mercadante and Rodriguez-Amaya, 1998) (see Figure 4). To restore the natural level in pigments in the processed products, extracted pigments are incorporated into the final food products (Britton, 1995; Burton and Ingold, 1984; Garcia-Viguera et al., 2000; Palozza and Krinsky, 1991). Similarly the preparation of fortified products requires the addition of pigments to the products (Smith, 1991). Regardless of the type of final products, the pigments are incorporated either as they naturally occur or as a chemically modified form (e.g., chlorophyllin or glycosylation of crocetin) (Dufresne et al., 1999). As a consequence of these additional needs, the demand in natural colorants has increased compared to synthetic dyes (Joppen, 2003; Pszczola, 1988). However, this increase cannot always be satisfied due to

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the limited supply of raw materials because the production of pigments using conventional plant cultivation methods is influenced by climatic conditions, plant cultivars, and varieties (Rodriguez-Amaya, 2000). Consequently part of plant pigment research is oriented to finding new sources of pigments. This quest is not only directed toward finding natural alternatives to synthetic dyes but also with the aim to discover new taxons and new procedures to produce pigments, for instance, from cell cultures (Table I). Pigment production by cell or microalgal cultures is especially interesting because these methods can be established independently of the climatic influences. In addition, yields can be predicted (Rodriguez-Amaya, 2000) and eventually improved by applications of elicitors or through genetic engineering (Ye et al., 2000). For all the reasons cited previously, pigment analysis constitutes a real analytical challenge. To take this challenge, one must have a diversified number of analytical procedures able to rapidly and precisely quantify the different pigment levels in samples. Addressing these issues is never straightforward. With some samples, like bottled waters, analysis is very simple. TABLE I EXAMPLES OF NEW SOURCES FOR PIGMENTS SHOWING POTENTIAL FOR INDUSTRIAL PRODUCTION

Pigment produced

New sources

References

Anthocyanins

Cacti

Carle and Stintzing (2000); Stintzing et al. (2000) Gu¨renc, and Karaali (2000) Aoki et al. (2000); Ravishankar and Venkataraman (1990); Zhong et al. (1991) Ozeki and Komamine (1985)

Sunflower (seed hulls) Cell cultures of Perilla

Carotenoids

Betalains

Cell culture of Daucus carota (carrot) Cell culture of Vitis vinifera (vine) Pumpkin Green microalgae

Cell cultures of Chenopodium rubrum Cell cultures of Phytolacca americana Cell cultures of Beta vulgaris (fodder beet)

Do and Cormier (1990); Hirasuna et al. (1991) Chaudry and Stich (2000) Cordevo et al. (1996); Kopecky et al. (2000); Ravishankar et al. (2000); Usha et al. (1999) Berlin et al. (1986) Sakuta et al. (1987) Ravishankar et al. (2000)


45

Other samples are highly complex mixtures, requiring careful processing and storage conditions to ensure an unchanged level of pigments and color. This is especially important in the case of fortified foods, which create nutritional expectations from both the health authorities and the consumers. The development of powerful analytical methods is also of prime importance in the control of quality. Obviously, this requires a precise knowledge of the pigment composition of original products (Mercadante et al., 1998; Minguez-Mosquera et al., 1995a). In this contribution, the molecular structures, the general physicochemical properties of plant pigments, and the mechanisms involved in pigment degradation are first described. Then follows a review on invasive and noninvasive methods used for pigment analysis, including the mechanisms involved in pigment degradation. Finally, several examples of analytical procedures are presented.

II. SPECTROSCOPIC, MOLECULAR STRUCTURES, AND CHEMICAL PROPERTIES A. COMMON PROPERTIES

The absorption of light by pigment molecules results from the presence of conjugated carbon–carbon double bonds, which form the chromophore. Inside the chromophore, the electrons can be considered as belonging to this system as a whole rather than to an individual atom. When light is absorbed by the chromophore, the whole energy of the quantum is communicated to it and the chromophore is lifted from its normal state of lowest energy (ground state) to an energy-rich state (excited state). According to the Bohr theory of molecular structure, a molecule can exist only in a series of discrete states of electronic energies, which correspond to the absorbance bands [for a full description of the absorbance phenomenon, see Rabinovich (1956)]. From the comparison of the absorbance spectrum obtained with molecules from the same family, it was concluded that only the modifications in the circuit of the electrons along the chromophore are reflected in the absorbance spectrum of the pigment and, therefore, can be detected by using spectroscopic methods. As an example, one can compare the tremendous modifications in the absorbance spectrum of chlorophyll a caused by the replacement of the methyl side-group of C7 by an aldehyde side-group (Figure 2). A molecule in an excited state can deexcite through several ways. If the molecule is well protected from the interactions with other molecules, the simplest pathway to rapidly lose its excitation energy is to emit a photon

46

B. SCHOEFS

FIG. 2 Spectral modifications in the absorbance spectrum of chlorophyll a ( full line) triggered by the replacement of the methyl side-group at the C7 position by an aldehyde side-group, yielding chlorophyll b (dashed line). The structures of chlorophyll a and chlorophyll b molecules are also presented.

back into the space. This is called fluorescence. Tetrapyrrole molecules can deexcite by emitting fluorescence. The chlorophyll fluorescence spectrum contains only one main band because the emission always originates from the first excited state. Fluorescence is in competition with other pathways that are internal conversion and transition to the triplet state. In the case of carotenoids, the fluorescence intensity is very weak (Koyama and Hashimoto, 1993) because the main deexcitation pathway occurs through the transition to the triplet state, which is nonfluorescent.


47

B. CAROTENOIDS

Carotenoids are responsible for most of the yellow to red-orange colors of fruits and vegetables (Table II). The basic structure of a carotenoid molecule is a symmetrical tetraterpene skeleton formed by tail-to-tail linkages of two geranylgeranyl diphosphate molecules (C20 unit). The end-groups of the basic structure are modified into six-membered rings yielding to monocyclic and dicyclic carotenoid such as b-carotene (Figure 1), the most ubiquitous carotenoid. The carotenoid group of molecules can be divided into two families of compounds: the carotenes, which are devoid of oxygen function (like b-carotene), and the xanthophylls, which contain at least one oxygen function (like violaxanthin; see Figure 5 for the structure). Carotenoids are lipophilic compounds and are usually water insoluble, although some xanthophylls may be water soluble because they harbor very strongly polar groups, such as polysaccharides. A good example is crocetin glycosyl

TABLE II COLOR OF THE MAIN TYPES OF PIGMENTS FOUND IN PLANTSa

Family

Subfamily

Color

Found in

Tetrapyrroles

Chlorophylls (E140)

Green

Phycocyanobilins

Blue or red

Carotenes (E160)

Yellow to red

Xanthophylls (E161)

Yellow to red

Annatto (E160b) Anthocyanins (E163) Flavonols Tannins Phytomelanins Curcumin (E100) Betalains

Blue to red

Indigo

Blue to pink

Vegetables, fruits, juices, oils, sweets Cyanobacteria, red algae, chocolates, food complements Vegetables, fruits, seeds, roots, sweets, juices, oils Vegetables, flowers, fruits, sweets, cheese, juices, oils Red fruits, vegetables, seeds, flowers, roots, cheese, wines, syrups Fruits, teas Wines Fruits, seed coats Curcuma Tissues originating from Caryophyllales and fodder beet Drinks

Carotenoids

Polyphenolic compounds

Alkaloids

a

Yellow to cream Brown Black to brown Yellow Yellow to red-violet

The Exyz number refers to the European Community nomenclature for food colorants (Anonymous, 1995).

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pigments that give the color to saffron stigmata. For a comprehensive description of the chemical properties of carotenoids, see the review by Britton et al. (1995). Solubilization of non–water-soluble carotenoids can be strongly improved by complexation of the pigment with cyclodextrin. Carotenoids can exist in two configurations depending on the relative disposition of the four substituents around carbon–carbon double bonds. The configuration may be important for color intensity (Coultate, 1996) and for carotenoid assimilation (Storebakken and No, 1992; Torrissen et al., 1989). C. TETRAPYRROLES

The family of tetrapyrroles can be arranged in two classes depending on whether they are closed like chlorophyll (typically green) or open like phycocyanobilins (red or blue) (Table II). Since the pioneer works by Willsta¨ter and Sto¨ll (1913) on the chlorophyll structure, more than 50 different chlorophyll-related molecules have been reported (Scheer, 1996). Basically, chlorophyll molecules are conjugated tetrapyrroles, to which a cyclopentanone ring has been added. The macrocycle is planar and binds into the center of an atom of Mg2þ. Release of this ion converts chlorophyll to pheophytin. When pheophytin binds one Cu2þ or Zn2þ ion, it is converted to Cu- or Zn-chlorophyll. All naturally occurring chlorophyll molecules have a propionic acid residue at position 17. The position 17(3) is generally esterified with a long-chain alcohol, usually phytol. Chlorophyll b differs from chlorophyll a by the presence of an aldehyde residue instead of a methyl residue at position 7 (Figure 2). Chlorophyll a and chlorophyll b are the most abundant pigments in land plants, in the skin of unripened fruits, and in green algae. Chlorophyll b can be considered typical for these organisms. The inner seed coat from Cucurbitaceae, though green, does not contain chlorophyll molecules but several protochlorophyllide esters (Schoefs, 2000a,b, 2001b). Besides chlorophyll a, brown algae and diatoms contain pigments similar to protochlorophyllide and protochlorophyll: the chlorophyll c and chlorophyll c esters (Garrido et al., 2000). Because the ring IV of chlorophyll c and chlorophyll c ester molecules is not reduced (between the C17 and C18 carbons), these molecules are not true chlorophyll molecules (Figure 3). In addition, chlorophyll c and chlorophyll c ester molecules differ from true chlorophyll molecules by the nature of the esterifying residue at position 17, which is an acrylic residue in chlorophyll c and chlorophyll c ester molecules and a propionic residue in true chlorophyll molecules. From the description of the structure, it is clear that photosynthetic tetrapyrroles—chlorophyll a, chlorophyll b, and chlorophyll c


FIG. 3

49

Diversity in the structure of closed tetrapyrroles.

esters—are made up of a hydrophilic part, the macrocycle, and by a hydrophobic part, the phytol chain (Figures 2 and 3). The most hydrophilic segments of the macrocycle are the cyclopentanone ring (Figure 3) and the propionic ester group (position 17). Therefore, nonesterified macrocycles are much more polar than the esterified ones. The nature of the other side-groups and their configuration modify the polarity of the molecules. Besides the family of closed tetrapyrrole molecules, there is a smaller group of pigments: the phycocyanobilin group, which is only abundant in cyanobacteria, red algae, and cryptomonad algae. The main forms of phycocyanobilins are the blue phycocyanobilin and the red phycoerythrobilin (Figure 4). In situ phycocyanobilins are covalently bound to particular proteins, forming different pigment–protein complexes, the so-called phycobiliproteins (Figure 4). These phycobiliproteins assemble to form phycobilisomes, which serve as light harvesters in the photosynthetic process. The regulation circuits of the biosynthetic and degradation pathways of phycobilisomes have been reviewed by Geiselmann et al. (2004). The pigment–protein complexes are stable at pH levels of 5 to 9 but may precipitate at a pH level lower than 5 (Sarada et al., 1999). The protein–phycoerythrin complexes found in red algae and some cyanobacteria are potentially very interesting for several food and nonfood industries (Le Jeune et al., 2003).

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FIG. 4

Diversity in the structure of open tetrapyrroles.

D. PLANT POLYPHENOLIC COMPOUNDS

Anthocyanins constitute the largest family of colored phenolic compounds. They are responsible for colors ranging from salmon and pink, through scarlet, violet to purple, and blue of a large variety of fruits, flowers, petals, leaves, and vegetables. Therefore, these tissues constitute commercially valuable sources of anthocyanins (Table II) (Counsell et al., 1981). Anthocyanin compounds have antioxidant and antimutagenesis properties (Furatu et al., 1998; Yoshimoto et al., 1999) and are responsible for the so-called French paradox (Renaud and Lorgeril, 1992), which says that despite a much higher consumption of saturated fats in France, the risk of arteriosclerosis and coronary heart diseases has a lower incidence of death in the French population than in other industrial countries. Anthocyanins are glycosides of polyhydroxy and polymethoxy derivatives of 2-phenylbenzopyrylium or flavilyum salts. The aglycones are known as anthocyanidins, which from the chemical point of view are flavonoids. There are six natural anthocyanidins (Coultate, 1996) (Table III). The various possibilities of glycosylation or acylation regarding both the position and the nature of the side-groups increase the number of anthocyanins by a factor 15 to 20 times (Harborne and Mabuy, 1982). As the data reported in Table IV show, the differences in glycosylation and acylation around ring B have a strong impact on color and tinctorial strength (Giusti et al., 1999b). An increase in the hydroxyl substitution around ring B results in a shift of


51

TABLE III STRUCTURE OF THE SIX NATURAL ANTHOCYANIDINS

Anthocyanin name

R1

R2

Pelargonin Cyanidin Peonidin Delphinidium Petunidium Malvidin

H OH OCH3 OH OCH3 OCH3

H H H OH OH OCH3

the absorption maximum to longer wavelengths, to yield a blue hue. The presence of additional acylation with cinnamic acids produces similar effects (Dangles et al., 1993). One crucial aspect of the chemistry of anthocyanins is the instability of color to pH changes (Hong and Wrolstad, 1990). Each cook knows that red cabbage turns blue-violet during cooking. This change can be reversed by the addition of some drops of vinegar or pieces of apple during cooking. At acidic pH levels, anthocyanins are red, whereas under basic conditions, they are blue-violet. An intensive search led to the discovery that anthocyanin molecules with complex patterns of glycosylation and acylation present remarkable stability to acidity, heat treatment, and light exposure (Dangles et al., 1993; Francis, 1992). The improved stability has been attributed to the intramolecular and intermolecular co-pigment, self-association, metal complexing, and presence of inorganic salts (Brouillard, 1983; Goto, 1987). The phenomenon of co-pigmentation and self-association plays an important role in color diversity that each single anthocyanin chromophore may generate (Gonnet, 1998). As it can be deduced from the description of the chemical structures of anthocyans and anthocyanidins, polyphenolic pigments are usually water soluble (Figure 1). Noticeable exceptions are the curcumin oils (yellow),

52

TABLE IV INFLUENCE OF THE AGLYCONE SUBSTITUTION ON THE PIGMENT COLOR PROPERTIES ( pH < 7)a

Type of substitution

5-glucosidic

3-glucosidic

3,5-glucosidic

513

> < ¼ 509

¼ < < 503

> < ¼ 500

< < < Bathochromic shift

L* Hue Chroma Position of the absorbance maximum (nm) a

For the position of carbon 3 and carbon 5, see Table III.

B. SCHOEFS

Aglycone

Acylation with cinnamic acid but not with malonic acid


53

which correspond to a second class of plant polyphenolic compounds. Curcumin consists of two vinyl guaiacol groups joined by a b-diketone unit. E. ALKALOIDS

The alkaloids are the last group of pigment molecules reviewed in this chapter. Alkaloids are secondary plant products, which very often exhibit pharmacological properties. Among the alkaloid family of compounds, only the betalain subgroup of molecules presents the unique property of being highly colored. In fact, betalain pigments are anthocyanin-like pigments (Figure 1) but represent an evolutionary divergence from the anthocyanidin-producing plants because betalain pigments are derived from different biosynthetic pathways. In the plant phylum, betalain molecules are synthesized in some families of the Caryophyllales order (Cactacae, Chenopodiacae, Amaranthaceae, Phytolaccaceae, and Basellaceae). The betalain family of compounds is divided into red to red-violet betacyanins and the yellow betaxanthins, which are acylated and nonacylated glycosides of aglycones, respectively. Betaxanthins differ from betacyanins by the conjugation of a substituted aromatic nucleus to the 1,7-diazaheptamethinium chromophore. These pigments maintain their appearance over a wide range of pH levels, from 4 to 7.

III. CHEMICAL MODIFICATIONS OCCURRING DURING FOOD TREATMENTS AND STORAGE Everyone has observed that fruits like banana, citrus, orange, tomato, paprika, and so on are first green and then turn colored, to yellow or red, during ripening. This change in color usually operates through the combination of chlorophyll degradation pathway and synthesis of carotenoids, which are induced by ethylene (Fraser et al., 1994; Gross, 1991). Consequently, the reduction of ethylene emission by stored fruits and vegetables is a common process used to keep fruits and vegetables green for a longer period. Conversely, the treatment of fruits with ethylene is used successfully to promote ripening of tomatoes (Iwahori and Lyons, 1970) and to improve the peel color of citrus. Although the chlorophyll degradation in fruits is not completely elucidated, it appears that the initial steps are similar to those observed during plant senescence (Adachi et al., 1992; Bertrand and Schoefs, 1999; Ma and Shimokawa, 1998; Matile et al., 1999). Similar color modifications, which reflect pigment degradation, have also been observed with anthocyanins (Markakis, 1982). These pigment degradations occur in situ. Extracted

54

B. SCHOEFS

pigments are even more sensitive to degradation, and stability of natural pigments is one of the major features of food industries. Several basic studies have been conducted to understand the ways of degradation and to increase the stability of pigments during food processing and storage. In the following sections, the principal factors responsible for pigment degradations are shortly reviewed. A. DEGRADATION DUE TO ENZYMATIC ACTIVITIES

Pigment degradations during processing may arise from the disruption of cellular structure. This is accompanied by the liberation of enzymes, such as lipoxygenase (Irvine and Anderson, 1953; Irvine and Winkler, 1950), peroxidase (Martinez Parra and Munoz, 1997), polyphenol oxidase (Kobrehel et al., 1972, 1974), and/or betalain oxidase. These enzymes are involved in the decrease of the xanthophyll content during durum wheat milling (Borrelli et al., 1999) or the pigment content of red beet (Martinez Parra and Munoz, 1997; Shih and Whiley, 1981), respectively. The enzyme activity can be stopped during blanching (see later discussion). For instance, red-palm oil fruits are sterilized immediately after harvest to inactivate lipase enzyme, which would otherwise promote rancidity. This process provokes a considerable increase in the proportion of cis-a- and cis-b-carotene (Trujillo-Quijano et al., 1990). B. DEGRADATION BY HEAT

Food processing often requires one or more heating steps, which may allomerize chlorophyll molecules (Minguez-Mosquera and Gandul-Rojas, 1995) and even transform the green chlorophyll in dull olive-green pheophytin and pheophorbide (pheophytin without phytol; see Figure 3 for the structure) [bean: Muftugil (1986); parsley: Berset and Caniaux (1983); pea: Buckle and Edwards (1970); Acze´l (1971); spinach: Schwartz and Lorenzo (1991); tea: Kohata et al. (1998)]. In situ, these reactions are catalyzed by enzymes (chlorophyllase, Mg-dechelatase, lipoxygenase) (Bertrand and Schoefs, 1999), which can be inactivated by heating or during extraction with organic solvents. In some solvents, chlorophyllase can remain active, and heat inactivation requires several minutes at 90 8C. Short steaming (20–60 seconds), like that used during processing of tea leaves, may not inactivate the enzyme. Kohata et al. (1998) report that inactivation is only reached during tea-leaf raffination, a process that heats the leaves at 120 8C for 30 minutes. In corn, lipoxygenases and peroxidases are inactivated after 6 and 8 minutes of blanching, respectively, whereas broccoli requires only 90 seconds (Barrett et al., 2000). The difference in the inactivation time may be


55

explained by the different heat-transmission capacities. Regardless of these considerations, the severity of the heating steps should be limited to maintain the color, texture, flavor, nutritional quality (Lim et al., 1989; Maccarone et al., 1996; Theerakulkait et al., 1995), and the pheophorbide content. In Japan, the amount of pheophorbide in the food products is regulated by the Food and Health Administrations (e.g., pheophorbide: 160 mg/100 g). One common way to avoid accumulation of pheophorbide is to let it rebind a metal. Under certain conditions, pheophytin molecules can rebind divalent metals, principally copper and zinc ions, regenerating the green color, which is usually brighter (Coultate, 1996; Jones et al., 1977; LaBorde and von Elbe, 1990). This phenomenon is called regreening. The replacement capacity of chlorophyll b derivatives is much less than that of chlorophyll a (LaBorde and von Elbe, 1990; von Elbe et al., 1986). The chlorophyll derivatives were found more stable than the original chlorophyll, especially to acids (Humphrey, 1980). Formation of metallochlorophylls has a potential use as a green colorant for beverages and as a means to avoid accumulation of chlorophyll degradation product during processing. Canjura et al. (1999) designed a method based on continuous aseptic processing to favor the replacement of the chlorophyll-Mg2þ ion by Zn2þ. Again, care should be taken not to exceed the permitted value (U.S. Food and Drug Administration [FDA]: 75 mg/kg). Alternatively, one may use a sodium-copper-chlorophyllin compound, which is more water soluble than regular chlorophyll, as chlorophyllin does not have a phytol chain. The Cu content of chlorophyllin makes it furthermore more resistant to heat (Coultate, 1996). Thermal treatments also promote carotenoid degradations (including isomerization and oxidation) and carotenoid–protein complex disruptions (Boskovic, 1979; Pereira et al., 1999; Rodriguez-Amaya, 1989). Both modifications decrease the product’s nutritive value. In fact, cis-carotenoid isomers are less assimilated than the corresponding trans-isomers (Storebakken and No, 1992; Torrissen et al., 1989). In addition, cis-b-carotene and cis-gcarotene have a reduced vitamin A activity (Zeichmeister, 1962). This is especially true when severe heat treatments, such as those required to achieve commercial sterility, are applied (Robertson, 1985; Schwartz et al., 1981). Chlorophyll degradation also occurs during storage at 4 8C, with chlorophyll a being more sensitive than chlorophyll b (Schwartz and Lorenzo, 1991). C. DEGRADATION DUE TO ACIDIFICATION

In this chapter, the instability of many plant pigments to acidity has already been mentioned. For example, many polyphenolic compounds change their color depending on the pH level (Ikan, 1991). Under acidic

56

B. SCHOEFS

conditions, the red flavylium ions prevail. This could represent a disadvantage for the use of polyphenols as a colorant because they are unstable and degraded under weak acidic or basic pH levels. This is exemplified by a study of the stability of anthocyanin-3-glucoside extracted from grape (i.e., delphinidin-3-glucoside, cyanidin-3-glucoside, petunidin-3-glucoside, peonidin-3-glucoside, and malvidin-3-glucoside). The effect of pH during accelerated storage studies, performed at 55 8C, showed that no pigment molecule remains after 4 days at a pH level of 3. The degradation is accelerated when the pH level is increased to 5 (Baublis et al., 1994; SarniManchado et al., 1996). Such pronounced degradation has a strong influence on the color, which turns from red-orange to yellowish. This change becomes visually noticeable only when the degradation of anthocyanins has reached 60%. A similar result has been found with other anthocyanin preparations. The degradation of anthocyanins extracted from grapes can be largely prevented by the addition of a 4-vinylphenol side-group (Sweeny and Iacobucci, 1983), but this modification causes a somewhat larger precipitation of the pigment. In contrast to the natural anthocyanins, the 4-vinylphenyl derivatives are protected against discoloration by SO2 (SarniManchado et al., 1996). Acylation of anthocyanins improves their stability during processing and storage (Rommel et al., 1992). In monoacylated anthocyanins, only one side of the pyrylium ring can be protected against the nucleophilic attack of water, and therefore, only a weak intermolecular effect might occur (Brouillard, 1983). Differences in glycosidic substituents at the C3 position of the aglycon and the position of the acyl group in the sugar moiety may explain the different stabilities (Table V). The mechanism

TABLE V INFLUENCE OF THE ACYL GROUP POSITION AT THE AGLYCONE ON THE ANTHOCYANIN STABILITY TO HEAT

Aglycon

Source

Malic acid Sophorose

Red radish

Rutinose

Red-fleshed potato

Stability (half-life week)

Acylation groups (position)

þ (5) (5)

p-coumaric acid þ (3) þ (3)

22 (25 8C) 120 (4 8C) 11 (25 8C) 110 (4 8C)

Reference

Rodriguez-Saona et al. (2000) Rodriguez-Saona et al. (2000)


57

of protection is still not determined, but specialists suggest that the acylating group is protecting the oxonium ion from hydration, thereby preventing the formation of the hemiketal or chalcone forms (Francis, 1989). Diacetylated anthocyanins can also be stabilized by a sandwich type of stacking caused by hydrophobic interactions between planar residues of the acyl groups and the positively charged pyrylium nucleus. This configuration would diminish the formation of the pseudobase (Brouillard, 1981; Goto and Kondo, 1991). Acids also induce chemical modifications of carotenoids. For instance, juice preparation requires acidic conditions, which trigger the spontaneous conversion of 5,6- and 5,60 -epoxide groups of violaxanthin, lutein epoxide, and antheraxanthin to 5,8- and 50 ,80 -furanoid epoxides (Coultate, 1996; Gross, 1991). This conversion has a dramatic effect on the color because furanoids are essentially colorless. To be convinced about this fact, it is enough to compare the flesh color of fresh (solid line) and canned (dashed line) pineapples (Coultate, 1996) (Figure 5).

FIG. 5 Effect of processing on the color of pineapple flesh. The structure and spectral modifications occurring during the processing are displayed.

58

B. SCHOEFS D. DEGRADATION DUE TO OXYGEN EXPOSURE

Exposure to oxygen is deleterious, particularly in dried food such as dehydrated carrot slices in which carotenoid oxidation and bleaching occur rapidly because of the formation of activated oxygen species (Coultate, 1996). E. DEGRADATION DUE TO LIGHT

Modifications of food color reflecting changes in pigment composition may also happen during storage. For instance, the appearance of pink in cheddar cheese, colored with annatto, when stored under fluorescent light used in grocery stores dairy cases, is well known (Boyd, 2000; Hong et al., 1995). Betalains are also degraded during storage. For instance, vulgaxanthin, which represents 5% of the colored molecules of red beet extract, is less stable than the major colorant betacyanin. Therefore, when stored, red beet extracts develop a bluish shade. The original color may be restored by adding carotenoids. F. DETECTION OF ADULTERANTS AND CONTROL OF QUALITY

The use of different colorants in food is regulated by the legislation of each country. These legislations are becoming progressively more restrictive, and the number of colorants allowed is limited and strictly controlled (Anonymous, 1995; Francis, 1987). In addition, many degradation products of natural colorants can impart flavor and odor (Tonnesen and Karlsen, 1985). For instance, the appearance of unwanted odor may result from the formation of b-ionone due to the breakdown of b-carotene molecules (Coultate, 1996). Therefore, an important aspect of food research is the search for adulterants and/or the presence of toxic compounds in preparations. High-performance liquid chromatography (HPLC)–electrospray ionization (ESI)–mass spectrometry (MS) was used to analyze anthocyanin extracts from red-fleshed potato. The HPLC profile presents five major peaks corresponding to nonacylated anthocyanins and anthocyanins acylated with either ferulic acid or p-coumaric acid (Giusti et al., 1999a). In addition, the ESI-MS profiles may reveal presence of several toxic alkaloids as in red-fleshed potato (Rodriguez-Saona et al., 1998). Nuclear magnetic resonance spectroscopy (NMRS) has also been used to detect adulteration in olive oil (Mannina et al., 2003). To improve the color of virgin olive oil, chlorophyll molecules are sometimes added. This addition may be detected by NMRS because chlorophyll and pheophytin, the regular tetrapyrrole pigments of olive oil, give a different spectrum.


59

IV. METHODS OF ANALYSIS: AN OVERVIEW A. GENERAL CASES

Because plant pigments have a chromophore made of conjugated double bonds, they react easily with acids, bases, oxygen, heat, and light (anthocyanins: Aoki et al., 2000; betalains: Carle and Stintzing, 2000; carotenoids: Anguelova and Warthesen, 2000; Martin et al., 1999; Pfander et al., 2000; tetrapyrroles: Bertrand et al., 2004; Salin et al., 1999). Special care should be taken when manipulating extracted pigments (see earlier discussion). This is important when a sample containing chlorophyll molecules is exposed to light because excited chlorophyll molecules efficiently transfer the excitation energy to the triplet state of oxygen, resulting in the formation of activated oxygen species, which are very reactive and can oxidize other organic molecules, including other pigments, lipids, or proteins. For this reason, care should be taken during extraction and analysis. The production of singlet oxygen by chlorophyll is not restricted to aqueous pigment extracts but also occurs in less polar solvents like in oils (Anguelova and Warthesen, 2000; Haila et al., 1997). For this reason, oils containing a nonnegligible amount of chlorophyll or chlorophyll-like molecules should be stored in the dark and at reduced temperature (Chen et al., 1997; Jung and Min, 1991), especially if the content in carotenoids is low, as it is the case in pumpkin seed oil (Schoefs, 2000a,b, 2001b). The presence of carotenoids reduces the formation and the action of activated oxygen species (Goulson and Warthesen, 1999; Palozza and Krinsky, 1991). B. SPECTROSCOPIC METHODS

The absorbance spectrum can be considered the fingerprint of pigments (for limitations, see later discussion). Therefore, absorbance spectroscopy constitutes the simplest way to identify and quantify the major pigments in a mixture (Schoefs, 2000b, 2002). Once identified, it is possible to use a set of equations to estimate their respective concentrations (Bertrand and Schoefs, 1997; Bertrand et al., 2004; Jeffrey and Humphrey, 1975). Because the environment of the pigment (solvent, temperature, ligation to protein, etc.) strongly influences the position and the shape of the spectrum, the crude absorbance spectrum of intact tissues is usually useless for direct measurements of the pigment concentration (Borsari et al., 2002). Accurate measurements of pigment concentration require pigment extraction with a solvent for which equations have been established or at least in which specific (or molar) absorbance coefficients have been determined. With these data, one can establish a new equation set, adapted to the particular

60

B. SCHOEFS

situation. The precision of the measurements depends on the type of the device used, the precision in the determination of the position of the absorbance maxima, and of course the accuracy of the absorption coefficient used for the calculation. For instance, Porra et al. (1989) reevaluated the extinction coefficients for chlorophyll a and chlorophyll b by comparing the concentration of chlorophyll standards as determined by the measure of magnesium amount by atomic absorption spectroscopy and by spectrophotometric determination. These data were used to derive new equation sets, which were found significantly different from those used previously. Therefore, it is advisable to regularly check the literature for new values and equation sets. The major limitations of pigment identification on the sole basis of the absorbance spectrum is the overlapping of the absorbance bands of the pigments in the mixture. This difficulty makes the method less efficient when the number of pigments is higher than three. For completeness, it is necessary to add that the cis-carotenoid isomers can be recognized, because the absorbance spectrum presents an additional band in the UV region (Figure 6). The position of the cis-double bound is reflected in the ratio of Acis/AMAX. The total phenols in an anthocyanin preparation can be routinely determined using the Folin-Ciocalteau procedure (Singleton and Rossi, 1965).

FIG. 6 Comparison of the ultraviolet-Vis absorbance spectra of all–trans-b-carotene ( full line) and cis-b-carotene (dashed line).


61

Fluorescence spectroscopy can be more diagnostically helpful because of the selective excitation of pigments but useless in the case of carotenoids, because their fluorescence is very weak (Koyama and Hashimoto, 1993). In some cases, other spectroscopic methods can be used to identify pigments on the basis of particular structural features. For instance infrared (IR) spectroscopy was used to reveal the presence of an allelic group in the carotenoids fucoxanthin, alloxanthin, and bastaxanthin (Britton et al., 1995). IR spectroscopy was also used to establish the details of the lightinduced oxygen-dependent bleaching of the food colorant chlorophyllin (Bertrand et al., 2004; Chenery and Bowring, 2003; Salin et al., 1999). In conclusion, spectroscopic methods usually permit crude identification of pigments in an extract, but in most cases, the specific composition remains obscure. Therefore, obtaining details on the composition of a mixture of pigments requires additional analyses, which often involve separation of the mixture into its components using methods such as chromatography. C. CHROMATOGRAPHIC SEPARATION

1. Open column Various phases such as powdered sucrose, DEAE-Sepharose, cellulose, or MgO/Hyflosupercel have been used to achieve chlorophyll and carotenoid separation (Omata and Murata, 1983; Strain et al., 1971; Wasley et al., 1970). The all–trans-b-carotene can be separated from 9-cis and 13-cis isomers using an open column filled with calcium hydroxide. For phycobilins, an hydroxylapatite column can be used (Siegelman and Kycia, 1978). Methods to separate anthocyanins using gel filtration have been proposed (Somers, 1966). Although today open-column chromatography is mostly used to clean extracts or for preparative purposes, it may still be used for analytical purposes. For example, Somers and Evans (1977) reported that the formation of aggregates of glucoside anthocyanins is typical for wine aging and is related to the color stability. This natural process can be strongly accelerated by adding acetaldehyde. Therefore, upon addition of this compound to a young wine, it can ‘‘appear’’ old. In an attempt to find an analytical method, which would establish the ‘‘real’’ age of a wine, Johnson and Morris (1996) used a silica gel column, together with a gradient of formic acid to separate the different glucoside anthocyanin aggregates from red wines. Using this technique, the anthocyanins were eluted into four fractions. The two first fractions contained free anthocyanins, whereas the two last ones contained polymerized and condensed anthocyanins (Datzberger et al., 1991). When the amounts of anthocyanins in each fraction from the nontreated and the treated wine were compared, a clear

62

B. SCHOEFS

difference appeared because the treated wines had more aggregates of monoglucoside anthocyanins than the nontreated ones. This may be explained by the fact that acetaldehyde accelerates preferentially the condensation of monoglucoside pigments, but only slightly diglucoside ones (Johnson and Morris, 1996). 2. Thin-layer chromatography Thin-layer chromatography (TLC) is often used because it is a fast, effective, and relatively inexpensive analytical method. Special care should be taken when the separation is done on silica gel, because its slight acidity may trigger pigment degradations such as epoxide-furanoxide rearrangement in carotenoids (Schiedt and Liaaen-Jensen, 1995) and chlorophyll pheophytinization. Therefore, it is necessary to neutralize the acidity of silica before pigment separation. Unfortunately, separation of compounds with similar structures is usually rather difficult using TLC. This is well illustrated by comparing the chromatograms obtained from the pigments extracted from pumpkin seed oil and separated by TLC (Figure 7A) or by HPLC (Figure 7B). Using TLC, three bands are found: The two green bands correspond to protochlorophyll (band 2) and protopheophytin (protochlorophyll molecules, which have lost their Mg2þ atom) (band 3) pigments, whereas the yellow band reflects the presence of carotenoids (band 1). When the same sample was analyzed by HPLC, 14 peaks were obtained. As a consequence, TLC methodologies have been progressively supplemented by more efficient separation techniques, such as HPLC (Braun and Zsindely, 1992). Nevertheless, both open-column chromatography and TLC methods are still useful for cleaning extracts or preparing large amounts of pigments (He et al., 1998). 3. High-performance liquid chromatography Photosynthetic pigments, chlorophylls, and carotenoids have a clear hydrophobic character and are usually analyzed by C18–reversed-phase (RP) columns. A C30-RP appeared on the market. The C30-RP is particularly efficient in the separation of carotenoids because the interactions of the pigments and the stationary phase are maximized by the similar size. With this phase, many cis isomers of the same carotenoid are separated from each other (Emenhiser et al., 1995; Lacker et al., 1999; Sharpless et al., 1996). This C30-RP has been successfully applied to the determination of saponified carotenoids in orange juice (RousseV et al., 1996). However, when a mixture is complex, coelutions may become rapidly limiting and less selective stationary phases, such as the C18-RP, are therefore preferably


63

FIG. 7 Chromatogram of the pigments from pumpkin seed oil. (A) Thin-layer chromatography: petroleum ether/acetone 80/20 V:V). (B) High-performance liquid chromatography: methanol, acetonitrile, methylene chloride (see Schoefs, 2000a).

used (Schoefs et al., 1995, 1996). The mobile phase used for the separation of hydrophobic molecules is usually made up of organic solvents, except when very polar molecules like glycosyl esters of carotenoid or chlorophyll c are present in the mixture. In this latter case, a polar organic solvent mixed with a small amount of water is recommended (Dufresne et al., 1999; Latasa et al., 2001).

64

B. SCHOEFS

To improve pigment separation, heating the column is sometimes proposed. Vanheukelem et al. (1994) found that carotenoids were optimally separated at 60 8C, whereas chlorophylls and chlorophyll-related molecules were best separated at 30 8C. This procedure is, however, not recommendable because heating can trigger carotenoid isomerization and chlorophyll epimerization and allomerization. These modifications are not detected using usual HPLC methods (Hyva¨rinen and Hynninen, 1999). Therefore, care should be taken to employ proper chromatographic conditions to ensure that pigments do not escape the analysis. To illustrate this possibility, the HPLC elution profiles of pigments from pumpkin seed oil using two RP HPLC methods recommended for the analysis of plant pigments (Ilik et al., 2000; Schoefs et al., 1995) are compared (Figures 7B and 8).

FIG. 8 High-performance liquid chromatogram of the pigments extracted from pumpkin seed oil. Elution program: solvent A: 100% methanol; solvent B: methanol-hexane (4/1 v/v). Linear gradient solvent B from 0% to 100% in 28 minutes and then isocratic elution for 6 minutes.


65

HPLC has also been used to evaluate the purity of commercially available chlorophyllin (Chernomorsky et al., 1997, Schoefs, 2001a, 2002). Although most of the methods used for the separation of photosynthetic pigments only require organic solvents, the elution mixtures necessary to separate more hydrophilic compounds, such as anthocyanins, are usually a mixture of organic and aqueous solvents (Sarni-Manchado et al., 1996). Before analysis of anthocyanins by HPLC, it is recommended to purify the extract on a C18-RP cartridge previously activated with acidified methanol (Hong and Wrolstad, 1990). Anthocyanins and other phenolics are adsorbed onto the phase, whereas sugars, acids, and other water-soluble molecules are washed away with 0.01% aqueous HCl. Addition of ethyl acetate elutes phenolic compounds other than anthocyanins, which are eluted using acidified methanol (0.01% HCl v/v) (Oszmianski and Lee, 1990). Using a similar protocol Takeoka et al. (1997) analyzed the anthocyanin content of seed coat from black bean by HPLC. Three anthocyanins were found: delphinidium 3-glucose, petunidin 3-glucose, and malvidin 3-glucoside. Because the AACYL/AVIS ratio reflects the molar ratio of the cinnamic acid and anthocyanin (Harborne, 1958), the use of a photodiode array detector allows determination of additional information about the acylation or the glycosylation patterns of anthocyanins (Hong and Wrolstad, 1990). For instance, absence of an absorbance peak between 300 and 350 nm in the absorbance spectra of the eluted pigments suggests that none is acylated with aromatic amino acids (Andersen, 1988). HPLC analyses of anthocyanins from radish established that the eight anthocyanins separated were acylated (Giusti and Wrolstad, 1996a). Comparison of the absorption spectra of acylated and nonacylated compounds revealed that acylation shifts the anthocyanin color to the shorter wavelengths (Giusti and Wrolstad, 1996a,b). HPLC elution programs for anthocyanins are constantly being improved, and today one can separate up to 15 anthocyanins within a single 30-minute run (Giusti et al., 2000). One difficulty, but not the least, with the determination and quantification of anthocyanins by HPLC is that there is a general lack of availability of pure anthocyanin standards. Supplementing HPLC with a diode-array detector and fast computers has greatly increased the analytical power of HPLC. Using such detectors, one can follow simultaneously elution on the full UV–Vis range (190–800 nm). This possibility guarantees that each pigment can be followed at its absorbance maximum, that is, with the maximum sensitivity. 4. Supercritical and subcritical HPLC Supercritical fluid extraction has been suggested as an alternative method for selective one-step isolation of carotenoids without degradations (but see Britton et al., 1995). For instance, Favati et al. (1988) isolated b-carotene

66

B. SCHOEFS

and lutein from leaf concentrates at 40 8C and pressure ranging between 29 and 70 MPa. From the analytical point of view, supercritical extraction is compatible with supercritical fluid chromatography because the two techniques can share the mobile phase and some devices, favoring the development of extraction and separation methodologies. Because the elution strength of pure CO2 in respect to the carotenoids is rather weak, it is necessary to add co-solvents to increase the carotenoid solubility in CO2. The solubility parameter theory predicts that the maximum solubility of a compound is reached when the solubility parameter of the solvents equals that of the solute. For instance, b-carotene can be extracted at 43 8C and approximately 70 MPa (Favati et al., 1988). Unfortunately, some equipments do not allow elution at such an elevated pressure. Therefore, new conditions of pressure and temperature should be found using a diagram connecting the variations of pressure to gas densities for different temperatures (Smith, 1988). At lower temperature, densities similar to those reached at higher temperatures can be obtained for lower pressures. These conditions are referred to as subcritical conditions. Using this technique, Ibanez et al. (1998) successfully separated b-carotene from lycopene in less than 10 minutes. The best separation was obtained at 10 8C under a pressure of approximately 35 MPa, with a home-packed capillary column containing deactivated silonal groups with an octamethyl-cyclotetrasiloxane reagent. This column cannot be used at higher temperatures because the structure of the phase is broken at a high temperature, forming O-Si-CH3 bonds with the silica phase. 5. Capillary HPLC Pigments with very similar structures might be difficult to separate using classic RP-C18 HPLC. This is the case of zeaxanthin and lutein. To overcome this difficulty, a particular HPLC method should be applied (Darko et al., 2000). Alternatively a capillary electrophoresis column can be used. This last method was successfully applied to the separation of zeaxanthin and lutein from eye humor (Karlsen et al., 2003). The good separation and the fast elution of the pigments obtained by these authors suggest that capillary electrophoresis is suitable for routine analysis of pigments contained in tiny samples. None of the methods described is entirely suitable for the elucidation of pigment structure. Although the spectral fingerprint is often sufficient to identify the chromophore, it does not contain enough information to determine the complete structure of the pigment (Schoefs, 2000b, 2001b). The missing information can be partly deduced from the chromatographic behavior and from the comparison of the obtained retention data with


67

literature. Other methods such as MS have to be applied to fully characterize the structure of the molecule (Bertrand et al., 2004; Schoefs, 2001a).

D. MASS SPECTROMETRY

Because of their high mass, low volatility, and thermal instability, chlorophylls and carotenoids have long presented special analytical challenges to MS. The use of newer methods, such as chemical ionization, secondary ion MS, fast-atom bombardment, and field-, plasma-, and matrix-assisted laser desorption—for which the necessity of sample vaporization before the ionization is suppressed—opened ways to the molecular ion detection and, thus, to direct molecular weight determination. Unfortunately, these methods produced additional signals in the region of lower masses, reflecting the presence of chlorophyll degradation products, such as 10-OH chlorophyll a (m/z 908) or pheophytin a (m/z 870) and additional signals at m/z 482, m/z 556, and m/z 615. From these results, whether the additional signals reflect molecules present in the sample before the analysis or whether their formation results from pigment degradation during sample preparation or analysis is difficult to determine (Bertrand et al., 2004; Schoefs, 2002). Most recent progress in MS analysis of tetrapyrroles has been obtained with the development of atmospheric ionization methods, that is, atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI). The APCI technique, in combination with reversed HPLC, has been shown to be efficient for detecting chlorophyll a and its nine degradation products on low nanogram levels. The procedure is approximately 1000 times more sensitive than the thermospray ionization (Eckhardt et al., 1991). To demonstrate the potential of electrospray MS in chlorophyll research, ESI. interface was employed with an ion trap mass spectrometer as mass analyzer (Fenn et al., 1989; Hutton and Major, 1995; Schoefs, 2001a). ESI is a mild ionization technology feature that can obtain a high proportion of the chlorophyll a–protonated molecular ion (M þ H)þ, m/z 893.5. It is remarkable that the spectrum was obtained with approximately 2 pmol of the compound. Further structural information has been obtained with the unique MSN capability of the ion trap analyzer, allowing consecutive dissociation of side-chain functional groups from the selected precursor ion (up to eight steps) (Schoefs, 2001a). The MSN procedure is highly selective and enables consecutive cleavage of at least seven functional groups around the porphyrin, providing valuable structural information about the tetrapyrrole. Using MS methods, chlorophyll allomers and their derivatives produced during fruit and vegetable processing (Minguez-Mosquera et al., 1995b) have been isolated and their structure elucidated (Hyva¨rinen and Hynninen, 1999).

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A C4-RP HPLC method coupled to ESI-MS has been described for the analysis of the different components of phycobilisome from the cyanobacterium Synechocystis PCC 6803 (Zolla and Bianchetti, 2001). Although anthocyanins have positive charges at low pH levels and are very soluble in water and alcohol because of their high mass, low volatility, and thermal instability, their high MW, which ranges from a few hundred to a few thousands (Terahara et al., 1990), made their analysis by MS methodologies difficult. As in the case of chlorophyll, the ESI method appears especially suitable for the ionization of this family of labile, nonvolatile polar compounds. Using this method, Giusti et al. (1999a,b, 2000) obtained structural information on a minor anthocyanin component of grape juice and red cabbage. A limitation of MS in the analysis of anthocyanins and alkaloids arises from its inability to differentiate between diastereoisomeric forms of sugars. Therefore, this method cannot provide information on the exact glycosidic substitutions other than the number of carbon atoms and the presence of methyl side-groups in the sugars. To get more information on the structure of these substitutions, it is necessary to use MS-MS technology. When applied to the anthocyanins from grape and red cabbage, typical patterns were obtained (Guisti et al., 2000). The MS-MS resulted in the cleavage of glycosidic bonds only between the flavilium ring and the sugar directly attached to it. In the case of acylated anthocyanins, the fragmentation pattern allows a rough determination of the localization of the acylating group (Giusti et al., 2000). ESI-MS has also been successfully applied to determination of the molecular structure of other non–water-soluble pigments such as the polyphenolic yellow molecules contained in turmeric extracted from the rhizome of Curcuma longa (He et al., 1998). All the methods described are invasive techniques that are time consuming and often expensive. To reduce sampling and analytical costs, and to speed up the analyses, noninvasive analytical procedures have been developed with the aims to characterize the pigment content of samples and, in the case of food products, to estimate the impact of a color change on the visual perception of the product.

V. PIGMENT IDENTIFICATION AND QUANTIFICATION: THE PROBLEM OF STANDARDS In the preceding sections, the different analytical methods that can be used to identify pigments were presented. One crucial point is the calibration of the signal detector. This question requires standards, ideally identical to each


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pigment under consideration. Those standards are not always commercially available and, therefore, should be prepared. Relative to this problem, Schiedt and Liaaen-Jensen (1995) have defined the minimum criteria for the identification of carotenoids: (1) The absorbance spectra in the UV-Vis region, obtained in at least two different solvents, should be in agreement with the chromophore suggested; (2) the chromatographic properties of putative pigment and standard must be identical in TLC (Rf) and HPLC (tR): both compounds should co-elute; and (3) a mass spectrum should be obtained, which allows at least confirmation of the molecular mass. Although such ‘‘rules’’ were not specified for the identification of other natural pigments, similar criteria are suggested. Even when the identification and detector calibration have been performed according to these rules, pigment quantification may not be straightforward. For instance, the amount of pigments in a sample may be apparently higher after a treatment, such as heating, than before! Such a paradox, which was frequently reported in blanched and cooked vegetables or fruits (Stahl and Sies, 1992), has been attributed to the greater extractability of the pigments after cooking. It is believed that heating triggers cell wall rupture, facilitating release of the pigments. A. NONINVASIVE METHODS

The success of noninvasive methodologies will lie in the development of remote and noncontact ‘‘sampling’’ methodologies. Several possibilities have been described (Andrews and Dallin, 2003). In this chapter, only some methodologies using changes in the light properties are developed. B. EVALUATION OF THE COLOR AND ITS PERCEPTION

The visual impression of a colored object mainly depends on the pigments in a sample. This feeling is affected by morphological factors such as the presence of epidermal hair or cuticular waxes and the shape and the orientation of the cells in the epidermis and the subepidermis. In fact, pigments and surface topography act together to selectively absorb, reflect, and refract the incident visible light, which will eventually be sensed by eyes. When light strikes the eye, it is detected by one of the three color sensors of the retina: a red, a green, or a blue one. It is known that the information is not sent as an individual color but as a red/green signal, a yellow/blue signal, or a black/ white signal. The signals, generated at the retina level, are transduced through the optic nerve to the brain and interpreted as color. When all the wavelengths are reflected by an object, the eyes see it as white, whereas when all are absorbed, the object appears black.

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Although it is out of the scope of this review to explain in detail the basis of color measurement (Wyszecki and Stiles, 1987), it is, however, necessary to briefly describe the principle of the color measurement to facilitate the understanding of the results. Each color can be described by a set of three parameters: (1) the hue, which is the dominant shade; (2) the saturation or chroma, which measures how much color is present; and (3) the lightness, which corresponds to the degree of darkness of a particular color. Therefore, for each unique set of illuminants or observed conditions, it is necessary to describe a different hue, saturation, and lightness. By using a standard illuminant and a standard observer, the amount of light reflected by an object can be converted into the hue, saturation, and lightness values. Then a sample can be compared to any standard with these three attributes. In 1976 the Commission Internationale de l’Eclairage (CIE) adopted a standard method of calculating color attributes, known as the CIE Lab Color Space. The lightness coefficient L* ranges from black (0) to white (100), whereas the coordinates a* and b* designate the color on a rectangular-coordinate grid perpendicular to the L* axis. The color at the grid origin is achromatic (i.e., gray; a* ¼ 0, b* ¼ 0). On the horizontal axis, positive and negative a values indicate the hue of redness (positive values) and greenness (negative values), whereas on the vertical axis, b* indicates yellowness (positive values) and blueness (negative values). The hue H* is defined as the arctang (b*/a*). The CIE Lab Color Space method has been used frequently to characterize the in situ color modifications occurring in fish flesh fed with various diets (Akhtar et al., 1999; Bjerkeng et al., 1997), during semolina milling (Borrelli et al., 1999), and anthocyanin production by Perilla cell culture (Aoki et al., 2000). In a study comparing the stability of natural colorants and dyes in gels with different levels of sugar, a good correlation between the sensory and instrumental ranking was found. This indicates that the values of L* and H* are good representatives of the lightness and hue of the gels, respectively. This study also established that the value of L* is solely dependent on the pigment concentration and that the sugar concentration has no influence on the L* value (Calvo and Salvador, 2000). It is necessary to emphasize that no change in color does not necessarily mean that there is no modification in the pigment composition. In fact, there are conflicting reports in the literature on the correlation between color measurement and pigment composition. This is especially true when several pigments have to be monitored (Lancaster et al., 1997). Several mathematical combinations of the parameters have been proposed to predict the pigment modifications occurring during food processing (Gomez et al., 1998; Ma and Shimokawa, 1998; Steet and Tong, 1996). Some authors also use the hue angle (for the definition, see Wyszecki and Stiles, 1987) to characterize color modifications (Akhtar et al., 1999; De Ell


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and Toivonen, 1999). Other methods, based on reflected and scattered light, were used to derive estimated pigment content (Kawashima and Nakatani, 1998) and to predict color (McClements et al., 1998), but they are less popular. C. USE OF CHLOROPHYLL FLUORESCENCE TO ESTIMATE THE FRESHNESS OR THE MATURITY OF PLANTS

The freshness of vegetables can be assessed using chlorophyll fluorescence kinetic measurements, which reflect the photosynthetic activity. It is out of the scope of this chapter to explain in detail the background of the development of this method, but the interested reader is referred to a review by Rohacek and Bartak (1999). In brief, the chlorophyll fluorescence yield depends on the reduction state of the photosystem II primary acceptor, QA. When all the QA acceptors are oxidized, for instance, after a period of complete darkness, the fluorescence yield, denoted F0, is minimal. In contrast, when all the QA acceptors are reduced, for example, during a saturating light pulse, the level of fluorescence, denoted Fm, is maximal. The state of the photosynthetic apparatus can be estimated using the Fv/Fm ratio with Fv ¼ Fm F0. Healthy plants exhibit an Fv/Fm ratio of approximately 0.8. When the photosynthetic process is inactivated, such as in senescent plants or in cold-stored plants, the ratio decreases (De Ell and Toivonen, 1999). Tian et al. (1996) showed that the ratio could be a sensitive indicator of responses of broccoli to hot water treatment, even before visual changes were noted. Other parameters can be obtained from chlorophyll fluorescence kinetic to characterize the physiological state of the plants (Rohacek and Bartak, 1999). With the development of the CCD camera, we now can visualize the global fluorescence emission of an organ such as a leaf or a fruit. This new dimension of measurement differs from the classic one in that it is not focused on one cell or a small group of cells, allowing fluorescence imaging (Buschmann et al., 2000). This technology has many potential applications. For instance, Nebdal et al. (2000) developed a strategy to measure the decrease of chlorophyll fluorescence from the lemon peel during ripening. On the basis of fluorescence data, they defined robust parameters allowing the prediction of damage at the lemon surface before the appearance of visible signs. This methodology can also be applied to trace chlorophyll in highly colored tissues such as redishing tomatoes. The development of chlorophyll fluorescence imaging in fields other than agriculture is also very promising, and it has already been used to follow pollution and even to visualize the stress caused at the leaf surface by an insect walking on the leaf (Bown et al., 2002).

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Other spectroscopic methodologies such as infrared and Raman spectroscopies should be developed to allow the determination of the presence of molecules in intact samples (Chenery and Bowring, 2003). VI. EXTRACTION AND ANALYSIS: CASE BY CASE It may be useful to have a set of analytical protocols specially formatted for particular samples. However, the complete extraction of pigments often requires several steps and may use a mixture of several solvents. This is especially true when the sample contains pigments of different polarities or pigments that are present in a complex matrix (Britton et al., 1995; Taungbodhitham et al., 1998). In rare cases, pigments can be extracted in a one-step process (Kopecky et al., 2000). The aim of the following discussion is not to make an exhaustive list of the extraction and analytical methods described in literature, but more to provide representative selected examples, which can then be used as reference (Table VI). A. PIGMENTS FROM INTACT FRUITS AND VEGETABLES

1. Chlorophyll and carotenoid molecules Chlorophyll and carotenoid molecules can be extracted with acetone using a homogenizer. To extract lycopene from tomatoes, Thompson et al. (2000) used a mixture of organic solvents composed of hexane, acetone, and ethanol (50:25:25 v/v/v). The extraction should be repeated until no color is observed. All fractions are pooled in a separatory funnel and partitioned with diethyl ether. NaCl solution (e.g., 10% w/v) can be added to help the transfer of the pigments in the nonpolar phase. The water phase is discarded and the pigmented layer is dried with anhydrous Na2SO4 (e.g., 2% w/v). When necessary, the pigments can be saponified with KOH-methanol (10% w/v) and left for some time with periodic shaking. The pigments are then transferred to diethyl ether by adding distilled water, and the organic phase is washed with water until neutrality is reached. Finally, the aqueous phase is removed. To dry the pigments, the organic phase is filtered on a bed of anhydrous Na2SO4. a. Determination of the pigments. TLC: Separation can be carried out on silica gel GF60 plates in a presaturated chamber. The elution mixture is composed of petroleum ether (bp: 65–95 8C), acetone and diethylamine (10:4:1 v/v/v) (Hornero-Meńdez and Minguez-Mosquera, 1998).

TABLE VI SUMMARY OF THE METHODS USED TO EXTRACT AND ANALYZE PIGMENTS FROM VARIOUS SOURCES

Pigment analyzed

Extraction

Fruits and vegetables

Carotenoids, tetrapyrroles Anthocyanins Anthocyanins Saffron Carotenoids Carotenoids

Various organic solvents

Seeds Flower Juices and drinks Oil

Pasta Vegetable puree Cheese Textile Pharmaceutical products Fish flesh Urine and plasma Humor eyes

Tetrapyrroles Carotenoids Carotenoids Tetrapyrroles Carotenoids Saffron Pigmentcyclodextrin complexes Astaxanthin Carotenoids Carotenoids

Pretreatment before analysis

Separation methods TLC, HPLC

Acetone, chloroform 1% HCl in methanol Cold water Tetrahydrofuran Dilution in an organic solvent

þ þ (þ)

HPLC HPLC HPLC TLC, HPLC Open column, TLC, HPLC

Various organic solvents Acetone

þ

Open column, TLC, HPLC TLC, HPLC

Water/tetrahydrofuran Water/pyridine Polar solvents for dilution

þ

HPLC HPLC HPLC

Various organic solvents Ethyl acetate/methanol —

þ þ


Products

HPLC HPLC HPLC

Note: HPLC, high-performance liquid chromatography; TLC, thin-layer chromatography.

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HPLC: Many HPLC methods have been described in the literature (Hornero-Meńdez and Minguez-Mosquera, 1998; Schoefs, 2002, 2003, 2004; Thompson et al., 2000). When the carotenoid composition is complex, as in passiflora fruit, which contains more than 10 carotenoids, it might be necessary to first separate the different groups of carotenoids. This can be done using an open column packed with alumina. Fraction 1, which contains carotenes and epoxi-carotenoids, is eluted with petroleum ether; fraction 2, which is composed of monohydroxy- and keto-carotenoids, is eluted with 70–90% diethyl ether in petroleum ether; and fraction 3, made up of polyhydroxy-carotenoids, is eluted with 0–30% ethanol in ether. The pigments contained in individual fractions can be further separated using particular TLC or HPLC methods (Mercadante et al., 1998). 2. Anthocyanins During extraction with acetone and chloroform (Giusti and Wrolstad, 1996a,b), it is advisable to prepurify the extract on acidic methanol-activated C18 minicolumns (Hong and Wrolstad, 1990) before analysis by HPLC. B. ANTHOCYANINS IN SEEDS

There are different methods to separate anthocyanins from seeds. Comparing the extraction efficiency of the different solvents, it appears that the best is 1% HCl in methanol (Gao and Mazza, 1996; Gu¨renc, and Karaali, 2000). C. WATER-SOLUBLE CAROTENOIDS FROM FLOWER TISSUES

An illustration is given by the stigma of saffron that are shaken during 30 minutes in cold water (4 8C). The solution is heated to 60 8C for 30 minutes and allowed to stand in the dark for 24 hours. The clear supernatant contains the pigments (Tsatsaroni et al., 1998). D. PIGMENTS FROM JUICES AND DRINKS

1. Carotenoids The juice is first mixed with tetrahydrofuran. The nonpolar pigments are transferred in petroleum ether. The water phase is discarded and the organic phase is washed with water. This is repeated until the water phase becomes colorless. The ether extracts are pooled and dried on anhydrous sodium sulfate. When the juice contains pulp, it is advisable to remove it


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by centrifugation. The isolated pulp is dispersed in distilled water and extracted as explained earlier (Arena et al., 2000). 2. Determination of the pigments Carotenoids can be analyzed using the methods previously described. When the extract is enriched in chlorophyll derivatives, the HPLC method described by Canjura et al. (1999) may be used. It consists of an isocratic elution (9 minutes) of a hexane/isopropanol (98.3:1.7 v/v) mixture, followed by a linear gradient (2 minutes) to yield a mobile phase with equal parts of hexane/isopropanol (98.3:1.7 v/v) and hexane/isopropanol (98.3/3.0 v/v). This mixture is held during 7 minutes to increase the hexane/isopropanol (98.3/3.0) fraction to 100%. E. PIGMENTS FROM OIL

Pigments can be separated from the uncolored molecules of oils using a silica gel column, TLC (Ellsworth, 1971), or HPLC (Goulson and Warthesen, 1999) before further analysis. Alternatively, the oil can be directly injected in an HPLC (Schoefs, 2000a). For a separate analysis of the fatty acid moiety, the pigments should be saponified with KOH-methanol (10% w/v) and left 10 minutes with periodic shaking at room temperature. Then the pigments are transferred by addition of water to diethyl ether. The aqueous phase is reextracted twice. The pigmented diethyl ether phase is dried and stored. F. CAROTENOIDS FROM RAW AND COOKED PASTA

The raw pasta is comminuted in a blender, followed by cylinder milling to achieve a powder passing a 40-mesh screen. The pigments are then extracted using a ternary mixture of hexane/acetone/ether (10:7:6 v/v/v), followed by a cold saponification for 1 hour (Pereira et al., 1999). The pigments are then transferred to petroleum ether. A similar procedure can be used to extract pigments from cooked pasta, but acetone is the preferred solvent and the pigments should not be saponified before analysis. A fast analytical method consists of the separation of b-carotene from the other carotenoids using an open column packed with MgO/hyflosupercel (1/2). b-Carotene is eluted with 4% diethyl ether in petroleum ether. The remaining adsorbed pigments are eluted together with acetone, transferred in petroleum ether and separated on TLC (3% methanol in benzene). The presence of carotenoid epoxide is seen by exposing TLC to HCl vapors, which triggers a change in the colored spots from yellow or orange to blue or green (Gross et al., 1972). For the determination of cis-isomers, a C30-RP-HPLC

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column is recommended (Schmitz et al., 1995). Alternatively, a calcium hydroxide column can be used. G. PIGMENTS FROM VEGETABLE PUREES

The pigments from vegetable purees are extracted with acetone and the solution is filtered through a filter paper. Then the pigments are dried over a bed of anhydrous Na2SO4. For tetrapyrroles, a method separating chlorophyll a and chlorophyll b from their degradation products is recommended. H. CAROTENOIDS FROM CHEESE

Cheese mostly contains the carotenoids bixin and norbixin. To extract these pigments, cheese is crushed in a solution of water/tetrahydrofuran (1:1 v/v) and centrifuged. The supernatant is biphasic: The aqueous phase contains norbixin, and the organic phase contains mainly bixin. Norbixin and bixin can be separated by HPLC using a linear gradient starting with 100% of water/acetone (3:2 v/v) to 100% of water/acetonitrile (1:4 v/v) in 15 minutes (Tricard et al., 1998). I. PIGMENTS FROM TEXTILE

Textiles may be colored with natural colorants such as saffron or curcumin. Saffron pigments can be extracted from cotton and wool fibers using a pyridine-water mixture (25/75 v:v). This method is, however, not able to extract curcumin from the same fibers (Tsatsaroni et al., 1998). J. PIGMENTS IN PHARMACEUTICAL PRODUCTS: THE PIGMENT–CYCLODEXTRIN COMPLEXES

A general way used to increase the stability of natural colorants is to complex them with cyclodextrins. Cyclodextrins are oligosaccharides composed of six, seven, or eight glucopyranose units (referred to as a-, b-, and g-cyclodextrin, respectively). These molecules have a doughnut shape with an interior cavity. The cavity is hydrophobic, whereas the external surface is hydrophilic. The unique property of cyclodextrin is to form an inclusion complex by incorporation of guest molecules in the cavity. As the external surface is hydrophilic, the guest molecule–cyclodextrin complex can be easily solubilized. For instance, the polyphenolic yellow pigments curcumin and


77

curcuminoids are nonsoluble in water unless they are complexed to cyclodextrin (Tonnesen et al., 2002). Stability studies have shown that the pigment–cyclodextrin complexes may be more stable than the noncomplexed pigments. For example, the stability of the red color of ketchup versus temperature and decomposition of rutin in water is improved by complexation with cyclodextrins. Because the diameter of a chlorophyll molecule exceeds the size of cyclodextrins, chlorophyll cannot be completely included into the cavity of cyclodextrins. Nevertheless, chlorophyll–cyclodextrin complexes show improved water solubility in cold and warm waters and color stability to light (Sato et al., 2000). A detailed study on the benefit for the solubility and stability of curcumin brought by cyclodextrin has indicated that substituents on the cyclodextrin molecule have little influence on photodegradation, whereas the interior of the cavity may be of some importance. Besides the positive effects of cyclodextrins, these compounds may trigger pigment fading, as observed with b-cyclodextrin-callistephin (Dangles et al., 1992a,b). This effect is, however, not observed with a-cyclodextrins (Lewis et al., 1995). Therefore, the type of cyclodextrin should be carefully chosen. Because the cyclodextrin–pigment complexes are much more soluble than the noncomplexed pigments, particular chromatographic methods should be used for their analysis (Cserhati and Forgacs, 2003). For instance, to analyze curcumin–cyclodextrin complexes, Tonnesen et al. (2002) used a C18-RP-HPLC (Nova Pack column). The mobile phase was citric acid and acetonitrile (60/40 v:v). K. PIGMENTS FROM ANIMAL TISSUES

A section on pigments in animal tissues may appear at first glance out of the scope of a chapter on plant pigments. However, when one looks carefully at the biochemistry of animal cells, it becomes clear that almost no plant pigment can be synthesized by these cells. On the other hand, some of them cross the intestine and are found in cells (Parkinson and Brown, 1981). This is especially the case for carotenoid molecules, which are eventually transformed. 1. Carotenoids from fish flesh Extraction: Akhtar et al. (1999) recommend to finely grind the sample with magnesium sulfate. The pigments are then extracted by stirring the sample/salt mixture with acetone for 1 hour, followed by paper filtration. The water is removed by passing the extract through anhydrous Na2SO4. Analysis: It is known that salmonids accumulate red carotenoids in their flesh. Several HPLC methods can be used to separate the red

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carotenoids (Schoefs et al., 2001). For instance, the method of Kopecky et al. (2000) separates astaxanthin from its esters and from canthaxanthin. The esterification state of the consumed astaxanthin, either free or esterified, is not important because only free astaxanthin is deposited in the flesh (Storebakken and No, 1992; Torrissen et al., 1989). On the other hand, the ingested astaxanthin stereoisomer molecules cannot be epimerized by the fish and are deposited unchanged in the same extent in the flesh (Foss, 1984). Thus, the composition of astaxanthin in flesh reflects the diastereoisomer composition of the meal feed. Consequently, the diastereoisomer composition of the fish flesh can be used to determine the origin of the fish. In fact, in the natural environment, the astaxanthin stereoisomer composition varies largely among taxa. For instance, Haematococcus pluvialis (green alga) accumulated the (3S,30 S)-isomer of astaxanthin, whereas crustaceans contain a highly racemized mixture of astaxanthin isomers (Matsuno et al., 1984; Rendstro¨m et al., 1981a,b). In fish farms, farmers can supplement the feed with either shrimp meal, with the red yeast Phaffia rhodozyma, or with Haematococcus pluvialis (Johnson and An, 1991; Nelis and De Leenheer, 1991). Because Phaffia accumulates exclusively (3R,30 R)-astaxanthin, the separation of the stereoisomers can be used to determine whether a fish is grown on a farm. The HPLC methods proposed by Aas et al. (1987) can be used to distinguish between astaxanthin stereoisomers in a fish flesh. These methods can separate the stereoisomers after transformation to their respective dicamphenate esters. However, to avoid coelution between 7,8-didehydro- and 7,8,70 ,80 -tetradihydro-astaxanthin and (3S,30 S)-cis isomers of astaxanthin, the astaxanthin fraction should be purified by TLC on alkaline plates (Bjo¨mland et al., 1989). A Pirckle covalent l-leucine column is preferable, especially for routine analyses, since the separation is achieved in 18 minutes (Turujman, 1993). In addition, this method is able to separate the cis and trans isomers. Noninvasive methods were also used to establish whether the absorption and deposition of astaxanthin are affected by feeding the fish with or without astaxanthin supplementation in all or alternated meals (Wathne et al., 1998). The redness a* of the salmon fed with astaxanthin in alternating meals was higher than that for fish fed with a mixture of colored and uncolored meals. A variation in the lightness L* was also observed. Wathne et al. (1998) found that the total carotenoid concentration in the flesh could be estimated using the value a*. However, the equation cannot be applied for the determination of the carotenoid content in other studies because the value of the coefficients depended on the fish physiological state and on the fish taxon.


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2. Pigments from urine and plasma One can find several methods in the literature for the detection of polyphenolic compounds and carotenoids in urine and plasma. The early methods used a aqueous-based matrix only (Heath et al., 2003). The major disadvantage of these methods was the absence of protein extraction before HPLC analysis. Heath et al. (2003) overcomes this difficulty by treating the samples with an extracting reagent composed of ethyl acetate and methanol (190/10 v:v) to transfer the carotenoid molecules from the aqueous medium to the organic phase, which can then be analyzed by a C18-RP-HPLC (Water Symmetry Shield column protected by a guard column) using a mobile phase composed mostly with organic solvents (actonitrile/methanol/water/acetic acid 41/23/36/1 v:v:v:v). The six major carotenoids found in human plasma are b-carotene, lycopene, b-cryptoxanthin, lutein, a-carotene, and zeaxanthin (Ford et al., 2003; Karlsen et al., 2003). A C18-RP-HPLC method was also applied to the detection of curcumin in plasma and urine from humans after absorption of pure curcumin (Heath et al., 2003). As food, curcumin is mostly taken from curry powders and is usually in a mixture with other yellow pigments such as demethoxycurcumin, bisdemethoxycurcumin, and dihydrocurcumin (Tang and Eisenbrand, 1992). To separate these compounds, He et al. (1998) used a C18-RP-HPLC similar to that described by Heath et al. (2003). In the absence of standards, the identification of the eluted molecules cannot be performed on the sole basis of the UV-Vis spectrum, and ESI-MS was used (He et al., 1998). Sharpless et al. (1996) used a C30-RP-HPLC column to differentiate the various carotenoid isomers contained in human plasma. 3. Pigments from eyes Carotenoids lutein and zeaxanthin have a protective role in the eyes (Krinsky et al., 2003). Sampling and handling of minute biological samples containing degradable pigments is an extremely challenging task. Emphasis should be placed on the sampling technique to prevent and avoid degradations during sampling and sample processing. This is especially the case with the detection of carotenoid molecules in eye humor as the starting volume is as small as 4 ml. To remove the proteins from the sample before analysis by HPLC, the sample is treated with a mixture composed of isopropanol and BHT (2,6-di-tert-butyl-4-methylphenol), which precipitates the proteins. The supernatant, which contains the carotenoid molecules, can be analyzed by HPLC or by capillary liquid chromatography equipped with a C30 column (mobile phase: water/methanol 2/98 v:v). Using this method, Yeum

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et al. (1995) found that human aqueous humor is much simpler than that of plasma because it contains only lutein and zeaxanthin.

VII. FUTURE TRENDS Research on plant pigments is fascinating because it involves many methods and a deep knowledge in several fields. Plant pigment studies should be continued because they may procure several immediate interesting impressions, as well as commercial and health advantages. For these advantages, one should have the most powerful methodologies to analyze pigment composition of samples. Most of the available methods are destructive, time consuming, and cost consuming. Therefore, the development of rapid and nondestructive methods should progress further. In establishing these tools, it is necessary to correlate physical parameters such as firmness, soluble solids, dry matter, and color with physiological data (e.g., respiration and photosynthesis). These methods could involve those techniques such as delayed luminescence (Triglia et al., 1998), infrared spectroscopies (Bakeev, 2003; Chenery and Bowring, 2003; Slaughter et al., 1996), NMRS (Borsari et al., 2002), noncontact sampling (Andrews and Dallin, 2003), and others, which in the past were more restricted to basic research.

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CHITIN, CHITOSAN, AND CO-PRODUCTS: CHEMISTRY, PRODUCTION, APPLICATIONS, AND HEALTH EFFECTS FEREIDOON SHAHIDI*,{ AND REEM ABUZAYTOUN{ *Department of Biochemistry { Department of Biology Memorial University of Newfoundland St. John’s, Canada

I. Introduction II. Chemistry A. Structure, Physical, and Chemical Properties of Chitin and Chitosan B. Chemical Reactions of Chitin and Chitosan C. Solubility D. Preparation of Chitin and Chitosan E. Preparation of Chitin and Chitosan Oligomers F. Chitinases, Chitosanases, and Their Functions III. Applications of Chitin, Chitosan, and Their Oligomers A. Medical Applications B. Food Applications of Chitin, Chitosan, and Their Oligomers C. Agricultural Applications D. Industrial Applications IV. Safety and Regulatory Status References

I. INTRODUCTION Chitin is derived from the Greek word chiton, which means a coat of nail. It is the major component of the exoskeleton of invertebrates, crustaceans, insects, and the cell wall of fungi and yeast (Knorr, 1984; Lower, 1984; Tan et al., 1996) in which chitin acts as a supportive and protective component. Chitin is the second most plentiful natural polymer on earth after cellulose (Brzeski, 1987; Ornum, 1992). At least 10 gigatons (1 1013 kg) of chitin ADVANCES IN FOOD AND NUTRITION RESEARCH VOL 49 ISSN: 1043-4526


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is produced and hydrolyzed each year in the biosphere (Muzzarelli, 1999). Chitin, poly-(164)-N-acetyl-d-glucosamine, is a cellulose-like biopolymer found in a wide range of products in nature (Shahidi et al., 1999). It was discovered in 1811 by a French scientist Henri Braconnot, who isolated it from mushroom (Winterowd and Sandford, 1995). In 1823 Odier found the same compound in the cuticles of insects (Muzzarelli, 1977; Winterowd and Sandford, 1995). The biosynthesis of chitin occurs in the membrane-bound protein complex chitin synthase. In arthropod outerskeleton and most of the fungi, uridine diphosphate-N-acetyl-d-glucosamine is polymerized into chitin by chitin synthase (EC 2.4.1.16) (Hirano, 1996). Chitosan, a copolymer of d-glucosamine and N-acetyl-d-glucosamine with b-(164) linkage, is obtained by alkaline or enzymatic deacetylation of chitin and is an abundant polymeric product in nature. Chitosan was first discovered by Rouget in 1859 when he heated chitin to the boiling point in a concentrated KOH solution (Dunn et al., 1997). Chitosan is found in different morphological forms such as a primary, unorganized structure, crystalline and semicrystalline forms. For different reasons, especially problems of environmental toxicity, these two biopolymers are considered interesting substances for producing polymers (Sorlier et al., 2001). Because of their unique structures, they possess high biological and mechanical properties as they are biorenewable, biodegradable, and biofunctional (Hirano et al., 2000). Two methods, namely chemical and enzymatic, are known for preparation of chitin, chitosan and their oligomers, with different degrees of deacetylation, polymerization, and molecular weight (MW). Chitin, chitosan, and their oligomers can be produced chemically using concentrated HCl followed by column chromatographic fractionation (Jeon et al., 2000). Three methods are known for modification of the process of isolation of chitin and chitosan oligomers (Jeon et al., 2000). These are acetolysis, fluorohydrolysis, and sonolysis. Meanwhile, chitin and chitosan oligomers can be prepared through microbiological and fungal treatments (enzymatic preparation). Certain enzymes are involved in the degradation of chitin and chitosan such as chitinases and chitosanases, respectively, and the process is environmentally friendly. Chitin, chitosan, and their oligomers have different applications such as medical uses as a wound-healing agent, dietary uses as hypocholesterolemic agents, antitumor, and antiulcer agents, and as a coating of artificial parts of the body such as leg, tooth, and arm, among others. They may also be used in food preservation such as for seafoods (Shahidi et al., 1999) and fruits (EL-Ghaouth et al., 1992c), as well as for acidity adjustment (Scheruhn et al., 1999), and as antibacterial and antifungal agents (Shahidi et al., 1999). The following section presents the chemistry and applications of chitin, chitosan and their oligomers.

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II. CHEMISTRY A. STRUCTURE, PHYSICAL, AND CHEMICAL PROPERTIES OF CHITIN AND CHITOSAN

Chitin is a high-molecular-weight polymer of N-acetyl-d-glucosamine (NAG) units linked by b-d (164) bonds, having 1000–3000 units (Lower, 1984). The chemical structure of chitin is the same as that of cellulose, with the hydroxyl group at position C2 replaced by an acetamido group (Figure 1). Chitin can be deacetylated to produce chitosan, which is soluble in dilute acidic solutions (Table I) and is highly viscous when dissolved; this

FIG. 1

The chemical structures of chitin, chitosan, and cellulose.

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TABLE I COMMON SOLVENTS FOR CHITIN AND CHITOSAN

Compound

Solvent

Chitin

Dimethylformamide/lithium chloride; diethylformamide/lithium chloride; hexafluoroisoacetone sesquihydrate; hexafluoroisopropanol (Capozza, 1975); 1,2-chloroethanol/sulfuric acid (Austin, 1975) Formic acid/water; acetic acid/water; lactic acid/water; glutamic acid/water, etc.

Chitosan

makes it distinctly different from chitin (Jeon et al., 2000; Shahidi et al., 1999). Chitosan has many useful applications in different fields, mainly because of the presence of amino groups at the C2 position, and because of the primary and secondary hydroxyl groups at the C3 and C6 positions, respectively (Furusaki et al., 1996; Kurita, 1986). Chitosan is the simplest and least expensive derivative of chitin (Ornum, 1992). Unlike with most polysaccharides, the presence of positively charged amino groups repeatedly placed along the chitosan polymer chain allows the molecule to bind to negatively charged surfaces via ionic or hydrogen bonding (Muzzarelli, 1973; Rha, 1984; Shahidi, 1995). The term chitosan is favored when the nitrogen content of the molecule is higher than 7% by weight (Muzzarelli, 1985) and the degree of deacetylation (DD) is more than 70% (Li et al., 1992). B. CHEMICAL REACTIONS OF CHITIN AND CHITOSAN

1. Neutralization Chitin and chitosan are weak bases. They go through the usual neutralization reactions of basic compounds. The non-bonding pair of electrons on the primary amino group of the glucosamine unit accepts a proton, and thus becomes positively charged (Winterowd and Sandford, 1995). 2. Nucleophilic reactions Chitosan is a strong nucleophile because of the presence of a nonbonding pair of electrons on its primary amino groups. Chitosan reacts readily with most aldehydes to produce imines (Kurita et al., 1988). It also reacts with acyl chlorides to form the corresponding acylated derivatives (Hirano et al., 1976) (Figure 2).


FIG. 2

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Reactions of chitosan with an aldehyde or acyl chloride.

3. Acid- or base-assisted hydrolysis Chitin and chitosan are labile to acid- or alkaline-assisted degradation. Under acidic or basic conditions, acetic acid can be freed as N-acetyl groups at the C2 positions of N-acetyl glucosamine units are released, leaving behind primary amine groups (Muzzarelli, 1977). In addition, presence of the primary amino groups in chitosan presents further potentials for modification of the molecule such as N-acylation, N-alkylation, and N-alkylidenation. Acidic conditions also cause some degree of depolymerization as degradation of the b-glycosidic bonds occurs (Madhaven and Ramachandran, 1974). Depolymerization under basic conditions occurs, but to a lesser extent, and chitosan can be hydrolyzed using nitrous acid (Allan and Peyron, 1989) (Figure 3). 4. Sulfation reactions Although most reactions of chitosan involve the primary amino groups, it is possible to selectively derivatize the hydroxyl groups. This can be done by protecting the amino groups via reaction with formic or acetic acid to produce a polysaccharide formate or acetate salt. The chitosan formate or acetate salt may then be reacted with an electrophile (Muzzarelli, 1977).

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FIG. 3

Hydrolysis of chitosan with aqueous nitrous acid.

The hydroxyl group at the C6 position is more active than that at the C3 position and is, therefore, derivatized favorably. Figure 4 shows the sulfation as a common example of this type of reaction. When this type of reaction (Figure 4) is conducted on chitin, the end products behave like heparin, an anti–blood-clotting agent (Wolform and Shen-Han, 1959). 5. Heavy metal complexes Chitin and chitosan are capable of forming complexes with many of the transition metals and some of those from groups three through seven of the periodic table (Muzzarelli, 1973). The heavy metal complexes are supposed to form as a result of donation of a nonbonding pair of electrons on the nitrogen and/or on the oxygen of the hydroxyl groups to a heavy metal ion. Cupric ion appears to form one of the strongest metal complexes with chitosan in the solid state (Domard, 1987; Kentaro et al., 1986; McKay et al., 1986). Koshijima et al. (1973) observed that ferrous ion has the ability of binding to chitosan. Under experimental conditions (100 mg of powdered chitosan mixed with a solution of ferrous nitrate [25 mg] in 50 ml of water, at 30 8C, and reaction time of 74 hours), about 28% of the ferrous ions were complexed with chitosan. The rate of formation and stability of these


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FIG. 4 Sulfation of chitosan in the presence of an organic acid such as formic or acetic acid.

complexes is affected by the presence of counterions, competing heavy metal ions, temperature, and pH of the solution, as well as particle size, crystallinity, and the degree of N-acetylation of chitin and chitosan (Winterowd and Sanford, 1995). C. SOLUBILITY

The most remarkable difference between chitin and chitosan is their solubility characteristics. There are few solvents for chitin, whereas almost all aqueous acids dissolve chitosan (Table I). Most solvents used for dissolution of chitin are toxic (Table I) and hence cannot be used in food processing applications. Nonetheless, when chitin is ground to a fine mesh, it could be used to increase viscosity of liquids. Solvents for chitosan are generally safe to consume, allowing the formation of solutions that are appropriate for gel production. Thus, chitosan is better matched to the viscosity of foods (Winterowd and Sandford, 1995). The solubility characteristics of chitosan are governed mostly by the extent of N-acetylation, the distribution of acetyl groups, the pH, and the ionic strength (Anthonsen et al., 1993). The amino group in chitosan has a pKa value of 6.2 to 7.0, which makes chitosan a

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polyelectrolyte at low pH values (Claesson and Ninham, 1992). It was reported that chitin molecules with a highly deacetylated chitin with a number of acetylated chitin joining each other in a block, and then a number of units with free amino groups in a block could produce products that are soluble in water (Kristbergsson et al., 2003). However, no details are available in the nonproprietary literature in this regard. The solubility problems associated with chitin and chitosan may limit their use in physiological and functional foods. The intestines of most animals lack the ability to produce chitinase and chitosanase. These two enzymes have the ability to hydrolyze chitin and chitosan, respectively. Therefore, they will be excreted unchanged in the feces. On the other hand, chitin and chitosan oligomers are considered to have more physiological functions because they are water soluble and their solutions are less viscous, so they are readily absorbed in the human intestine (Jeon et al., 2000). 1. Molecular weight of chitin and chitosan The MW of natural chitin is normally higher than 1,000,000 Da and that of commercially available chitosan is around 100,000–1,200,000 Da (Li et al., 1992; Lower, 1984). Numerous forces during commercial production may influence the MW of chitosan. Factors such as high temperature (>280 8C thermal degradation of chitosan occurs and the polymer chains quickly break down), dissolved oxygen concentration, and shear stress may cause these changes to occur (Li et al., 1992; Muzzarelli, 1977). D. PREPARATION OF CHITIN AND CHITOSAN

Two hydrolytic methods were reported to prepare chitin and chitosan. These are acid hydrolysis (chemical treatment) and enzymatic hydrolysis (Shahidi et al., 1999). 1. Chemical treatment The normal procedure for preparation of chitin from crustacean shells includes the use of NaOH, HCl, and decoloring agents to remove the remaining proteins, calcium, and color, respectively. The chitin that is produced can then be deacetylated with sodium hydroxide to produce chitosan (Tsai et al., 2002). Jaworska and Konieczna (2001) reported that chitosan can be prepared via chemical means using concentrated hydroxides (40–50%) at high temperatures (100–130 8C). Oh et al. (2001) reported that deacetylation proceeds rapidly during the first hour of treatment with 50% NaOH at 100 8C and the product is 68%


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deacetylated. This is followed by a slower step, and by the end of 5 hour, about 78% deacetylation is achieved. Increasing time does not deacetylate chitin any further, but it lowers the MW of the product. The resulting chitosans from both chemical and enzymatic methods are different with respect to their DD, distribution of acetyl groups, chain length, and conformational structure of chitin and chitosan molecules. These factors together will affect the characteristics of chitin and chitosan. Optimum conditions for chitosan pretreatment (deacetylation by 45% alkali solution for 1 hour) were studied by investigating the coagulation efficiencies of chitosan prepared under different conditions (Huang et al., 2000). The procedure involved crushing crab shells to a powder and isolating the chitin. The next step included deacetylation of chitin using NaOH at 100 8C (Kamil et al., 2002) followed by rinsing the product several times with deionized water to reach a pH level of 7, and finally drying at 80 8C for 48 hours. The resulting chitosan was dissolved in different concentrations of acetic acid and hydrochloric acid, stirring at room temperature, until it was completely dissolved. It was noticed that as the concentration of acid increased, the viscosity of dissolved chitosan coagulants decreased due to binding of positively charged chitosan to the negatively charged acid anion in the solution. The conformation of chitosan polymers changes and becomes more compact in the acidic solution and thus lowers the viscosity of the solution; the best solution was obtained at a pH level of 2. a. Effect of degree of acetylation, degree of deacetylation, and molecular weight of chitosan and its activity. Chitin can be found with varying degrees of acetylation (DA), ranging from fully acetylated to totally deacetylated. The degree of acetylation is very important because of its effects on physical properties of chitin. For example, as the degree of acetylation increases, the degree of solubility in different solvents decreases. Oh et al. (2001) reported that the DD of chitosan is affected by the concentration of alkali, temperature, reaction time, previous treatment of chitin, particle size, and chitin concentration. Heux et al. (2000) found that after partial deacetylation ( hydroxyl ( OH) > amino group ( NH2). They also concluded that colloidal chitin binds with chitosanase as a competitive inhibitor because of their similar structure. a. Chitin deacetylase. Chitin deacetylases are found in several fungi and insects (Tsigos et al., 2000). They hydrolyze chitin by acting on N-acetamido bonds to produce chitosan. The use of these enzymes offers a controlled, non-degradable process leading to the production of novel well-defined chitosan oligomers and polymers (Tsigos et al., 2000). Win and Stevens (2001) studied shrimp chitin as a substrate for chitin deacetylase for fungus Absidia coerulea. The chitin was exposed to physical and chemical treatment to obtain a better accessibility of its acetyl groups for deacetylation process. Chitin was exposed to physical treatments such as heating, sonicating, and grinding. None of these treatments increased the enzymatic deacetylation efficiency. Partially deacetylated shrimp chitin was produced before and after grinding of chitin. Chitin was treated with 50% NaOH for 20 hours at 40 8C and subsequently ground to various particle sizes (75–250 mm). Another sample of chitin was ground first and then treated with 50% NaOH. The results showed higher enzyme activity with decreasing particle size in both treatments. Chitin was exposed to different chemicals such as succinic anhydride, hot phosphoric acid, and 2-propanol. Solvent effect studies revealed that the best results were obtained when chitin was dissolved in CaCl2.2H2O and methanol. A good substrate for chitin deacetylase from A. coerulea was obtained when mixing superfine chitin with formic acid, followed by neutralization. The MW of chitin was reduced from 2 105 to 1.2 104 kDa. The pretreated chitin could be deacetylated by enzyme up to a DD of 80–90% (Win and Stevens, 2001). Furthermore, this process was scaled up without the use of NaOH using a four-step procedure. Ten steps were involved and led to production of superfine chitin by (1) dissolving chitin in a solution of CaCl2.2H2O and methanol as a solvent; (2) dissolving the superfine chitin in 18% formic acid; (3) adjusting the pH to an optimum level for the enzyme; and (4) enzymatic deacetylation with Absidia CDA.

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III. APPLICATIONS OF CHITIN, CHITOSAN, AND THEIR OLIGOMERS A. MEDICAL APPLICATIONS

1. Wound-healing agent Chitin and chitosan have been tested to have both material and biological properties that might be beneficial to enhance wound repair. In addition, both of them have great influence on different stages of wound healing in experimental animal models (Howling et al., 2001). Howling et al. (2001) found that chitosan polymers can interact with and modulate the migration behavior of neutrophils and macrophages modifying subsequent repair process such as fibroplastica and reepithelialization. It was reported that chitin and chitosan have both stimulatory and inhibitory effects on proliferation of human dermal fibroblasts and keratinocytes (Howling et al., 2001). They also have an enhancing effect on the survival function of osteoblasts and chondrocytes (Lahiji et al., 2000). The procedure for promoting wound healing by chitosan was tested as follows: Chitosan was coated onto plastic coverslips that had been filled into 24-well plates. Human osteoblasts and articular chondrocytes were seeded on either uncoated or chitosan coated coverslips. The incubation temperature of the culture was 37 8C, 5% CO2 for 7 days. By using a fluorescent molecular probe, cell viability was judged. Reverse transcriptase polymerase chain reaction and immunocytochemistry were used for phenotyping expression of osteoblasts and chondrocytes. The results showed that the chondrocytes and osteoblasts appeared spherical and refractile of the chitosan-coated coverslips, whereas 90% of the cells on the plastic coverslips were elongated and spindle shaped after this period of incubation (Lahiji et al., 2000). It was reported that the wound recovering material composed of polyelectrolytic complexes of chitosan and sulfonated chitosan that speeded up wound healing and afforded a good-looking skin surface (Lahiji et al., 2000). Chitosan has the ability to promote wound healing; this is due to the tendency to form polyelectrolyte complexes with polyanion heparin, which possesses anticoagulant and angiogenic properties (Lahiji et al., 2000). By forming a complex with heparin and acting to lengthen the half-life of growth factors, chitosan supports tissue growth and helps wound healing. Other studies have examined the effect of chitin and chitosan samples with different deacetylation levels and polymer chain length on the proliferation of human dermal fibroblasts in vitro (Howling et al., 2001). It was found that chitosans with a high DD strongly motivated fibroblast proliferation; meanwhile, samples with lower degrees of deacetylation showed less activity.


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Cho et al. (1999) used water-soluble chitin (WSC) prepared at room temperature through depolymerization by ultrasonication after alkaline treatment of chitin. The DD and MW were controlled. Chitin with DD of 8.60%, chitosan with DD of 83.9% and WSC were embedded to the wounded backs of rats after full thickness skin cuts. It was noticed that the WSC had the highest efficiency in recovering strength of the wounded skin due to the hydrophilicity and high biodegradability of WSC that maximized its activity as a wound-healing accelerator. In addition, the arrangement of the collagen fibers in the wound was the same as that of the normal skin. Hirano and Zhang (2000) described the preparation of a novel blend fiber. This fiber is a mixture of cellulose with each of hyaluronate (HA), heparin (Hep), chondroitin 4-sulfate, chondroitin 6-sulfate, and a chitin-chondroitin 6-sulfate blend using an aqueous 10% sulfuric acid solution containing 40–43% ammonium sulfate as a coagulating solution. These blend fibers could be used as covering materials for the wound-healing tissues of animals and plants. Recently, bandages made of chitosan were investigated in the field of military in the new war in Iraq (Brown, 2003). Z-Medica, a small company, supplied these products to the U.S. ground troops in Iraq and Afghanistan (Becker, 2003). These bandages are used immediately after injury to control bleeding and were found to save numerous lives (Becker, 2003). Arterial bleeding was stopped in about a minute when these bandages were applied with pressure to a wound (Brown, 2003). The use of such bandages was approved by the Food and Drug Administration (FDA) in November 2002 (Mientka, 2003). They called it ‘‘shrimp’’ bandage that contains chitosan. This bandage can stop capillary bleeding and stanch severe arterial hemorrhaging (Mientka, 2003). Mientka (2003) reported that chitosan bandages had the ability to stop bleeding at a rate of 600 ml/min. Moreover, there was no sign of allergenicity for use of these bandages in soldiers who were allergic to shrimp (Mientka, 2003). 2. Dietary applications Chitosan may be considered as a dietary supplement for reducing body weight in humans. Industrial production of chitosan tablets (Muzzarelli et al., 2000) and chitosan dietary fibers (Hughes, 2002) has occurred. Furthermore, Schiller et al. (2001) reported that a rapidly soluble chitosan (LipoSan Ultra that has a higher density and solubility than chitosan itself ) facilitated weight loss and reduced body fat. This effect was due to the fact that this chitosan was able to prevent dietary fat absorption in overweight and mildly obese individuals that consumed a high-fat diet.

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a. Chitosan tablets. Although the reactivity of chitosan toward lipids is not clear, it is claimed that chitosan, because of its cationic nature, binds to appropriate bile and fatty acids and brings about their excretion (Muzzarelli et al., 2000). This claimed efficacy of chitosan in reducing the body weight, hypercholesterolemia, and hypertension stimulated production of chitosan tablets. Muzzarelli et al. (2000) studied the capacity of chitin, chitosan, N-lauryl chitosan, and N-dimethylaminopropyl chitosan on sequestering steroids. They reported that chitin might be more effective in holding olive oil and enriching the retained oil fraction with steroids sequestering than chitosan. In addition, chitin derivatives were able to distinguish between different lipids. These results put into question the need for high cationity for sequestering cholesterol. The use of chitosan monomer glucosamine sulfate for joint building is also commonplace. b. Chitosan dietary fibers. Dietary fibers have many health advantages such as lowering low-density-lipoprotein (LDL) cholesterol levels and hence reduce heart disease and lower the risk of colon cancer. Moreover, dietary fibers are involved in weight loss. They increase the feeling of fullness (satiety) after meals and slow the sugar absorption from the gut (as brush buries), balancing blood sugar and insulin levels (Hughes, 2002). Chitosan is considered a dietary supplement, fat-blocking fiber that can be used in weight management by slowing sugar absorption (Hughes, 2002). The fat-absorbing mechanism of chitosan has been explained by Hughes (2002); the chitosan with its positively charged amino groups ( NHþ 3 ) is attracted to the anionic carboxyl groups of fatty acids and bile acids forming films passing through the digestive system undigested (Hughes, 2002; Ylitalo et al., 2002). Different results concerning the role of chitosans in weight loss have been recorded. One study proved the ability of chitosan in reducing weight without controlled diets (Hughes, 2002). Others suggested that the effect of chitosan could be related to a calorie-restricted diet. An Italian clinical study used chitosan supplements with a low-calorie diet to achieve weight loss (Hughes, 2002). Chitosans have also been used to prevent body weight increase in animals (Hughes, 2002). Meanwhile, negative results were recorded regarding chitosan effectiveness in this field (Hughes, 2002). During a high-fat diet and chitosan supplementation, no increase in fecal fat content was noticed (i.e., chitosan had no effect on fat absorption) (Hughes, 2002). Some researchers do not recommend the use of chitosan in the diet of individuals who are allergic to crustaceans (Ylitalo et al., 2002).


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3. Antihypercholesterolemic agent Chitosan has been reported to cause significant hypocholesterolemic activity in different experimental animals (Hirano et al., 1990; Sugano et al., 1978, 1980; Ylitalo et al., 2002). Sugano et al. (1988) noted that chitosan oligomers did not exhibit this effect. The studies were carried out on rat groups fed on a diet rich in cholesterol to find the effect of chitosan hydrolysates with different MWs and viscosity on the hypocholesterolemic activity. The lower the MW of chitosan, the better its cholesterol-lowering potential. The mechanism of antihypercholesterolemic activity of chitosan has been described by Ylitalo et al. (2002). In the stomach, and because of the acidic condition, the ( NH2) groups of chitosan accept protons (Hþ) to form positively charged amino groups ( NHþ 3 ). Consequently chitosan becomes a soluble salt in the presence of hydrochloric acid (HCl). Fats, fatty acids (oleic, linoleic, palmitic, stearic, and linolenic acids), and other lipids, as well as bile acids, due to their negative charge (X-COO ), attach themselves strongly to the positively charged amino groups ( NHþ 3 ) of chitosan. This binding might inhibit their absorption and recycling from the intestine to the liver. However, this interruption of enterohepatic circulation of cholic acid and other bile acids can lead to an increase in the biosynthesis of cholic acid from cholesterol in the liver. The cholesterol content of liver cells is thus decreased, which may lead to activation of LDL-receptor expression and could further increase LDL uptake via LDL receptors in the liver (Ylitalo, 2002). Shahidi et al. (1999) reported that production of dietary cookies, potato chips, and noodles enriched with chitosan is commonplace in certain countries. The products enriched with chitosan have high hypocholesterolemic effects. In addition, vinegar products containing chitosan are produced and sold in Japan because of their cholesterol-lowering ability (Shahidi et al., 1999). 4. Antitumor activity Suzuki (1996) reported that chitin and chitosan oligomers can act as inhibitors of growth tumor cells via their immunoenhancing effects. Suzuki et al. (1985) found that chitin oligomers from (GlcNAc)4 to (GlcNAc)7 have strong attracting responses to peritoneal exudate cells in BALB/c mice. However, chitooligosaccharides from (GlcN)2 to (GlcN)6 did not exhibit such an effect. With regard to hexamers, both (GlcNAc)6 and (GlcN)6 were reported to process growth inhibitory effects against allogenic and syngeneic mouse

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systems (Suzuki et al., 1986a). These results indicate that the effect was not by direct cytocidal action on tumor cells but was host mediated. 5. Antiulcer agent Ito et al. (2000) reported that chitosans with different MWs had ulcerhealing actions. The effects of low MW (LMW) chitosan, high MW (HMW) chitosan, and chitin on ethanol-induced gastric mucosal injury and on the healing of acetic acid–induced gastric ulcers in rats were compared. It was found that orally administrated LMW chitosan could prevent ethanol-induced gastric mucosal injury. Repeated oral administration of LMW chitosan in a dose-dependent manner accelerated the gastric ulcer healing. The effects of HMW chitosan and chitin on gastric ulcer healing were less than those of LMW chitosan. 6. A coating agent for prosthetic articles (artificial parts of the body) Muzzarelli et al. (2000) described a method for coating prosthetic articles with chitosan-oxychitin. Plates of titanium (Ti) and its alloys were plasma sprayed with hydroxyapatite and glass layers, and subsequently a chitosan coat was deposited on the plasma-sprayed layers using chitosan acetate. These layers were treated with 6-oxychitin to form a polyelectrolytic complex. This complex was optionally contacted with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide at 4 8C for 2 hours to form amide links between the two polysaccharides, or acetylation with acetic anhydride in methanol to obtain a chitin film. In all cases, the modified coats were insoluble, uniformly flat, and smooth. Prosthetic materials coated with chitosan– oxychitin were capable of provoking colonization by cells, osteogenesis, and osteointegration. There were two main reasons behind the selection of chitosan-oxychitin– coated orthopedic plates. First, chitosan enhances the integration of the implant, and second, chitosan stimulates bone regeneration. B. FOOD APPLICATIONS OF CHITIN, CHITOSAN, AND THEIR OLIGOMERS

New applications of chitin and its oligomers led to more than 50 patents in the 1930s and the early 1940s. However, commercialization of these products was hindered by inadequate manufacturing services and competition from synthetic polymers (Averbach, 1981). However, after the 1970s, industrial use of chitin and its oligomers increased (Kaye, 1985). Furthermore, improvement in research and small-scale production of chitin and chitosan


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has extended the number and varieties of potential applications of chitinous materials. In addition, environmental problems and cost for disposal of shellfish processing discards have increased the urgency for development of environmentally safe alternatives for numerous plastic or polymeric products (Ashford et al., 1976; Berkeley, 1979; Shahidi and Synowiecki, 1991). Some food applications of chitin, chitosan, and their oligomers are summarized in Table II. Chitin, chitosan, and their derivatives offer a wide range of applications including bioconversion for the production of value-added food products, preservation of foods from microbial spoilage, formation of biodegradable films, recovery of waste material from food processing discards, purification of water, and clarification and deacidification of fruit juices (Shahidi et al., 1999) (Table II). 1. Antimicrobial activity Chitin, chitosan, and their derivatives have antimicrobial activity against bacteria, yeast, and fungi (Yalpani et al., 1992). The exact mechanism of antimicrobial action of chitin and chitosan and their derivatives remains illusive, but different mechanisms have been proposed (Shahidi et al., 1999). Chitosan has the ability to produce phytoalexins, cell wall phenols and callose (Tsai et al., 2002). Chitosan is considered to be a soluble chelating agent and activator due to the presence of a positive charge on the C-2 of its glucosamine monomer at pH values less than 6. This characteristic gives it a higher antimicrobial activity than chitin (Chen et al., 1998). A leakage of proteineous and intercellular components occurs due to the interaction between the positively charged chitosan molecules and the negatively charged microbial cell membranes (Chen et al., 1998; Papineau et al., 1991; Sudharashan et al., 1992; Young et al., 1982). This is affected by the MW of chitosan (Tsai et al., 2002). Being a chelating agent, chitosan has the ability to selectively bind trace metals, which prevents production of toxins and microbial growth (Cuero et al., 1991). Chitosan is also an activator for several defense processes in the host tissue (EL-Ghaouth et al., 1992b), having the ability to bind water and inhibit various enzymes (Young et al., 1982). Tsai et al. (2002) studied the effects of DD and preparation methods for chitin and chitosan on their antimicrobial activity. It was found that chemically (ch-chitin) and microbiologically prepared chitin (MO-chitin) could undergo further chemical deacetylation to produce chitosan with different DDs. However, MO-chitin that was deacetylated by various proteases had no antimicrobial activity (Tsai et al., 2002). However, for chitosan, as the DD increased, its antimicrobial effect on bacteria increased, even to a greater extent than that on fungi.

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TABLE II FOOD APPLICATIONS OF CHITIN, CHITOSAN, AND THEIR DERIVATIVES

Area of application

Examples

Antimicrobial agent

Bactericidal Fungicidal Measure of mold contamination in agricultural commodities Controlled moisture transfer between food and the surrounding environment Controlled release of antimicrobial substances Controlled release of antioxidants Controlled release of nutrients, flavors, and drugs Reduction of oxygen partial pressure Controlled rate of respiration Temperature control Controlled enzymatic browning in fruits Reverse osmosis membranes Clarification and deacidification of fruit juices Natural flavor extender Texture adjusting agent Emulsifying agent Food mimetic Thickening and stabilizing agent Color stabilization Dietary fiber Hypocholesterolemic agent Livestock and fish feed additive Reduction of lipid absorption Production of single cell protein Antigastritis agent Infant food ingredient Recovery of metal ions, pesticides, phenols, and PCBs Removal of dyes, radioisotopes Seed and fruit covering Fertilizer Fungicide Skin and hair products Artificial skin Surgical structures Contact lens Treating major burns Blood dialysis membranes Artificial blood vesicles Enzyme immobilization Encapsulation of nutraceuticals Chromatography Analytical reagent Synthetic fiber Chitosan-coated paper Manufacturing material for fiber Film and sponges

Edible film

Food additive

Nutrition

Water treatment Agriculture

Cosmetics Biomedical and pharmaceutical materials

Others


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Genetically, chitosan can enter the nuclei of a microorganism and bind with DNA. This binding inhibits the mRNA and protein synthesis (Hadwiger et al., 1985; Sudharashan et al., 1992). a. Antibacterial agent. Several studies have examined the effect of concentration of chitosan to complete inactivation of certain types of bacteria (Shahidi et al., 1999). Wang (1992) observed that a much higher concentration of chitosan (1–1.5%) was required for complete inactivation of Staphylococcus aureus after 2 days of incubation at pH 5.5 or 6.5 in the medium. Furthermore, Chang et al. (1989) found that chitosan concentrations of 0.005 or more were sufficient to elicit complete inactivation of S. aureus. This was in accordance with the findings of Darmadji and Izumimoto (1994) on the effect of chitosan in meat preservation. Simpson et al. (1997) studied the effect of different concentrations of chitosan on the growth of different cultures of bacteria on raw shrimp. They found that Bacillus cereus required chitosan concentrations of 0.02% for antibacterial effect, whereas Escherichia coli and Proteus vulgaris exhibited minimal growth at 0.005% and growth was inhibited at 0.0075% more. Numerous studies have shown the effect of different concentrations of chitosan on E. coli growth. Complete inhibition was observed by Wang (1992) after 2 days incubation with 0.5% or 1% chitosan at a pH level of 5.5. It was also reported that if chitosan concentration increased by about 1% in the broth, it could afford complete inactivation. However, Darmadji and Izumimoto (1994) reported that growth inhibition of E. coli required a 0.1% chitosan concentration. Simpson et al. (1997) found that only 0.0075% chitosan was required to inhibit the growth of the same species. The observed variations are possibly due to the existing differences in the degree of acetylation of chitosans employed (Shahidi et al., 1999). Iida et al. (1987) and Nishimura et al. (1984) have reported that if chitin is partially deacetylated, especially at 70%, it has the ability to stimulate nonspecific host resistance against E. coli and Sendai virus infection in mice. Meanwhile, chitin and chitosan have the ability to increase the number of mouse peritoneal exudate cells that generate reactive oxygen intermediates and then display candidacidal activities (Suzuki et al., 1984). Suzuki et al. (1986) reported that chitin hexamer (GlcNAc)6 had a strong candidacidal activity. b. Antifungal agent. Chitosan decreased the in vitro proliferation of many fungi with the exception of Zygomycetes (Allan and Hadwiger, 1979). Chitosan acts as an antifungal agent via the formation of gas-permeable coats, interference with fungal growth, and stimulation of many defense processes, including accumulation of chitinases, production of proteinase

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inhibitors, and lignifications and stimulation of callous synthesis (Bai et al., 1988; EL-Ghaouth et al., 1992a). EL-Ghaouth et al. (1992b) studied the antifungal influence of chitosan on in vitro growth of common postharvest fungal pathogens in strawberry fruits. These authors found that chitosan with 7.2% NH2 significantly decreased the eradial proliferation of Botrytis cinerea and Rhizopus stolonife, with a greater impact at higher concentrations. In an in vivo study, EL-Ghaouth et al. (1992b) observed signs of infection in chitosan-covered fruits after 5 days of storage at 13 8C compared with 1 day for the control treatment. After 14 days of storage, chitosan coating (at 15 mg/ml) decreased spoilage of strawberries induced by the same fungi by more than 60% and noticed that coated fruits were grown normally and did not show any clear sign of phytotoxicity. Fang et al. (1994) reported the preservative influence of chitosan on lowsugar candied Kumquat (fruit). Chitosan (at 0.1–5 mg/ml) inhibited the growth of Aspergillus niger, whereas chitosan at less than 2 mg/ml was ineffective in inhibiting mold proliferation and aflatoxin synthesis by Aspergillus parasitius. Cuero et al. (1991) conducted a similar study and observed that N-carboxymethylchitosan decreased aflatoxin formation in A. flavus and A. parasitius by more than 90% while fungal growth was decreased to less than half. Furthermore, Savage and Savage (1994) reported that apples coated with chitosan reduced the rate of molds occurring on them over a period of 12 weeks. Cheah and Page (1997) found that chitosan coating of carrot with a 2% or 4% chitosan solution considerably reduced their Sclerotinia rotting from 28% to 88%. El-Katatny et al. (2001) reported the characterization of a chitinase and endo-b-1,3-glucanase from Trichoderma harzianum strain Rifai T24. These two enzymes are the key enzymes in the lyses of cell walls during their mycoparasitic effect against plant diseases caused by fungi, including S. rolfsii. The chitinase from T. harzianum was purified in two steps using ammonium sulfate precipitation followed by hydrolytic interaction chromatography. SDS-PAGE showed that the enzyme exhibited a single band at 43 kDa. The b-1,3-glucanase was purified in three steps using ammonium sulfate precipitation, hydrophobic interaction chromatography, and gel filtration, showing a molecular mass of 74 kDa. The optimum pH level of both enzymes was 4.5. The optimum temperature of the T24 chitinase was 40 8C, whereas the optimum temperature of b-1,3-glucanase was 50– 60 8C. Both the T. harzianum T24 chitinase and b-1,3-glucanase were strongly inhibited by Hg2þ, suggesting that sulfhydryl groups are involved in the catalytic reaction (El-Katatny et al., 2001). The pure form of the two enzymes from T. harziaanum T24 inhibited the growth of S. rolfsii in an additive manner showing a promising effective dose of 50% (ED50) at a 2.7-g/ml concentration.


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A mixture of these enzymes, which showed thermostability and low effective dose (ED50) values against S. rolfsii, may be considered a potential tool for controlling of plant’s pathogens. i. Chitin as a measure of mold contamination of agriculture commodities and food products. Chemically, when chitin in the cell wall of fungi is determined, it has a benefit that indicates the total content of mycelium based on chitin (Bishop et al., 1982; Donald and Mirocha, 1977). Bishop et al. (1982) used chitin to evaluate the presence of mold in tomato products, ketchup, paste, and puree. They noticed variations in chitin content among different fungal species depending on the cultural age and growth conditions; values ranged from 5.7 to 43 mg of glucosamine per milligram of dry weight. 2. Preservation of foods Chitosan can be used for food preservation to inhibit the growth of spoilage microorganisms in mayonnaise (Oh et al., 2001). By treating crude chitin with various NaOH concentrations (45%, 50%, 55%, and 60% w/v), four kinds of chitosans were prepared (chitosan-45, -50, -55, and -60, respectively) (Ylitalo et al., 2002). Four species of food spoilage microorganisms were treated by chitosans to examine their effects on microbial activity (Oh et al., 2001). These were Lactobacillus plantarum, Lactobacillus fructivorans, Serratia liquefaciens, and Zygosacchaomyces bailii. Chitosan had a biocidal effect; the number of cells grown was clearly reduced. It has been found that after an extended phase, some strains recovered and started to grow. As the concentration of chitosan increased, the activities of these strains increased. It was noticed that chitosan-50 had the most efficiency against L. fructivorans; meanwhile, the inhibition of L. plantarum growth was mostly by chitosan-55 and no difference was found among the chitosans against S. liquefaciens and Z. bailii. Thus, for mayonnaise, during its storage at 25 8C, the addition of chitosan decreased the viable cell counts of L. fructivorans and Z. bailii. a. Preservation of seafood and meats. Tsai et al. (2002) found that 1% chitosan solution with a high DD can be added to certain kinds of fish to increase their shelf life from 5 to 9 days. Kamil et al. (2002) showed that chitosans prepared from snow crab shells had different viscosities, closely correlated to the time of deacetylation. Different viscosity chitosans (14, 57, 360 cP chitosans) were prepared and used to examine the impact of chitosan covering on fish quality during refrigerated storage. This study showed the potential of chitosan as a protective coating for herring and

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cod in decreasing or preventing moisture loss, lipid oxidation, and microbial growth. Cod samples coated with 57 and 360 cP chitosans demonstrated a considerably ( p < .05) lower relative moisture loss in comparison with those of uncoated samples and those coated with 14 cP chitosan throughout the storage period. Furthermore, crab chitosan showed a medium to high viscosity-dependent protective effect in both fish model systems. In general, 360 cP chitosan exhibited a better preservative effect in comparison with 57 and 14 cP chitosans in both systems at 4 1 8C. In this study, the effects of different viscosity chitosans on lipid oxidation of cooked comminuted fish were also tested. The chitosan showed antioxidant activity in cooked comminuted fish model system, as revealed in their peroxide value and content of 2-thiobarbituric acid–reactive substances (TBARS), which were reduced in a concentration-dependent manner. However, the antioxidant efficiency of relatively high viscosity chitosan in both model systems was lower than that of the low-viscosity chitosan at the same concentration. The mechanism of action appears to be a result of chelation of metal ions found in fish muscle proteins, gas exchange adjustment (particularly oxygen) between fish meat and the surrounding environment, and the bactericidal effect of chitosan itself. Thus, chitosan as an edible coating would enhance the quality of seafoods during storage (Jeon et al., 2002). Weist and Karel (1992) studied the effect of using chitosan powders in a fluorescence sensor for monitoring lipid oxidation in muscle foods. The efficiency of chitosan powders was explained to be due to the ability of the primary amino groups of chitosan to form a stable fluorosphere with volatile aldehydes such as malonaldehyde, which is derived from the breakdown of fats (Weist and Karel, 1992). On the other hand, chitosan was used to improve the preservation of vacuum-packaged processed meats stored under refrigerated conditions (Quattar et al., 2000). These authors used chitosan matrix to produce antimicrobial films by adding acetic or propionic acid (with or without addition of lauric acid or cinnamaldehyde) to this matrix (Quattar et al., 2000). These films were applied to bologna, regular cooked ham, or pastrami. The amounts of antimicrobial agents found in the chitosan matrix were measured several times during storage. It was found that within the first 48 hours of application, propionic acid was nearly completely released from the chitosan matrix. Whereas, 2–22% of acetic acid remained in chitosan even after 168 hours of storage. With regard to the presence of lauric acid, but not cinnamaldehyde, it was found that the release of acetic acid was reduced significantly and more limited to bologna than to ham or pastrami (Quattar et al., 2000). In another study, Li et al. (1996) found that addition of 3000 ppm of N-carboxymethylchitosan to cooked pork was sufficient to prevent the oxidative rancidity of the product.


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b. Fruit preservative agent. Chitin, chitosan, and their derivatives have been used as food wraps, because of their film-forming properties. The chitosan film controls moisture movement between food and the surrounding environment. The presence of the film results in a decreased rate of metabolism, respiration, and high impermeability to certain substances such as fats and oils, in addition to temperature. These would lead to a delay in ripening of fruits (Shahidi et al., 1999). The coating of fruits with chitosan delays the rate of ripening and the occurrence of decay in tomato (EL-Ghaouth et al., 1992c), bell pepper, cucumber (EL-Ghaouth 1991b), and strawberries (EL-Ghaouth et al., 1991a). The control of disease in fruits by chitosan could account for the antifungal activity of chitosan and its capacity to provoke defense enzymes and phytoalexins in the plant tissue or a combination of both (EL-Ghaouth et al., 1992a). Chitosan (7.2% NH2) inhibited the growth of postharvest pathogens, namely B. cinerea, A. alternata, C. gloesporioides, and R. stolonifer (EL-Ghaouth et al., 1992b). Among the fungi examined, R. stolonifer was least affected by chitosan (EL-Ghaouth et al., 1992b). Whereas chitin did not influence the growth of any of the fungi tested, the growth delay of fungi provoked by chitosan increased with increasing DD (EL-Ghaouth et al., 1992a). The inhibitory effects of chitosan correlated with its cationic nature and the size of the polymers. Moreover, the importance of cationic groups and the length of the polymer chain was demonstrated by the low fungicidal activity displayed by N,O-carboxymethyl chitosan compared to that of chitosan, and by the improved activity of chitosan with increasing levels of deacetylation. The antifungal effects may, in part, account for the capacity of chitosan to enhance membrane permeability and result in cellular leakage. Three mechanisms may be involved in the action of chitosan as an antifungal agent in the preservation of postharvest crops. First, the treatment of potato with chitosan, challenged with Erwinia carotovora (the soft rot pathogen of potato), showed a declined count of bacteria and tissue maceration, thus resulting in an increase in cell viability. Second, potatoes treated with chitosan showed an inhibition in bacterial reproduction and secretion of pectic enzymes (produced by pathogenic bacteria capable to attack the plant tissue). The third mechanism of chitosan action is by controlling the pH level. The pathogenic bacteria that cause decay of crops after harvesting such as potatoes secrete macerating enzymes (negatively charged proteins), leading to an outflow of protons and cations from the cell wall of the plant and hence a pH increase in the cell and cell wall. c. Acidity adjusting agent. Chitosan could be used for deacidification of fruit juices because chitosan salts carry a strong positive charge that could interact with proteins and hence act as dehazing agents in fruit juice (Shahidi

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et al., 1999). Scheruhn et al. (1999) reported that treating coffee drinks with chitosan increased the pH level and decreased the acid content of the coffee drinks because of the acid-binding properties of chitosan in the coffee. This treatment depended on the concentration of chitosan and the acid content of the drinks, as well as the raw material of chitosan and its processing (Scheruhn et al., 1999). d. Antioxidant agent. Muscle food products containing a high content of unsaturated lipids are highly labile to off-flavor and rancidity development. Warmed-over flavor (WOF) is developed in cooked poultry and uncured meat upon storage, resulting in the loss of attractive meaty flavor (Shahidi et al., 1999). Darmadji and Izumimoto (1994) noticed that 1% chitosan added to meat resulted in a decline of 70% in the 2-thiobarbituric acid (TBA) values after 3 days of storage at 4 8C. St. Angelo and Vercellotti (1989) reported that N-carboxymethylchitosan was effective in preventing the formation of WOF over a broad range of temperatures. Moreover, ground beef treated with 5000 ppm of N-carboxymethylchitosan exhibited 93% inhibition of TBA values and a 99% reduction in hexanal content. Furthermore, Shahidi et al. (1999) reported that N,O-carboxymethylchitosan (NOCC) and its lactate, acetate, and pyrrolidine carboxylate salts were effective in controlling the oxidation and off-flavor development in cooked meat stored for 9 days at refrigerated temperature. The mechanism by which this inhibition occurred was thought to be related to the chelation of free iron, which was released from hemoproteins during heat processing. These results were further confirmed by Li et al. (1996) who found that addition of 3000 ppm N-carboxymethylchitosan to cooked pork was sufficient for inhibiting the development of oxidative rancidity in the product. C. AGRICULTURAL APPLICATIONS

1. Retention of nutrients and nutrient cycle in the soil Smither-kopperl (2001) found that chitin exhibits several functions, including retention of nutrients, in the soil. Chitin contributes to their cycling of nutrients such as nitrogen. When chitin decomposes, it produces ammonia, which takes part in the nitrogen cycle. Furthermore, chitin is a main constituent in geochemical recycling of both carbon and nitrogen. Fungi, arthropods, and nematodes are the major contributor of chitin in the soil. Among these, the fungi provide the largest amount of chitin in the soil (6–12% of the chitin biomass, which is in the range 500–5000 kg/ha). In another study, Kokalis-Burelle (2001) reported that chitin contributes significantly to soil enrichment. It was found that chitin could control plant


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pathogens and pathogenic nematodes and provoke the development of host plant resistance against these pathogens. Chitin led to an increase in microorganism population; this sharp increase could shift and prompt their action as anti-plant pathogens in two ways. First, the microorganism may act as parasite for plant pathogens. Second, they can kill or inhibit these pathogens through production of toxins or metabolites or enzymes. Furthermore, the increase in microorganism numbers increases the number of nonparasitic nematodes, which results in a decline in the number of pathogenic nematodes. D. INDUSTRIAL APPLICATIONS

1. Purification of water There is an increasing demand for treatment of industrial wastewater before their use or disposal because of the environmental and health difficulties associated with heavy metals and pesticides and their deposit through the food chain (Shahidi et al., 1999). Traditional methods for the elimination of heavy metals from industrial wastewater may be inefficient or costly, particularly when metals are present at low concentrations (Deans and Dixon, 1992; Volesky, 1987). Recovering of metal ions from discards can be achieved using a chelation ion exchange process. Biopolymers, such as chitin and chitosan, have the ability to lower the concentration of transition metal ions to parts per billion levels. These biopolymers should be ecologically safe and commercially available and bear a number of functional groups, such as hydroxyl and amino groups in their backbones (Deans and Dixon, 1992). Chitosan can be used as a means for treatment of wastewater because it has a good sorption capacity (Jeuniaux, 1986). In Japan, chitin and chitosan have been used for water purification because of their ability to complex metal ions via their amino groups (Simpson et al., 1994). Chitosan powder and dried films of it have free amino groups above the pKa of their NH2 groups. Therefore, chitosan powder and dried films have potential use in complexing metal ions (Tirmizi et al., 1996). The U.S. Environmental Protection Agency (USEPA) has approved the use of commercially available chitosan for wastewater treatment up to a maximum level of 10 mg/L (Knorr, 1984). Muzzarelli et al. (1989) demonstrated the efficiency of cross-linked N-carboxymethylchitosan in removing lead and cadmium from drinking water. Micera et al. (1986) showed that chitosan has a high binding capacity with metals such as copper and vanadium. Deans and Dixon (1992) observed that unfunctionalized chitosan was efficient in eliminating Cu2þ, but not Pb2þ. Thome and Daele (1986) examined the ability of chitosan to remove polychlorinated biphenyls (PCBs) from polluted stream water. The authors

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showed that chitosan was highly effective, compared to activated charcoal, for purification of PCB-polluted water. Use of chitosan for purification of potable water is also in practice. IV. SAFETY AND REGULATORY STATUS Chitosan has many industrial, agricultural, pharmaceutical, and cosmetic applications. Consequently, safety and toxicological studies have been performed on chitosan to address issues related to its regulatory status. Rao and Sharma (1997) reported no toxicity for 2% chitosan solution in acetic acid, when applied on punctured bleeding capillaries in mice, rabbits, and guinea pigs. These researchers further observed that eye irritation tests in rabbits and skin irritation tests in guinea pigs did not produce any toxic effect due to chitosans. Similar results were obtained by Mou et al. (2003), who reported no obvious toxic reaction using a mixture of polylactic acid and chitin as a basic scaffold material in tissue engineering. Chitosan received the ‘‘Generally Recognized as Safe’’ (GRAS) status by the FDA in the United States in 1983 for use as animal feed component; its use in pet food was also reported by Shepherd et al. (1997). The use of chitosan for purification of potable water was approved by the USEPA, up to a maximum concentration of 10 mg/L (Knorr, 1986). In 1992, Japan’s health department approved the use of chitin and its derivatives as functional food ingredients. Based on its definition of functional foods, chitin and chitosans possess most of the required attributes related to enhancement of immunity, prevention of illness, delaying of aging, recovery for illness, and control of biorhythm (Subasinghe, 1999). Thus, the use of chitosan in foods such as potato chips has been in commercial practice for some time. Therefore, regulatory status of chitosan varies from country to country and its use in food requires further studies to address issues of concern (Lenz and Hamilton, 2004). REFERENCES Allan, C.R. and Hadwiger, L.A. 1979. The fungicidal effect of chitosan on fungi of varying cell wall composition. Exp. Mycol. 3, 285–287. Allan, G.G. and Peyron, M. 1989. The kinetics of the depolymerization of chitosan by nitrous acid. In ‘‘Chitin and Chitosan: Sources, Chemistry, Biochemistry, Physical Properties, and Applications’’ (G. Skjak-Braek, T. Anthonsen, and D. Sandford, eds), pp. 443–466. Elsevier Applied Science, New York. Anthonsen, M.W., Varum, K.M., and Smidsrod, O. 1993. Solution properties of chitosans: Conformation and chain stiffness of chitosans with different degrees of N-acetylation. Carbohydr. Polym. 22, 193–201. Ashford, N.A., Hattis, D.B., Murray, A.E., and Seo, K. 1976. Industrial applications of chitin and chitin derivatives. Inter. Ocean 76, 1160–1170.


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A REVIEW OF THE APPLICATION OF SOURDOUGH TECHNOLOGY TO WHEAT BREADS CHARMAINE I. CLARKE*,{ AND ELKE K. ARENDT* *Department of Food and Nutritional Sciences National University of Ireland Cork, Ireland { National Food Biotechnology Centre National University of Ireland Cork, Ireland

I. Introduction A. History of Sourdough Technology B. Use of Sourdough in Rye Products C. Use of Sourdough in Wheat Products II. Microflora of Sourdough A. Sources of Lactic Acid Bacteria for Sourdough B. Classification of Sourdoughs III. Positive Effects of Sourdough on Wheat Bread Quality A. Nutritional Quality B. Microbiological Spoilage C. Flavor D. Bread Characteristics E. Staling IV. Understanding the Technological Functionality of Sourdough Application A. Primary Effects of Acidification B. Secondary Effects of Acidification C. Proteolysis during Sourdough Fermentation V. Effect of Sourdough Incorporation on Bread Dough Structure A. Interaction between Sourdough and Dough Additives B. Use of Lactic Acid Bacteria Metabolites to Replace Additives VI. Conclusion References



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I. INTRODUCTION A. HISTORY OF SOURDOUGH TECHNOLOGY

Bread in its many forms is one of the most staple foods consumed by humans (Cauvain, 1998a). The art and craft of bread making existed at the outset of recorded history and predates it, as documented by excavations undertaken in many parts of the world (Spicer, 1975). Depictions of the activities involved in the baking of bread have been found in tomb paintings from ancient Egypt, and one of the most detailed accounts of baking dates back to the reign of Seti I (1303–1290 b.c.) (Ezzamel, 1997). The purpose of bread making is to present cereal flours to the consumer in an attractive, palatable, and digestible form (Chamberlain, 1975). The earliest breads were likely unleavened or flat (Quail, 1996), but the first major technical innovation was the introduction of leavening, which yielded breads of superior palatability (Chamberlain, 1975). Early dough fermentation would probably have relied on a mixture of naturally occurring yeasts and lactic acid bacteria (Oura et al., 1982; Williams and Pullen, 1998). The underlying functionality of such an adventitious microbial population is that a dough formed by the addition of water to ground cereals will with time be fermented by the microorganisms naturally present to become a sourdough characterized by acid taste, aroma, and increased volume due to gas formation (Hammes and Ga¨nzle, 1998). The use of the sourdough process as a form of leavening is one of the oldest biotechnological processes in food production (Ro¨cken and Voysey, 1995). To facilitate continuous production, one could save a portion of ripe sourdough dough to seed subsequent doughs, a process that continued into the nineteenth century (Williams and Pullen, 1998). In addition to the yeasts naturally present on the cereal grains, brewers’ yeast was often added to enhance the fermentation process (Oura et al., 1982; Ro¨cken and Voysey, 1995; Williams and Pullen, 1998), but the sourdough procedure predominated in bread making until specially prepared baker’s yeast became available in the nineteenth century (Pederson, 1971). B. USE OF SOURDOUGH IN RYE PRODUCTS

The availability of baker’s yeast has not eliminated the use of sourdoughs in rye bread making in which a reduction in pH is necessary to achieve suitability for baking (Hammes and Ga¨nzle, 1998; Oura et al., 1982; Salovaara, 1998). This results from the inability of rye doughs to form a gluten network, which in wheat doughs provides the water-binding and gasretaining properties. In rye, these functions are taken over by pentosans, whose solubility and swelling increase with a decrease in pH (Hammes and

TO WHEAT BREADS SOURDOUGH TECHNOLOGY

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Ga¨nzle, 1998). Sour conditions also partially inactivate the increased enzyme activity in rye flour, particularly amylase activity (Seibel and Bru¨mmer, 1991). This is an important aspect, because the starch in rye gelatinizes at a relatively low temperature, 55–70 8C, which coincides with the temperature range for maximal a-amylase activity (Cauvain, 1998b). An excessive amount of a-amylase in rye flour produces not only a sticky crumb, but at higher levels, a very open grain, a reduction in loaf volume, and in some instances, cavitation of the loaf (Reed, 1966). The acidification also exerts positive effects on the structure of starch granules, leading to increased water-binding capacity (Hammes and Ga¨nzle, 1998). Acidification of rye doughs improves their physical properties by making them more elastic and extensible and confers the acid flavor notes so characteristic of rye breads. C. USE OF SOURDOUGH IN WHEAT PRODUCTS

Whereas sourdough is an essential ingredient for ensuring baking properties of doughs containing more than 20% rye flour, its addition to wheat doughs remains optional (Ro¨cken, 1996). However, a vast array of traditional products rely on the use of sourdough fermentation to yield baked goods with particular quality characteristics. Some examples include the wellknown Italian products associated with Christmas, Panettone, which originated in Milan (Sugihara, 1977), and Pandoro originally from Verona (Zorzanello and Sugihara, 1982) or their counterpart, Colomba, which is traditionally associated with Easter (Sugihara, 1977). San Francisco sourdough French breads (Kline et al., 1970) and soda crackers (Sugihara, 1985) are other examples of wheat products that rely on the process of souring. The same process is also used in the production of a number of flat breads, a typical example of which is the Egyptian baladi bread (Qarooni, 1996). Further to these traditional varieties of baked goods, the use of lactic acid bacteria and yeasts in the form of sourdough is well established in Italy (Corsetti et al., 2001), Germany (Seibel and Bru¨mmer, 1991), Spain (Barber and Ba´guena, 1989a), and France (Infantes and Tourner, 1991). The use of sourdough in wheat breads has gained popularity as a means to improve the quality and flavor of wheat breads (Bru¨mmer and Lorenz, 1991; Corsetti et al., 2000; Stear, 1990; Thiele et al., 2002).

II. MICROFLORA OF SOURDOUGH By one definition, sourdough has been described as ‘‘a dough made of cereal products (and other ingredients, if required), liquids, and microorganisms (such as lactic acid bacteria and yeasts) in an active state. Acidification

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(fermentation) produced by these substances is a continuous process. The activity of the microorganisms is never interrupted. Microorganisms contained in the flour can also be activated in the course of this process’’ (Seibel and Bru¨mmer, 1991). It is considered that the occurrence of lactic acid bacteria and yeasts in sourdoughs and the association between acidification and bacterial metabolism was first demonstrated in 1894 (Hammes and Ga¨nzle, 1998). Numerous species of lactic acid bacteria occur naturally in wheat flour, including members of the genera Lactobacillus, Pediococcus, Enterococcus, Lactococcus, and Leuconostoc (Hammes and Vogel, 1997). Likewise, numerous species of lactic acid bacteria, mainly belonging to the genus Lactobacillus, have been isolated from sourdoughs (Corsetti et al., 2001; Ottogalli et al., 1996). Lactobacilli are gram-positive, non–sporeforming rods or coccobacilli with complex nutritional requirements. They are found where rich carbohydrate-containing substrates are available such as plants or material of plant origin and in human-made habitats such as that of fermenting food (Hammes and Vogel, 1995). Their optimum growth temperature is 30–40 8C (Bergey, 1994). Lactic acid is the principal product of carbohydrate fermentation by sourdough lactic acid bacteria (Seibel and Bru¨mmer, 1991). Different species, however, can vary greatly in the manner in which they can metabolize carbohydrates and thus may be broadly classified as those that produce lactic acid as the sole (homofermentative) or as a major (heterofermentative) end product of fermentation. Considerable amounts of acetic acid are also formed by those heterofermentative species (Oura et al., 1982). From a microbiological point of view, the definition of sourdough given earlier in this chapter makes reference not only to the presence of lactic acid bacteria but also to the presence of yeasts. Associations of yeasts and lactic acid bacteria are often encountered or used in the production of beverages and fermented foods (Gobbetti, 1998). The vast majority of yeasts found in sourdoughs have been allotted to the species Candida milleri, Candida holmii, Saccharomyces exiguus, and Saccharomyces cerevisiae (Hammes and Ga¨nzle, 1998). There are many trophic and nontrophic interactions between the associations of lactic acid bacteria and yeasts found in sourdough (Gobbetti, 1998). A. SOURCES OF LACTIC ACID BACTERIA FOR SOURDOUGH

Reliance on the fortuitous microorganisms of flour to ‘‘spontaneously start’’ the fermentation process is probably the oldest method used for sourdough production (Spicher, 1983). These organisms may originate from the cereal itself, from contaminants of baker’s yeast, or from the milling or baking environment (Hammes and Ga¨nzle, 1998). In this context, a dough prepared


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from equal quantities of flour and water allowed to stand for 24 hours at 26–35 8C will begin to ferment when gram-negative enteric bacteria present in the flour initiate the process. With repeated additions of flour and water, the dough will become more acidic as the microflora becomes dominated by lactic acid bacteria (Ro¨cken and Voysey, 1995). It is evident from sourdoughs that have been propagated for some time that selection occurs during propagation, leading to the establishment of, usually, one or two species at numbers three or four orders of magnitude above those of the adventitious microbial flora (Hammes and Ga¨nzle, 1998; Hammes et al., 1996; Meroth et al., 2003). The addition of a portion of ripe sour, for example, seed sour, from a previous batch and continuous propagation of the same is another method by which a sourdough may be started (Spicher, 1983). To minimize the variations between sourdoughs where the composition of the microflora is not critically controlled, the application of defined starter cultures for sourdough production has been developed. The commercial availability of single and multiple strain preparations of lactic acid bacteria means that continuous propagation is not necessary and that a high standard of bread quality can be consistently maintained (Hammes, 1990). B. CLASSIFICATION OF SOURDOUGHS

Based on the technology applied for their production, sourdoughs have been classified into three groups (Bo¨cker et al., 1995). Most traditional sourdoughs can be classified as type I doughs. Doughs of this type are characterized by continuous propagation to maintain the activity of the microflora, and this is typically achieved by the use of a multistage process. Lactobacillus sanfranciscensis is the dominant organism isolated from these sourdoughs of this type, and Lactobacillus pontis may also be found. The organisms occurring in doughs of this type are sensitive to low pH levels, so if the sourdough is maintained at ambient temperature and acidification continues, more acid-resistant species will become dominant. In keeping with the requirements of modern baking technology, more efficient fermentation processes are emerging within the field of sourdough applications. Type II sourdoughs are those that are produced by continuous propagation and extended fermentation times. This type of sourdough fermentation originates from the demand for continuous production of pumpable sourdoughs in industrial applications in bread factories, bakeries, and producers of sourdough products (Meuser et al., 1987). Type II doughs can be produced in large volumes and stored for up to 1 week. In contrast to type I doughs, type II sourdoughs exhibit higher dough yields, that is, softer and increased fermentation temperature. In view of the fact that a single

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fermentation period of 15–20 hours is employed, gas formation by the lactic acid bacteria is strongly reduced and baker’s yeast must be applied to the dough for the purposes of leavening. Microorganisms found in wheat sourdoughs of this type belong to the species L. pontis, Lactobacillus panis, Lactobacillus reuteri, and Lactobacillus fermentum (Vogel et al., 1999). Type III sourdoughs can be regarded as artificially composed dried sourdoughs in that lactic acid starter bacteria have been selected with respect to their robustness for drying. They are added as a souring enhancer to sourdoughs for bread dough production. Isolates from these sourdoughs matching the desired properties can be allotted to the species Lactobacillus plantarum, Lactobacillus brevis, and Pediococcus pentosaceus (Bo¨cker et al., 1995). Applying lactic acid bacteria in a freeze-dried state is another method used to initiate sourdough fermentation. Bacterial isolates from, for example, a mature sourdough or other natural environment are selected and tested for their suitability for being employed as sourdough starters and their viability after drying. Freeze-dried strains of Lactobacillus delbrueckii, L. brevis, L. plantarum, and Lactobacillus fructivorans, for example, have been described (Hammes and Ga¨nzle, 1998). In contrast to the type I sourdough starters, these strains are not necessarily well adapted to the cereal environment, so frequent inoculation is advised (Ro¨cken and Voysey, 1995).

III. POSITIVE EFFECTS OF SOURDOUGH ON WHEAT BREAD QUALITY There is considerable consensus regarding the positive effects conferred on bread products by the use of sourdough. From a consumer perspective, the use of sourdough confers a natural image on the product (Salovaara, 1998). Lactic acid bacteria have a long history of use in food and are ‘‘generally regarded as safe’’ organisms (Magnusson et al., 2003). A. NUTRITIONAL QUALITY

It has been observed that bread containing lactic acid produced during sourdough fermentation or added directly can lower the postprandial glucose and insulin responses in humans (Liljeberg and Bjo¨rck, 1994; Liljeberg ¨ stman et al., 2002). The presence of sourdough acids has et al., 1995; O also been reported to have a positive effect on the formation of resistant starch (Liljeberg and Bjo¨rck, 1994; Liljeberg et al., 1996). Furthermore, the nutritional quality of sourdough baked goods is improved with regards to mineral availability (Larsson and Sandberg, 1991; Lopez et al., 2001, 2003;


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Salovaara and Goransson, 1983). Phytate is present in all cereals and forms insoluble complexes with the minerals in flour, consequently reducing its bioavailability, so excessive amounts of it in the diet can have a negative effect. The low pH values associated with chemically or microbiologically acidified wheat doughs lead to solubilization of the phytate complex, thus increasing mineral bioavailability. In addition, it has been reported that exopolysaccharides produced by L. sanfranciscensis may improve the nutritional properties of sourdough fermented products in view of the fact that they may be metabolized by bifidobacteria (Korakli et al., 2002). In addition to these nutritional advantages, sourdough technology may have the potential for the production of special sourdough type of breads with a low content of gliadin peptides toxic for those with celiac disease. This is in view of the fact that selected sourdough lactic acid bacteria have been shown to have hydrolyzing activities toward prolamin peptides involved in human cereal intolerance (Di Cagno et al., 2002). B. MICROBIOLOGICAL SPOILAGE

The general trend to reduce the use of preservatives and treatments that might affect healthy attributes of food has led to attempts to improve bread quality and shelf-life through formulation with compounds naturally occurring in foods (Barber et al., 1992). There has been much interest in the potential application of lactic acid bacteria as a means of biopreservation, that is, control of one organism by another (Magnusson et al., 2003). In addition to the control and inhibition of spoilage organisms during fermentation due to the low pH values (Hammes and Ga¨nzle, 1998; Salovaara, 1998), positive effects of the use of sourdough on the mould-free shelf-life of wheat bread have been reported (Barber et al., 1992; Lavermicocca et al., 2000; Salovaara and Valjakka, 1987). Prevention or limitation of the growth of rope-producing spores of Bacillus subtilis has also been achieved through the use of sourdough or certain strains of sourdough isolates in bread (Pepe et al., 2003; Ro¨cken and Voysey, 1993; Rosenquist and Hansen, 1998). With respect to deterioration in the quality of bread during shelf life, mould growth is the most common cause of microbial spoilage. In addition to the economic losses associated with spoilage of this nature, another concern is the possibility that mycotoxins produced by the moulds may cause public health problems (Legan, 1993). Certain sourdough lactic acid bacteria and their components have been shown to have an antifungal effect against various fungal species isolated from flour and bakery products, some of which are toxin producers (Lavermicocca et al., 2000, 2003). The same effect has been demonstrated in the context of sourdough wheat breads (Lavermicocca et al., 2000).

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It is evident that the antifungal phenomenon is not only due to the development of organic acids during the sourdough fermentation process, despite that an improvement in the shelf life of sourdough baked products was initially attributed to the production of organic acids, particularly acetic acid, by lactic acid bacteria (Ro¨cken, 1996). Barber et al. (1992) reported no correlation between bread shelf-life and pH level, but these authors concluded, however, that the type and amount of acid present may have an effect on other microstatic agents. Furthermore, Corsetti et al. (1998b), employing an agar–well-diffusion assay, found that the individual organic acids (acetic, caproic, propionic, butyric, n-valeric, and formic) produced by L. sanfrancisco CB1 gave no halos of inhibition against Fusarium graminearum. These authors did, however, find that a mixture of all six organic acids had a strongly inhibitory effect. In the same vein, Magnusson et al. (2003) found that those lactic acid bacteria that did not exhibit an antifungal effect actually produced more lactic acid than those strains that had highly active antifungal activities. The same author (Magnusson, 2003) does, however, note that the antifungal activities of lactic acid bacteria are complex and that the presence of organic acids may indeed play a role. Other substances contributing to activity of this nature may include reuterin, hydroxy fatty acids, proteinaceous compounds, cyclic dipeptides, 3-phenyllactic acid, caproic acid, and diacetyl hydrogen peroxide (Magnusson, 2003). There is great divergence among lactic acid bacteria in terms of their antifungal activity. One study that evaluated more than 200 strains of sourdough lactic acid bacteria, using a well-diffusion assay, reported that the antimould activity varied greatly among the strains and was mainly detected within obligately heterofermentative Lactobacillus species (Corsetti et al., 1998b). Lavermicocca et al. (2000) screened a number of strains isolated from sourdough breads and also found that the rate of inhibition of a number of fungal species was highly strain dependent. These authors found that L. plantarum 21B, which is facultatively heterofermentative but typically homofermentative, had the greatest inhibition spectrum. C. FLAVOR

Taste and flavor of bread can be improved with optimal use of sourdough (Seibel and Bru¨mmer, 1991). The flavor of sourdough wheat bread is richer and more aromatic than in wheat bread, a factor that can be attributed to the long fermentation time of sourdough (Bru¨mmer and Lorenz, 1991). Studies on the influence of lactic acid bacteria on the aroma of wheat bread revealed a positive influence, particularly on the crumb aroma (Hansen and Hansen, 1996). The concentration of 2-phenylethanol, one of the most potent odorants of wheat bread crumb (Grosch and Schieberle,


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1997), was increased in sourdough-enriched bread crumb (Gassenmeier and Schieberle, 1995). It has been demonstrated that the production of volatile flavor components in sourdough is strongly dependent on the starter culture, but the role played by the flour used has also been recognized (Hansen and Hansen, 1994a). Few volatile compounds have been identified in chemically acidified doughs compared to sourdoughs (Hansen and Hansen, 1994b), and the intensity of flavor was found to be greater in breads prepared with biologically acidified preferment than in those that were chemically acidified (Thiele et al., 2002). The main influence of microorganisms on sourdough flavor has been identified as their ability to enhance or reduce the amount of specific volatiles already present in the flour (Czerny and Schieberle, 2002). An increase in the level of amino acids in doughs, especially ornithine, has also been associated with improved bread flavor (Thiele et al., 2002). D. BREAD CHARACTERISTICS

Loaf-specific volume is a primary quality characteristic of bread (Maleki et al., 1980). The application of sourdough to wheat breads has a positive impact on bread volume (Barber et al., 1989b, 1992; Clarke et al., 2002; Collar et al., 1994a; Corsetti et al., 1998a, 2000; Crowley et al., 2002). The rate of application is important, however, because optimum levels of sourdough must be applied to achieve optimal bread quality (Barber et al., 1992; Collar et al., 1994a; Crowley et al., 2002). The nature of the acidification process may also be key. Regarding biological acidification, it has been reported that chemical acidification, in the absence of any considerable fermentation period, does not improve loaf-specific volume (Clarke et al., 2002). A chemically acidified dough fermented for more than 3 hours, however, was found to yield breads with greater volume than any of their counterparts fermented with lactic acid bacteria, a finding attributed to yeast metabolism being favored by acidic conditions (Corsetti et al., 2000). In keeping with the findings of Maleki et al. (1980) who reported that larger loaf size produced softer bread, sourdough breads have been shown to have lower crumb firmness values (Clarke et al., 2002; Collar, 1994a; Corsetti et al., 2000; Crowley et al., 2002). Crumb grain, described as the exposed cell structure of the crumb when a loaf of bread is sliced, is another important bread quality characteristic affected by the addition of sourdough. It is generally acknowledged that holes of relatively small size (1 or 2 mm) are required in bakery products, whereas large voids or irregular crumb distributions are undesirable (Cauvain, 1998a). An increase in the mean cell area, within the range that is still desirable, has been demonstrated via addition of 20% sourdough (Crowley et al., 2002).

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C. I. CLARKE AND E. K. ARENDT E. STALING

Bread is generally viewed as a perishable commodity, which is best consumed when ‘‘fresh.’’ The loss of perceived freshness is due to a number of factors, which may generally be categorized as those attributable to microbial spoilage and those that are due to a series of complex processes collectively known as staling (Pateras, 1998). Staling has been defined as ‘‘a term which indicates decreasing consumer acceptance of bakery products caused by changes in crumb other than those resulting from the action of spoilage organisms’’ (Bechtel et al., 1953). Despite extensive study, bread staling has not been eliminated and remains responsible for huge economic losses (Gray and Bemiller, 2003). Although a complex series of events occur during staling, including changes in the crystallinity of the starch during storage (Cauvain and Young, 2000), bread staling is mainly associated with the firming of the crumb (Gray and Bemiller, 2003; Pateras, 1998). The application of lactic acid bacteria in the form of sourdough has been reported to have positive effects on bread staling. One such effect is an improvement in loaf-specific volume, which is associated with a reduction in the rate of staling (Axford et al., 1968; Maleki et al., 1980), as has been demonstrated by a reduction in crumb softness for sourdough breads during shelf life (Clarke et al., 2002; Corsetti et al., 2000; Crowley et al., 2002). A decrease in the staling rate as measured by differential scanning calorimetry has also been reported for breads containing sourdough (Barber et al., 1992; Corsetti et al., 1998a, 2000). It has been noted, however, that the antistaling effect seen for sourdough is strain specific, involving dynamics other than those associated with the degree of acidification. Activities associated with bacterial hydrolysis of starch and the proteolysis of gluten subunits have been proposed (Corsetti et al., 1998a).

IV. UNDERSTANDING THE TECHNOLOGICAL FUNCTIONALITY OF SOURDOUGH APPLICATION Despite its long tradition and the well-documented positive effects conferred on bread products by its use, various details about sourdough technology have not been fully understood. This remains the case not only regarding sourdough microbial ecology and physiology, despite much progress in this regard (Brandt and Hammes, 2001; Gobbetti, 1998; Hammes and Ga¨nzle, 1998), but also regarding the influence of sourdough on the structure of dough and bread. The mechanisms at work in sourdough and its application are complex and numerous (Hammes and Ga¨nzle, 1998). Various flour characteristics and process parameters contribute to exercising very


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particular effects on the metabolic activity of the sourdough microflora. During fermentation, biochemical changes occur in the carbohydrate and protein components of the flour due to the action of microbial and indigenous enzymes. The rate and extent of these changes greatly influence the properties of the sourdough and ultimately the quality of the final baked product. A number of hypotheses have been put forward that can help explain the effects of sourdough on dough and bread quality, including those that are related to the direct impact of pH on dough structure, those corresponding to the effect of acid on cereal enzymes, and those that are related to the effect of the microorganisms alone. A. PRIMARY EFFECTS OF ACIDIFICATION

The pH of a ripe sourdough varies with the nature of the process and starter culture used, but for wheat sourdoughs, it ranges from 3.5 to 4.3 (Clarke et al., 2002; Collar et al., 1994a; Thiele et al., 2002; Wehrle and Arendt, 1998). The nature of the flour, in particular its ash content, has a considerable effect on acidification characteristics (Collar et al., 1994b). Depending on the rate of addition, the pH of the bread dough will also vary. Given a typical application rate of approximately 20%, dough pH values ranging from 4.7 to 5.5 have been reported (Clarke et al., 2002; Collar et al., 1994a). The acidification of the sourdough and the partial acidification of the bread dough will no doubt have a direct impact on structure-forming components like gluten, starch, and arabinoxylans. It was reported almost a century ago (Osborne, 1907) that the presence of acid increased the solubility of the glutenin fraction extracted from wheat flour. The swelling of gluten in acid is a well-known effect (Axford et al., 1979; Zeleny, 1947), and mild acid hydrolysis of starch in sourdough systems has been hypothesized (Barber et al., 1992). Acids strongly influence the mixing behavior of doughs, whereby doughs with lower pH values require a slightly shorter mixing time and have less stability than normal doughs (Hoseney, 1994). The direct influence of organic acids on the rheological properties of dough has been examined intensely using both empirical (Maher Galal et al., 1978; Tanaka et al., 1967; Tsen, 1966; Wehrle et al., 1997) and fundamental techniques (Clarke et al., 2002, in press; Wehrle et al., 1997). Several studies directly focused on the influence of added organic acids and sodium chloride on rheological properties measured using the farinograph (Maher Galal et al., 1978; Tanaka et al., 1967; Wehrle et al., 1997) and extensograph (Tanaka et al., 1967; Tsen, 1966). The farinograph is commonly used to provide empirical information regarding the mixing properties of dough (Spies, 1990). The water absorption of flour, as determined using the farinograph, is an important factor influencing the handling properties and

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machinability of dough in large mechanized bakeries and is related to the quality of the finished baked product (Catterall, 1998). Studies of wheat dough using the farinograph showed that water uptake or consistency was increased by added organic acids in the absence of salt (Maher Galal et al., 1978; Tanaka et al., 1967). The addition of organic acids also substantially decreased mixing time and weakened the dough (Maher Galal et al., 1978; Wehrle et al., 1997). Maher Galal et al. (1978) put forward the hypothesis on a molecular level, which stated that in an acidic environment, there is a sizable positive net charge and protein solubility is increased. The increased intramolecular electrostatic repulsion leads to an unfolding of the gluten proteins and an increased exposure of hydrophobic groups, but the presence of strong intermolecular electrostatic repulsive forces prevents the formation of new bonds. The net effect of these events is a weakening of the structure and thus a softening effect. Such a hypothesis is further supported by Osborne (1907) and Takeda et al. (2001) who reported increased solubility of the constituent gluten proteins at acidic pH values. This disentanglement of the gluten protein network upon the addition of acid is quite in keeping with the results obtained from empirical measurement of dough properties using the extensograph, which found that the addition of acid, in the presence of salt, resulted in doughs with increased resistance and decreased extensibility (Clarke et al., 2002; Tanaka et al., 1967; Tsen, 1966). An explanation of the dough response during this test, given in terms of the entangled protein network model, is that when a piece of dough is subjected to elongation, it will tear when the network between the two entangled regions reaches its full extension; thus, the more entangled the network, the higher its resistance to deformation (Masi et al., 2001). Fundamental rheological studies on chemically acidified doughs have also been performed (Clarke et al., 2002; Wehrle et al., 1997). Wehrle et al. (1997) reported that under optimal mixing conditions, the addition of acids leads to dough with lower phase angle values and thus more elastic behavior. The phase angle ranges from 0 degrees (ideally elastic material, hookean solid) to 90 degrees (ideally viscous material, newtonian liquid). For all viscoelastic materials, the phase angle is between 0 and 90 degrees, and the lower the values, the more elastic the material. Clarke et al. (2002) reported that the direct addition of an organic acid slightly decreased the absolute value of the complex modulus (i.e., dough firmness). The effect of acid addition on the rheology of a sourdough preferment has also been determined at the outset of the fermentation period (Clarke et al., 2004) at which point the changes directly associated with acidic pH values were seen to be an increase in elasticity and a simultaneous decrease in viscosity. A fundamental rheological evaluation of the effect of acid and salt on model gluten systems


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was also indicative of an increase in both the softness and the elasticity of gluten in the presence of acid (Schober et al., 2003). B. SECONDARY EFFECTS OF ACIDIFICATION

In addition to the direct impact of decreasing pH values on dough characteristics, secondary effects of acidification and fermentation time may include changes in the activity of cereal or bacterial enzymes associated with changes in the pH of the environment during the fermentation period. Kawamura and Yonezawa (1982) described wheat flour proteases that have optimal activity around a pH level of 4. In addition, Bleukx et al. (1997) detected proteolytic enzymes with acidic pH optima in vital wheat gluten. In terms of the effects of acidic pH values on dough characteristics, Wu and Hoseney (1989) showed for cracker sponges that a pH value of 4.1 was most effective in reducing the resistance to extension during a 12-hour fermentation period. In the same vein, Thiele et al. (2002) found a greater increase in the concentration of particular amino acids in an acidified relative to a nonacidified dough system during a 50-hour fermentation period. These authors concluded that the most important factors governing the levels of amino acids in wheat dough were dough pH value, fermentation time, and the consumption of amino acids by the fermentative microflora. Another study, using both empirical and fundamental techniques to compare the rheological properties of chemically and biologically acidified doughs, found that the addition of biological acidified material resulted in major changes in the structure of the dough not comparable to those attributable to the presence of acid alone (Clarke et al., 2002). This study did not employ a fermentation period for the chemically acidified treatment and thus concluded that in contrast to the limited time frame during which enzyme activity could have an impact on the structure of the chemically acidified dough, the fermentation period of the biologically acidified treatments could allow for an extended period of enzymatic activity further enhanced by acidic pH values. C. PROTEOLYSIS DURING SOURDOUGH FERMENTATION

The effect of proteolysis on the structure-forming components of dough is another well-documented aspect of sourdough fermentation (Corsetti et al., 2000; Di Cagno et al., 2002; Kawamura and Yonezawa, 1982). Despite that proteolysis and the proliferation of amino acids by enzymatic release during sourdough fermentation is well documented (Collar and Martinez, 1993; Collar et al., 1992; Di Cagno et al., 2002; Gobbetti et al., 1994; Thiele et al., 2002, 2003), proteolysis during sourdough fermentation remains unclear.

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This is due to the nature of the sourdough system where the effects of acidification and the endogenous microbial and cereal proteases all contribute to a complex set of dynamics. It is further complicated by the fact that there may be divergence between studies in terms of the particular lactobacilli used given that proteolytic activity is strain dependent. The enhanced proteolysis seen during sourdough fermentation has been attributed to both the proteolytic activity of lactic acid bacteria and that of cereal proteases. With regards to the bacteria, the level of proteolytic activity on wheat flour fractions has been found to be very strain dependent (Corsetti et al., 1998a; Di Cagno et al., 2002; Gobbetti et al., 1996). Certain bacterial strains have the specific capacity to hydrolyze albumin, globulin, and gliadin fractions of wheat flour (Di Cagno et al., 2002). Using free amino acid concentration as a measure of proteolysis, these authors reported that although chemical acidification resulted in an increased concentration, those doughs fermented with strains of L. alimentarius, L. brevis, L. sanfranciscensis, and L. hilgardii showed a greater concentration with some differences in the amino acid pattern. Further, two-dimensional electrophoresis revealed that both biological and chemical acidification caused a marked modification of the polypeptide pattern with respect to a nonacidified dough. The strain dependency of proteolysis was demonstrated by the fact that almost all of the polypeptides were hydrolyzed by the strains of L. alimentarius, L. brevis, and L. sanfranciscensis used, whereas the strain of L. hilgardii employed showed much less capacity for hydrolysis. The effect of the proteolytic activity of some of these strains on dough rheology measured using empirical techniques has also been shown to be more extensive than that seen for a chemically acidified equivalent (Di Cagno et al., 2002). In addition to the proteolytic activity of lactic acid bacteria, the role of cereal proteases has been explored. Thiele et al. (2002) observed that the presence of lactobacilli had little effect on total amino acid concentrations in wheat sourdoughs when compared to acid aseptic doughs, thus concluding that the proteolytic activity of lactobacilli was negligible in comparison with that of wheat flour. The reduction in pH brought about by the lactic fermentation did, however, enhance proteolysis relative to neutral sterile doughs. These findings were confirmed in another study (Thiele et al., 2003) that used fluorescence labeling of wheat protein fractions to determine the degree of gluten hydrolysis and depolymerization during sourdough fermentation, which found that compared to the degradation of gliadin and glutenin proteins in aseptic acidified doughs, the additional proteolytic activity of microbial enzymes was small. This study did, however, report that microbial fermentation affected the size distribution of the peptides resulting from proteolytic degradation of wheat proteins in so far as the presence of lactobacilli promoted a decrease in the concentration of larger peptides and


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an increase in that of smaller molecules such as dipeptides and amino acids. These authors concluded that proteolytic degradation of gluten proteins and depolymerization of the gluten macropolymer observed during sourdough fermentation were in the main attributable to dough pH and cereal enzyme activity. The contribution made by cereal proteases to the structural changes seen during sourdough fermentation has been further explored via the use of a sterile nonacidified dough preferment (Clarke et al., 2004). In comparing the rheological properties of this dough with those of chemically acidified or biologically acidified dough preferments, this study showed that those changes associated with time were the main influence on the properties of all three preferments during a 24-hour fermentation period. There was a reduction in elasticity and firmness for all treatments during the fermentation period, at the end of which there was little difference between the rheological characteristics of the treatments, irrespective of the presence of acid. Relative to the neutral treatment, the biological and chemical acid treatments were more degraded at the end of fermentation, indicating that the structural changes were further enhanced by the presence of acid, in keeping with the descriptions of wheat flour proteolytic enzymes with acidic pH optima described earlier. Proteolytic activity attributable to wheat flour cereal proteases has also been reported by Kawamura and Yonezawa (1982). Using SDS-PAGE, these authors reported that the mode of action of these cereal proteases was relatively specific for a high-molecular-weight subunit of glutenin (90,000 Da) and that this subunit disappeared as a function of time while new protein bands in the region of 26,000–28,000 Da and a new protein band of 68,000 Da appeared. In the same vein, Bleukx et al. (1997) identified a range of proteolytic activities associated with vital wheat gluten, which also led to a disappearance of high- and low-molecularweight glutenin subunits with the formation of new protein bands in the 30,000–33,000 Da region. Bleukx and Delcour (2000) identified a second aspartic proteinase associated with wheat gluten, which did not hydrolyze gluten when incubated with gluten alone. It was hypothesized, however, that such a proteinase might act synergistically with other proteases in gluten breakdown.

V. EFFECT OF SOURDOUGH INCORPORATION ON BREAD DOUGH STRUCTURE Given that the rheology of wheat doughs and the resulting loaf volume are mainly determined by gluten proteins, any changes associated with proteolytic degradation during sourdough fermentation will no doubt have an

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impact on the nature of the bread dough when prefermented material is incorporated. From a rheological point of view, it is well established that as fermentation progresses, there is a change in nature of the elements contributing to dough structure such as the decrease in the viscosity described for a gluten solution (Kawamura and Yonezawa, 1982). Using empirical techniques to measure the rheology of fermented doughs, Di Cagno et al. (2002) found a decrease in resistance to extension and an increase in both extensibility and degree of softening. Clarke et al. (2004) using fundamental techniques, reported that sourdough preferments became softer and less elastic as fermentation progressed. This study hypothesized that with time, large protein aggregates responsible for the dough’s structural integrity are broken down into small protein aggregates by cereal proteases, resulting in a softer and less elastic system. The structural implications of these changes were examined using confocal laser-scanning microscopy, which revealed that during the course of fermentation, the gluten of a biologically acidified preferment underwent a transition from having a distinct structure in the form of strands to becoming more amorphous, a change consistent with the hypothesis that the protein is partially degraded during fermentation. It was inferred that a reduction in the quantity of polymeric proteins (i.e., glutenins) present in the preferment was effected by degradation, thus resulting in a less elastic system. It was apparent from the subsequent rheology data for the dough that the incorporation of 20% of the flour in the form of a preferment, be it sterile or acidified, reduced elasticity and firmness, thus yielding a significantly softer, less elastic dough than the control containing no added preferment. Confocal laser-scanning microscopy revealed that the effect of the incorporation of biologically acidified material could also be seen with regards to dough microstructure. Relative to the fine well-oriented network of the control, the gluten of the dough with added preferment had a more amorphous nature and there were greater areas of aggregated material composed of thicker proteinaceous strands in evidence. This scenario is resonant of that described by Kieffer and Stein (1999) for relaxed and reshaped wheat systems, where the presence of thicker strands could allow for a greater increase in loaf volume. From a technological point of view, it was hypothesized that the incorporation of an optimally degraded preferment may be the principal reason for the achievement of doughs that yield better loaf quality characteristics when sourdough is optimally applied. In addition to the impact of sourdough on the structure and rheology of the constituent gluten proteins making up the framework of the dough, its effect on gas formation must also be considered because gas formation by microorganisms is necessary to obtain leavened bread. In the case of sourdough breads, carbon dioxide is produced by both lactic acid bacteria and yeast and the contribution of each group to the overall gas volume differs


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with the type of starter culture and the dough technology applied (Hammes and Ga¨nzle, 1998). For both types of microorganisms, the rate of production is dependent on a number of ecological factors including the nutrient supply, which depends in turn on the degradation of macromolecules such as protein and starch. The gas-holding capacity of the dough system, on the other hand depends on the physicochemical structure of the dough, which in the case of wheat dough, is mainly governed by the gluten network (Finney, 1943; Lookhart, 1997). In terms of evaluating the interactions between the two types of microorganisms using a rheofermentometer, Gobbetti et al. (1995) found that in comparison to that seen in sourdough produced with yeast alone, yeast fermentation was faster in the presence of heterofermentative lactic acid bacteria, whereas that with homofermentative bacteria was slower and produced more carbon dioxide. Hammes and Ga¨nzle (1998) documented that for traditional (type I) sourdoughs, the contribution made by heterofermentative lactic acid bacteria to gas production is substantial and may even be decisive but that in the case of sourdoughs where baker’s yeast is also applied during dough formation (type II sourdoughs), the quantity of gas produced by the sourdough microflora is only of minor importance. This effect was demonstrated in a report that used response surface methodology to examine the effects of sourdough fermentation time and yeast quantity on loaf quality parameters (Clarke et al., 2003). An increase in loaf-specific volume associated with an increase in sourdough fermentation time was observed when no baker’s yeast was applied. In the presence of yeast, however, the same trend was not in evidence, once more highlighting the overriding effect of gas production by yeasts relative to lactic acid bacteria. This same effect was also demonstrated by evaluation of the gaseous release characteristics of a range of doughs, all of which contained baker’s yeast (Clarke et al., 2002). This study found that there were no significant differences between the total amounts of carbon dioxide produced, lost, or retained by biologically acidified doughs relative to nonbiologically acidified dough as measured using a rheofermentometer. From a technological point of view, it may, therefore, be hypothesized that it is the gas retention, and not the gas production properties of the dough, that is improved when sourdough is applied. A. INTERACTION BETWEEN SOURDOUGH AND DOUGH ADDITIVES

In addition to reliance on the integral components of dough, there is an increasing trend for the use of additives in the baking industry to achieve optimal functionality in terms of dough-handling properties and bread quality attributes, including shelf life (Rosell et al., 2001). The interaction

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between sourdough and a number of additives such as exogenous enzymes and nonstarch polysaccharides has been evaluated (Corsetti et al., 2000; Di Cagno et al., 2003). With respect to the rate of starch retrogradation during storage, Corsetti et al. (2000) used crumb firmness measures and differential scanning calorimetry experiments to determine the contribution made by the addition of a-amylase, protease, pentosans, and pentosanases to the rate of staling observed in sourdough breads prepared using strains of S. cerevisiae, L. sanfranciscensis, and L. plantarum. These authors reported that compared with a control bread, the positive effect seen for the sourdough bread was further enhanced by the addition of a-amylase. In breads where pentosans alone or a mixture of pentosans, endoxylanase, and a strain of L. hilgardii was added, an even greater delay in bread firming and staling was observed. These authors concluded that the use of a lactic acid bacterial strain with particular characteristics may be a fundamental prerequisite in the retardation of bread crumb firmness. A combined effect of sourdough lactic acid bacteria and pentosans was also proposed. Di Cagno et al. (2003) studied the interactions between sourdough lactic acid bacteria and exogenous enzymes to optimize the effects on the microbial kinetics of acidification, acetic acid production, and textural properties of sourdough during the fermentation process. The enzymes used included glucose-oxidase, lipase, endoxylanase, a-amylase, or protease, enzymes that are typically applied to improve dough functionality. These authors found that of the 11 species of lactic acid bacteria used, only three were positively influenced by the addition of enzymes with regards to the rate and extent of lactic acidification. The use of enzymes in the context of sourdough may be difficult because the acidic environment may interfere with their activity. It was reported, however, that in some cases, the combined use of sourdough and enzymes could reduce the risk of dough weakening and the loss of gas-retention properties (Di Cagno et al., 2003). B. USE OF LACTIC ACID BACTERIA METABOLITES TO REPLACE ADDITIVES

The production of exopolysaccharides by lactic acid bacteria during food fermentation is another interesting aspect of sourdough technology with the potential for the replacement of hydrocolloids. These compounds, commonly named gums, are used as texturizing, antistaling, or prebiotic additives in bread production (Tieking et al., 2003). Exopolysaccharides are microbial polysaccharides secreted extracellularly, the amount and structure of which depend on the particular microorganism present and the available carbon substrate (Korakli et al., 2001). Studies of the application of exopolysaccharide-forming starter cultures have focused primarily on


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heteropolysaccharides from lactobacilli in dairy fermentations (Tieking et al., 2003). The production from sucrose of a levan type of fructan in wheat doughs has, however, been described by Korakli et al. (2001). In view of the fact that the amount of exopolysaccharide produced corresponded to 0.5– 1.0% on a flour weight basis, these authors concluded that the quantity would be sufficient to effect changes in the rheological properties of the dough, as well as in the textural and shelf-life parameters of the bread. In addition to the production of fructan, Tieking et al. (2003) also described the in situ production of glucan by sourdough bacterial strains in the context of wheat flour sourdough at levels ranging from 0.5 to 2 g/kg of flour. In view of the findings of Rosell et al. (2001) that the addition of 0.5% hydrocolloid had an impact on dough rheology and bread quality, Tieking et al. (2003) assumed that the amount of exopolysaccharide produced by lactic acid bacteria in the context of sourdough to be technologically relevant. That these substances can be metabolized by bifidobacteria is also of nutritional advantage.

VI. CONCLUSION Preservation of foods by fermentation is a widely practiced and ancient technology. In view of their unique metabolic characteristics, lactic acid bacteria are involved in many fermentation processes including cereals and in particular sourdough. It is clear that the application of sourdough to wheat bread production does indeed present a complex set of circumstances for food scientists and technologists. There exist myriad microbial, technological, and processing dimensions that must be considered to produce cereal products of optimal quality. It is also clear that a great deal of research has been conducted in relation to the application of sourdough to baked goods, including a development of the understanding of the roles played by the principal endogenous and process parameters. Significant advances have been made in understanding the contributions made by the presence of acid, the fermentation period, and the role played by cereal proteases in terms of fundamental changes in dough rheology and quality characteristics. There has also been much progress in the development of tools that allow for the selection of key sourdough microorganisms for particular activities such as those concerned with enzymatic, antifungal, antimicrobial, nutritional, and additive replacement aspects. These developments are important from the point of view of both basic research and industrial applications. The application of sourdough confers many advantages on the quality of the baked goods produced. The prospect of increasing quality, shelf life, safety,

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and the nutritional value of breads is of considerable economic impact and favorable from a consumer’s perspective. Given that there already exists a considerable body of knowledge regarding the role and activity of the microflora concerned with sourdough, the future challenges for research and development in the area must include the further improvement of reliability and product quality through optimization of starter culture performance and the elimination of those factors that impede the fermentation process. In this regard, it is to be anticipated that the considerable resources that have been devoted to the ‘‘biotechnology of lactic acid bacteria’’ over the past number of years will deliver results with respect to these objectives. There remains a need, however, for the continued use of an interdisciplinary approach that will ensure that biotechnological advances can indeed yield positive outcomes in terms of industrial applications and consumer satisfaction. This is particularly the case given that the prospect of metabolic engineering of strains of lactic acid bacteria to generate derivatives with new attributes is now a real one. REFERENCES Axford, D.W.E., Kolwell, K.H., Cornfor, S.J., and Elton, G.A.H. 1968. Effect of loaf specific volume on rate and extent of staling in bread. J. Sci. Food Agric. 19, 95–101. Axford, D.W.E., McDermott, E.E., and Redman, D.G. 1979. Note on the sodium dodecyl sulfate test of breadmaking quality: Comparison with Pelshenke and Zeleny tests. Cereal Chem. 56, 582–583. Barber, B., Ortola´, C., Barber, S., and Fernańdez, F. 1992. Storage of packaged white bread. III. Effects of sour dough and addition of acids on bread characteristics. Z. Lebensm. Unters. Forsch 194, 442–449. Barber, S. and Ba´guena, R. 1989a. Microflora of the sour dough of wheat flour bread. XI. Changes during fermentation in the microflora of sour doughs prepared by a multi-stage process and of bread doughs thereof. Rev. Agroquı´m. Tecnol. Aliment. 29, 478–491. Barber, S., Baguena, R., and Benedito de Barber, C. 1989b. Microbiological and physicochemical characteristics of sour doughs and their relation with the characteristics of bread doughs and breads thereof. In ‘‘Trends in Food Science’’ (W.S. Lien and C.W. Foo, eds), pp. 187–191. Singapore Institute of Food Science & Technology, Singapore. Bechtel, W.G., Meisner, D.F., and Bradley, W.B. 1953. The effect of crust on the staling of bread. Cereal Chem. 30, 160–168. Bergey, D.H. 1994. In ‘‘Bergey’s Manual of Determinative Bacteriology’’ (J.G. Holt, N.R. Krieg, P.H.A. Sneath, J.T. Staley, and S.T. Williams, eds), 9th Ed. Williams and Wilkins, Baltimore. Bleukx, W. and Delcour, J.A. 2000. A second aspartic proteinase associated with wheat gluten. J. Cereal Sci. 32, 31–42. Bleukx, W., Roels, S.P., and Delcour, J.A. 1997. On the presence and activities of proteolytic enzymes in vital wheat gluten. J. Cereal Sci. 26, 183–193. ¨ kosystem Sauerteig und zur Bo¨cker, G., Stolz, P., and Hammes, W.P. 1995. Neue Erkenntnisse zum O Physiologie der sauerteigtypischen Sta¨mme Lactobacillus sanfrancisco und Lactobacillus pontis. Getreide Mehl. Brot. 49, 370–374.


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DETECTION OF INSECT INFESTATION IN STORED FOODS SOMIAHNADAR RAJENDRAN Food Protectants and Infestation Control Department Central Food Technological Research Institute Mysore, India

I. Introduction II. Insect Pests of Stored Foods A. Types B. Effects on Foods III. Detection of Insects in Samples A. Visual Inspection B. Sampling and Sieving C. Flotation Methods D. Fragment Count or Acid Hydrolysis Method E. Staining Techniques F. CO2 Analysis G. Uric Acid Determination H. Imaging Techniques I. Serological Techniques J. Other Methods IV. Detection in Storage Facilities A. Visual Inspection B. Trapping Methods C. Acoustic Method V. Conclusion Acknowledgments References

I. INTRODUCTION Food materials of agricultural and animal origin are stored in diVerent types of storage structures for future consumption or trade purposes. During storage, pest organisms such as birds, rodents, insects, mites, and microbes ADVANCES IN FOOD AND NUTRITION RESEARCH VOL 49 ISSN: 1043-4526


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attack these commodities. More than 600 species of beetles and 70 of moths among the insects, 355 species of mites, 40 species of rodents, and 150 species of fungi have been reported to be associated with various stored products, including food commodities (Rajendran, 2002). Annual postharvest pest losses resulting from the attack of these biological agents, world over, have been estimated at 10% (Boxall, 1991). Weight losses (dry matter loss) in the range of 0.5–17% in cereals and up to 50% in pulses have been reported (Snelson, 1987). In stored dried fish in the tropics, losses up to 50% have been reported (Proctor, 1977). Inputs in the form of people power and finances invested in the production of food commodities will go to waste unless the materials are protected from depredating agents during storage. Insect pest activity in agricultural produce may start at any stage from harvest to consumption; in some cases, the infestation occurs in the standing crop itself. Insects such as Sitotroga cerealella (on paddy rice), Sitophilus zeamais, Prostephanus truncatus (on maize), Hypothenemus hampei (on coVee), Carpophilus spp. (on dried fruits), Caryedon serratus (on peanuts), and Callosobruchus chinensis (on many pulses) commence their pest activity in the standing crop or before storage (Rossiter, 1970; Tigar et al., 1994). Similarly, in some of the animal food products, insect pest attack has been observed during the processing stage itself. In dried fish, damage occurring to the fish in the drying yard due to blowflies (Chrysomya megacephala, C. albiceps, C. chloropygaputoria, C. regatis, and Lucilia cuprina) is a serious problem in countries in Africa and Southeast Asia. Furthermore, in cured fish when the moisture content is high at the time of processing, other types of flies including the filth fly, Discomyza maculipennis (Soans and Adolf, 1971), house fly, Musca domestica, and the cheese skipper, Piophila casei (Madden et al., 1995) are attracted. Insect infestation causes qualitative and quantitative losses of food commodities. Insects produce excrement and frass during their grain-boring and oviposition activities. Insects like Cryptolestes spp., Trogoderma granarium, and Plodia interpunctella preferentially feed on the germ that is soft and highly nutritious, rather than the hard endosperm of food grains. Insect contaminants such as excreta (uric acid), exuviae (cast skins) and dead bodies, webbing, and secretions in food commodities pose a quality-control problem for food industries. These contaminants are responsible for health risks to humans (Phillips and Burkholder, 1984). The occurrence of insect fragments in processed foods is an important quality-control problem of concern in processed food industries (Gentry et al., 2001). There are reports on the occurrence of various levels of insect fragments in diVerent kinds of processed foods in Brazil (Correia et al., 2000; Graciano et al., 1998; Rodrigues et al., 1998; Zamboni et al., 1988) and Italy (Bonafaccia et al.,

INFESTATION DETECTION IN STORED FOODS

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1999; Khoury et al., 1996; Locatelli et al., 1993, 2000). Pest infestation of dried fish leads to higher levels of uric acid and bacterial counts that are unacceptable to consumers and traders (Solanki, 1985). Toxicological studies revealed that excess levels of uric acid in the diet could induce hyperuricemia with associated nephropathy in rats (Starvic et al., 1969). Secretions from the adults of Tribolium spp. and Rhyzopertha dominica impart unacceptable oV-odors to foodstuVs. Processing and end-use qualities of food commodities are also aVected by insect infestation, as are cash value and marketability of diVerent commodities. There are national tolerance limits such as the maximum quantity of substances including pesticides and natural or unavoidable defects (live or dead insects, insect fragments, and related contaminants) allowable in a food. For instance, in the United States, the Food and Drug Administration (FDA) has established Defect Action Levels for live insects at two insects per kilogram and insect-damaged grains at 32 kernels/100 g in food grains; in wheat flour, there is a limit of 75 insect fragments/50 g, and in macaroni and noodle products, it is 225 fragments in a 225-g sample (Jeon, 2002). On the other hand, in India, according to the Prevention of Food Adulteration Act, the uric acid level in food commodities should not exceed 100 mg/kg and the number of weevil-damaged grains should not exceed 10% by count (Anon, 2001). In countries like Canada and Australia, there is zero tolerance for insects in food grains (White, 1995), and a similar standard is followed in international trade for food grains (Fleurat-Lessard, 1997). There has been a growing concern throughout the world about contaminants such as pests and pesticides in food commodities. Quality maintenance by way of reduction in insect contaminants to meet the requirements of International Standards Organization (ISO) standards and Hazard Analysis Critical Control Points (HACCP) is important for marketing the produce. Detection of insect infestation is, therefore, necessary (1) to ensure a supply of wholesome food to the consumers, (2) to assess eVectiveness of fumigation and other pesticide treatments, and (3) to serve as an early warning for taking appropriate control measures. Any delay in detection may result in pest outbreaks, causing severe contamination of food materials and quantitative loss. In addition, the detection of insect infestation in stored food commodities or on storage premises is the foremost step in pest management in food industries (Mueller, 1998). Detection methods applicable for commodity samples and in situ detection and monitoring in bulk storage and food processing facilities have been reviewed (Cotton and Wilbur, 1982; Fleurat-Lessard et al., 1994; Milner, 1958; Pedersen, 1992; Semple, 1992). Rajendran (1999) also studied infestation detection in stored foods and storage premises.

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II. INSECT PESTS OF STORED FOODS A. TYPES

The major insect pests attacking stored food commodities are either beetles (order: Coleoptera) or moth (order: Lepidoptera) pests; psocids (order: Psocidae) are also found in most of the stored produce (Cotton and Wilbur, 1982). Food commodities of agricultural origin are often infested by beetle pests belonging to the families Anobiidae, Anthribidae, Bruchidae, Bostrichidae, Cucujidae, Curculionidae, Nitidulidae, Ptinide, Silvanidae, and Tenebrionidae, as well as moth pests belonging to Gelechiidae and Pyralidae, whereas beetles belonging to Chrysomelidae, Cleridae, and Dermestidae attack animal food products (Rees, 1996; Sedlacek et al., 1996). Whole grains, milled products, and diVerent types of processed foods are infested (Table I). The beetle pests are relatively small, with the average adult size being 3–5 mm, and they are cryptic in nature and hence go unnoticed when present in food materials in low numbers. Insect pests of stored food commodities are highly prolific in that several generations can occur in a year depending on climatic conditions and availability of food. These insects have varied food habitats and can breed on foodstuVs containing less than 2% carbohydrate (e.g., Lasioderma serricorne and Tribolium confusum), on dried fish with 20% salt (e.g., Dermestes maculatus), on dry fruits having up to 60% sugar (e.g., Carpophilus spp.), and on tree nuts with 50–70% fat (e.g., Ephestia cautella). B. EFFECTS ON FOODS

Insect pest infestation causes losses in quantity and quality of food commodities and changes in chemical composition, aVecting the nutritive value of the produce (Howe, 1965; Scott, 1991; Swaminathan, 1977). Insect activity also leads to contamination of the produce (Table II). The flour beetles (Tribolium spp.) contaminate foodstuVs with their secretions, which contain 2-ethyl 1,4-benzoquinone and 2-methyl-1,4-benzoquinone. T. castaneum quinone secretions not only impart oV-odors to food commodities but are also considered to cause liver and spleen tumors in mice (El-Mofty et al., 1992). However, Hodges et al. (1996) demonstrated that unlike in wheat flour, the accumulation of quinone secretions of T. castaneum adults in rice was negligible at less than 1 ppm, and hence, they claimed that T. castaneum infestation in rice is not likely to be a health risk. Males of R. dominica secrete aggregation pheromones (dominicalures) that contribute to the characteristic sweetish or musty odor in grain infested with R. dominica (Khorramshahi and Burkholder, 1981). However, Seitz and Sauer (1996)


TABLE I INSECT PESTS OF STORED FOOD COMMODITIES

Scientific name

Common name

Cereals Corcyra cephalonica Cryptolestes spp. Ephestia cautella Liposcelis spp. Nemapogon granella Oryzaephilus surinamensis Paralipsa gularis Prostephanus truncatusa Plodia interpunctella Rhyzopertha dominicaa Sitophilus granariusa Sitophilus oryzaea Sitophilus zeamaisa Sitotroga cerealellaa Tenebrio spp. Tenebroides mauritanicus Tribolium spp. Trogoderma granarium

Rice moth Grain beetles Tropical warehouse moth Psocids European grain moth Saw-toothed grain beetle Stored nut moth Larger grain borer Indian meal moth Lesser grain borer Granary weevil Rice weevil Maize weevil Angoumois grain moth Meal worm Cadelle beetle Flour beetles Khapra beetle

Pulses Acanthoscelides obtectusa Callosobruchus chinensisa Callosobruchus maculatusa Zabrotes subfasciatusa

Dried bean beetle Adzuki bean weevil Cowpea beetle Mexican bean weevil

Oilseeds and Oilcakes Araecerus fasciculatus Caryedon serratusa Elasmolomus soriddus Ephestia cautella Necrobia rufipes Oryzaephilus mercator O. surinamensis Tribolium spp. Trogoderma granarium

Coffee bean weevil Groundnut borer Lygaeid bug Tropical warehouse moth Red legged ham beetle Merchant grain beetle Saw-toothed grain beetle Flour beetles Khapra beetle

Dry fruits and tree nuts Amyelois transitella Carpophilus spp. Corcyra cephalonica Cydia pomonellaa Ephestia calidella Ephestia cautella

Navel orange worm Dried fruit beetle Rice moth Codling moth Oasis dates moth Tropical warehouse moth (continued )

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TABLE I (continued ) Scientific name

Common name

Ephestia figulilella Ephestia elutella Lasioderma serricorne Oryzaephilus mercator Oryzaephilus surinamensis Paralipsa gularis Plodia interpunctella Stegobium paniceum Spectrobate ceratoniae Tribolium spp. Vitula edmandsae serratilinella

Raisin moth Tobacco moth Cigarette beetle Merchant grain beetle Saw-toothed grain beetle Stored nut moth Indian meal moth Drugstore beetle Carob moth Flour beetle Dried fruit moth

Beverage crops Araecerus fasciculatusa Ephestia cautella Ephestia elutella Hypothenemus hampeia Lasioderma serricorne Oryzaephilus mercator Ptinus tectus

CoVee bean weevil Tropical warehouse moth Tobacco moth Coffee berry borer Cigarette beetle Merchant grain beetle Australian spider beetle

Spices Araecerus fasciculatusa Lasioderma serricorne Stegobium paniceum Tribolium spp. Rhyzopertha dominica Ephestia cautella Plodia interpunctella

Coffee bean weevil Cigarette beetle Drugstore beetle Flour beetles Lesser grain borer Tropical warehouse moth Indian meal moth

Processed foods Cryptolestes spp. Ephestia cautella Ephestia kuehniella Lasioderma serricorne Liposcelis spp. Oryzaephilus surinamensis Plodia interpunctella Stegobium paniceum Tribolium spp.

Grain beetles Tropical warehouse moth Mediterranean flour moth Cigarette beetle Psocids Saw-toothed grain beetle Indian meal moth Drugstore beetle Flour beetles

Tubers (potato, cassava) Araecerus fasciculatusa Lasioderma serricorne Phthorimaea operculellaa Prostephanus truncatusa

CoVee bean weevil Cigarette beetle Potato tuber moth Larger grain borer (continued )


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TABLE I (continued ) Scientific name

Common name

Animal products Dermestes frischii Dermestes lardarius Dermestes maculatus Tribolium spp. Lasioderma serricorne Necrobia ruficollis Necrobia rufipes

Hide beetle Larder beetle Hide beetle Flour beetles Cigarette beetle Red-shouldered ham beetle Red-legged ham beetle

Others (Dried mushrooms) Othocis auriculariae Cis asiaticus Cis chinensis

Ciid beetles

a

Complete part of their life cycle inside grain, fruit, or other commodities.

stated that the odor is actually acrid or urinous, according to grain handlers and inspectors in the United States. Insect infestation in food commodities has health implications as well. The processing, cooking quality, and organoleptic properties may also be aVected in infested produce (Smith et al., 1971; Venkatrao et al., 1960b). Insects also play a significant role in the dissemination and proliferation of microorganisms including mycotoxigenic fungi in food commodities. In national and international trade, the channels of commodities that are infestation free are essential to avoid rejections. Smith et al. (1971) studied the baking and taste properties of insectinfested wheat flour. Changes in color, volume, size, and loaf characteristics and unacceptable taste and oV-odors in bread prepared from flour infested with Tribolium castaneum and T. confusum were observed. However, in the case of Tenebrio molitor and O. surinamensis, a similar eVect was observed, only with a higher level of infestation. Flour from wheat infested with R. dominica showed changes in rheological properties including dough stability, dough development times, water absorption, and mixing stability; bread prepared from the flour was darker, with poor crumb characteristics, and had an unacceptable oV-odor (Sanchez-Marinez et al., 1997). However, in studies on the eVects of Sitophilus granarius and R. dominica infestation in two varieties of durum wheat, Domenichini et al. (1994) did not find any significant changes in the rheological features of wheat flours and the quality of semolina prepared from wheat with a lower level of infestation.

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TABLE II EFFECTS OF INSECT INFESTATION ON STORED FOOD COMMODITIES

Effect

Insect

Commodity

Reference

Chemical composition altered

Rhyzopertha dominica

Wheat

Sitophilus oryzae, Tribolium castaneum, Trogoderma granarium Sitophilus oryzae

Wheat

Saxena and Singh (1994) Sharma et al. (1979)

Liposcelis paetus Trogoderma granarium, Rhyzopertha dominica

Milled rice Maize, sorghum, wheat Sorghum

Tribolium castaneum Several species Callosobruchus maculatus

Callosobruchus chinensis

Milled rice

Sorghum Cowpea, bombara groundnut Cowpea

Green gram Chickpea Green gram, Red gram Red gram

Acanthoscelides obtectus

Pulses

Corcyra cephalonica, Oryzaephilus surinamensis, Tribolium castaneum, Necrobia rufipes Stegobium paniceum, Lasioderma serricorne

Peanut

Araecerus fasciculatus, Rhyzopertha dominica, Sitophilus oryzae Araecerus fasciculatus

Turmeric, coriander powder Cassava

Coffee

Sudhakar and Pandey (1987) Pike (1994) Jood et al. (1992, 1993a,c, 1995, 1996) Pant and Susheela (1977) Arora et al. (1993) Emefu et al. (1992)

Ojimelukwe and Ogwumike (1999) Singh et al. (1982) Modgil and Mehta (1996) Modgil and Mehta (1994) Daniel et al. (1977) Regnault-Roger et al. (1994) Kadkol et al. (1957)

Gunasekaran et al. (2003) Padmaja et al. (1994); Premkumar et al. (1996) Narasimhan et al. (1972) (continued )


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TABLE II (continued ) Effect

Insect

Commodity

Reference

Changes in nutritional quality

Trogoderma granarium, Rhyzopertha dominica

Wheat

Jood and Kapoor (1992a,b); Jood et al. (1993b) Jood et al. (1993b) Nirmala and Kokilavani (1980) Jood and Kapoor (1992b) Ojimelukwe and Ogwumike (1999); Ojimelukwe et al. (1999) Rajan et al. (1975)

Sitophilus oryzae

Callosobruchus maculatus

Callosobruchus chinensis Off-odors

Rhyzopertha dominica, Tribolium castaneum Rhyzopertha dominica

Changes in end-use products/quality Sitophilus granarius, Rhyzopertha dominica Trogoderma granarium, Rhyzopertha dominica Tribolium confusum, Ephestia kuehniella Tribolium castaneum Tribolium confusum, Tribolium castaneum, Tenebrio molitor, Trogoderma granarium, Oryzaephilus surinmensis Oryzaephilus surinamensis, Tribolium castaneum Callosobruchus maculatus

Sorghum Finger millet Wheat, maize, sorghum Cowpea

Cowpea, maize Sorghum Wheat Wheat Wheat, maize, sorghum Wheat flour

Seitz and Sauer (1996) Sanchez-Marinez et al. (1997) Fogliazza et al. (1993) Jood et al. (1993c)

Wheat flour

Fogliazza et al. (1993) Venkatrao et al. (1960b) Smith et al. (1971)

Wheat flour

Pagani et al. (1996)

Cowpea

Ojimelukwe and Ogwumike (1999) Emefu et al. (1992)

Wheat flour

Cowpea, bombara groundnut Peanut

Ephestia cautella, Corcyra cephalonica, Oryzaephilus surinamensis, Tribolium castaneum Peanut Caryedon serratus, Tribolium castaneum, Trogoderma granarium

Srivastava (1970)

Davey et al. (1959)

(continued )

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TABLE II (continued ) Effect

Insect

Commodity

Reference

Sitophilus oryzae Trogoderma glabrum

Wheat (granary) Cereal

Kleine-Tebbe et al. (1992) Gorham (1989)

Tribolium castaneum

Wheat flour

Plodia interpunctella, Ephestia cautella Plodia interpunctella

Wheat

Sitophilus oryzae

Maize

Sitophilus zeamais

Maize

Sitophilus oryzae, Tribolium castaneum Sitophilus oryzae, Tribolium castaneum Sitophilus oryzae, Rhyzopertha dominica, Tribolium castaneum Ephestia kuehniella Ephestia cautella, Stegobium paniceum, Tribolium castaneum, Oryzaephilus surinamensis Corcyra cephalonica Chrysomya spp.

Maize Wheat, maize

EM-Mofty et al. (1992) Demianyk and Sinha (1981) Abdel-Rahman et al. (1969) Ragunathan et al. (1974) Dharmaputra et al. (1994) Sinha and Sinha (1992) Sinha (1994)

Rice

Pande et al. (1989)

Health implications 1. Inhalant allergy 2. Ulcerative colitis 3. Carcinogenic quinones 4. Association with toxigenic fungi or pathogenic microbes

Maize

Wheat flour Cravedi et al. (1993) Peanut, cumin, Srinath et al. (1976) wheat flour, copra Peanut Dried fish

Asaf et al. (1977) Garg (1977)

Venkatrao et al. (1960b) noted that T. castaneum infestation in wheat flour resulted in deterioration in the quality of gluten; in addition, the bread prepared from infested flour had low loaf volume, poor organoleptic quality (i.e., bitter taste), and oV-odor problems. Similar eVects were also reported in wheat flour prepared from wheat damaged by insect pests in the field itself. Adverse eVects on the processing quality of flour including slimy gluten and reduced volume of bread were caused by certain true bugs (Insecta: Pentatomidae) that feed on wheat maturing on the plant. The wheat bugs including Eurygaster austriaca, Aelia rostrata, Chlorochroa sayi, and Sitodiplosis mosellana in the United States, Europe, and Syria and Nysius huttoni in New Zealand damage the wheat in the developing plant by their feeding activity, and some of the proteinases in insect saliva


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are believed to be responsible for poor end-use quality of the flour (Every et al., 1998; Hariri et al., 2000; Swallow and Every, 1991). Insects are directly or indirectly associated with the occurrence of molds and increased mycotoxin levels in food commodities (Dunkel, 1998). Increased insect activity results in heating and higher moisture content, favoring mold growth. Moreover, insects themselves are involved in disseminating mold in food grains and other commodities. Higher levels of mold/mycotoxins as a consequence of increased insect activity in maize (Dharmaputra et al., 1994; Sinha, 1994; Sinha and Sinha, 1992), almonds (Schatzki and Ong, 2001), pistachio nuts (Doster and Michailides, 1999), and yam (Morse et al., 2000), and wheat have been reported (Table II).

III. DETECTION OF INSECTS IN SAMPLES Various methods are used to detect insect infestation in commodity samples (Table III). The choice of method depends on whether the infestation is inside or outside food grains, in the surrounding premises or inside bulk grain, availability of equipment facilities, and required sensitivity. These methods are deployed for diVerent types of food commodities in diverse storage situations, and the accuracy of detection varies. Most of the methods have the objective of detecting the presence of live insects either directly or indirectly. Free-living (external) insects are detected by visual inspection, sampling, sieving, and heat-extraction methods, whereas hidden (internal) infesters are detected by radiography, staining techniques, and near-infrared and fragment count methods. Infestation can also be detected indirectly by determining uric acid or CO2 level. Methods for the detection of both living and dead insects are rather limited (fragment count and enzyme-linked immunosorbent assay [ELISA] methods). In general, these methods are mainly concerned about insect detection in cereals only; nevertheless, they are also useful for other commodities such as pulses and oilseeds. A. VISUAL INSPECTION

Several clues indicate the presence of insect infestation in stored foods (Table IV). The presence of eggs of pulse beetles such as Callosobruchus spp. can be easily seen in infested pulses with the naked eye. Similarly, the exit holes of internal infesters such as Sitophilus spp., R. dominica, Prostephanus truncatus, and S. cerealella are clearly visible in infested food grains. In the case of khapra beetle (T. granarium) infestation, the exuviae of the larvae are indicators of the presence of the pest. Infestation by moth pests including E. cautella, Plodia interpunctella, and Corcyra cephalonica is

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TABLE III INSECT DETECTION METHODS APPLICABLE FOR COMMODITY SAMPLES

Test method

Applicability

Comments

Whole grains, milled products Whole grains, milled products Whole grains Whole grains Whole grains

Qualitative; only high-level infestation detected Hidden infestation not detected; commonly practiced Adults and larvae detected Active stages are detected Time consuming

Whole grains Whole grains, milled products Whole grains

Prohibitive capital cost Rapid, expensive, can be automated

Serological techniques

Whole grains, milled products

Uric acid determination CO2 analysis

Whole grains, milled products Whole grains

Specific gravity method

Whole grains

Cracking and flotation method Fragment count

Whole grains

Highly sensitive, species specific; shows infestation from unknown past to till date Shows infestation from unknown past to till date Simple, time consuming; indicates current level of infestation; not suitable for grains having >15% moisture Simple and quick; not suitable for oats and maize Variable results noted

Physical methods Visual inspection Sampling and sieving Heat extraction Acoustic Breeding out Imaging techniques: X-ray method Near infrared spectroscopy Nuclear magnetic resonance

Less sensitive

Chemical methods

Staining techniques: Egg—plugs Ninhydrin method

Whole grains, milled products

Highly variable results noted; shows infestation from unknown past to till date

Whole grains Whole grains

Specific for Sitophilus spp. Eggs and early larvae not indicated

marked by the presence of webbing or silken strands and large fecal particles in a sample of grain or grain product. Doster and Michailides (1999) reported that in stored pistachio nuts in processing plants in California, shell discoloration could be a visible indicator of infestation by the navel orange worm, Amyelois transitella, and associated fungal decay. Visual examination of grain for ‘‘exit holes’’ or ‘‘windows,’’ which are rounded holes with smooth edges having a minimum diameter of about 0.8 mm, has


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TABLE IV VISIBLE INDICATORS OF INSECT INFESTATION IN STORED FOOD COMMODITIESa

Indication

Commodity

Insect species

Eggs on grain surface Pupal cases on shells Exit holes

Pulses (whole)

Acanthoscelides obtectus, Callosobruchus spp., Zabrotes subfasciatus Caryedon serratus

Shell discoloration Silken strands present

Peanut in shell Cassava Coffee seed Peanut in shell Pulses (whole) Spices (whole) Wheat, rice, maize, paddy Pistachio nuts Cereals (whole or milled) Cocoa Oilseeds, oilcakes/meals Dry fruits, tree nuts

a

Araecerus fasciculatus Araecerus fasciculatus Caryedon serratus Acanthoscelides obtectus, Callosobruchus spp., Zabrotes subfasciatus Stegobium paniceum, Lasioderma serricorne Rhyzopertha dominica, Sitophilus spp., Sitotroga cerealella Amyelois transitella Corcyra cephalonica, Ephestia cautella Plodia interpunctella, Ephestia cautella, Ephestia elutella Ephestia cautella, Plodia interpunctella Ephestia cautella, Ephestia calidella, Plodia interpunctella

Adapted from Rajendran (1999).

been proven as a rapid method for detecting internal infestation (Nicholson et al., 1953). These workers noted that the correlation coeYcient between visual examination for exit holes and cracking test (described in Section III.C.2) was 0.73. Visual examination of food grains for insect exit holes is an indirect measure of detecting internal infestation and it is a qualitative test only. B. SAMPLING AND SIEVING

In both developing and developed countries, for insect pest detection in food grains, the sampling and sieving method is commonly practiced. The method is labor intensive and indicates the current infestation instantaneously (Hagstrum, 1994). Hidden infestation of eggs, larvae, and pupae, however, is not detected. In this simple method, representative samples are drawn from a stock of static bulk grain or grains in transit, are sieved, and are visually checked for the presence of insect pests. There are no international standards for the sampling and sieving method; yet there are national standards for use locally (Wilkin et al., 1994). Some of the world’s major

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agencies that deal with food grains (i.e., the U.S. Federal Grain Inspection Service, the Australian Wheat Board, the Canadian Grain Commission, and the Food Corporation of India) rely on sieving and inspection for living insects in the grains to detect infestation. Various types of grain samplers are used including the bag samplers (triers), deep bin cups, pelican samplers, Ellis cup, mechanically operated vacuum probes, and grain diverters. These are used for taking grain samples (0.5–1 kg) from diVerent kinds of storages (bag-stacks, bins, elevators) and transport containers (railcars, barges, and ships). The grain samples are sifted to recover the active free-living adults and larvae. Sieving is done either manually or mechanically depending on the number of samples to be examined or on the availability of facilities. Manual sieving is, however, very common, and the sieve mesh size varies according to the size of the grain or its product. For food grains, standard sieves of No. 10 or 12 having about 2-mm mesh size are used for separating the insects. The sampling and sieving method has been proven reliable, provided that the infestation is high at more than five insects per kilogram and when there is homogeneous distribution of insects in the grain mass (Wilkin and Fleurat-Lessard, 1991). The accuracy and reliability of grain sampling has been reported to be dependent on factors such as (1) the frequency and distribution of samples, (2) frequency and distribution of insects in the commodity, and (3) the eYciency of removal of insects from the samples (Hagstrum, 1991). It has been noted that insects such as S. zeamais, which are not uniformly distributed, are likely to be missed during sampling (Hodges et al., 1985). Tests were conducted simultaneously in France and the United Kingdom to compare the performance of two types of conventional sampling spears: a single compartment gravity spear that can collect 200-g lots and a multicompartmented (8 or 11 compartments) spear capable of collecting 300-g grain samples. Wheat samples were drawn at three depths using the spears from metal bins containing 20 tons of grains to which a known number of dead and live insects (S. granarius and O. surinamensis) had been added and checked for infestation level. It was observed that there was no significant diVerence between the two spear types in getting representative samples containing insects (Wilkin and Fleurat-Lessard, 1991). In experiments in the United Kingdom, wheat samples to which were added adults of O. surinamensis or S. granarius at one and five insects per 500-g sample and sieved (2-mm mesh sieve used) manually, there was complete recovery of the insects, confirming the accuracy of the method (Wilkin et al., 1993). White (1983) used an inclined plane sieve for pest detection in wheat grains in 30-kg samples. It was noted that by this method, it was possible to recover about 90% of T. castaneum and R. dominica added to wheat in 4–5 minutes. However, about seven runs of repeated sieving were necessary for complete recovery of T. castaneum, four


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for S. oryzae, and three for R. dominica. The presence of dockage in the commodity delayed the recovery of the insects due to blockage of holes of 1.6 mm in the wire mesh. Pereira et al. (1994) compared sampling and sieving with that of a probe trap method for insect detection in 1200 tons of wheat in a silo in Brazil. Observations made every 15 days during 7 months revealed that the data from the sieve method were not consistent when compared with that of the probe trap method. The total number of insects such as Ahasverus advena, Cryptolestes spp., Alphitobius spp., T. castaneum, Sitophilus spp., R. dominica, and Typhaea stercorea collected by trapping was significantly higher than that obtained by the sampling and sieving method. However, it was noted that, unlike the sieve method, it was diYcult to interpret the trap catch. Mechanical sieves have also been used in some of the developing countries for detecting insect pests in stored grains. A prototype machine capable of recovering insect pests occurring even at a low density of 0.2 insects/kg in 10 kg of wheat (and barley) in a short period of 1.8 minutes has been developed in the United Kingdom (Wilkin et al., 1994). The sampling and sieving method, in spite of its variable eYciency, is still popular and widely used in the trade due to time constraints and the advantage of getting results within a few minutes (Hagstrum and Subramanyam, 2000). C. FLOTATION METHODS

Flotation methods deployed for detecting internal or hidden infestation in whole cereals and pulses are of two kinds: (1) the specific gravity method wherein a whole grain sample is directly tested with a suitable salt solution having a specific gravity less than that of the grain, and (2) the crackingflotation technique in which whole grain is coarsely ground and treated with a mixture of an alcohol solution and light mineral oil so that the exposed insect particles, which are lighter than the grain, float on the top surface. 1. Specific gravity method White (1957) developed the specific gravity, or densitometric, method for detecting hidden infestation in whole grains for use by grain elevator operators in the United States. This method has also been used for the detection of a dipteran, Rhagoletis mendax in blueberry fruit, Vaccinum angustifolium (Dixon and Knowlton, 1994). The larvae of stored grain insects such as Sitophilus oryzae, S. granarius, S. zeamais, Callososbruchus spp., and Zabrotes subfasciatus feed inside the grains, creating a cavity and thus reducing grain density. This diVerence in the density between sound (uninfested) grain kernels and infested grains is exploited for the detection of infestation

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S. RAJENDRAN

using salt solutions. Accordingly, when a mixture of uninfested and infested grains is immersed in a salt solution of appropriate density (normally 1.19 g/cm3), for about 10 minutes, the heavier uninfested grains sink to the bottom while the lighter infested ones float. The flotation media include salt solutions like sodium silicate and sodium chloride or glycerol in water (ISO, 1987). The selection of salt solution of suitable density is very critical for infestation detection. Richter and Tchalale (1994) examined the importance of the density of the flotation medium on the eYciency of pest detection. The sensitivity and accuracy have been noted to be dependent on the use of product-adapted solutions. An adaptation coeYcient, Qa, has been suggested for preparing suitable floating media: Qa ¼

Mean density of the product Density of the salt solution

If the Qa is closer to 1.0, the adaptation of the flotation medium has been found better and the results have less error. The authors tested salt solutions (sodium chloride and sodium nitrate) of diVerent densities to detect S. oryzae infestation in wheat and Zabrotes subfasciatus in legumes, and it was noted that the optimum density of the solutions for detecting infested grain was 12–13% below the average density of uninfested grains (corresponding to a Qa value of 1.04). The kernel density may vary even according to variety and cultivar of the grain. The suggested density values for the floating medium for wheat is 1.15; for sorghum, 1.19; and for rice and peas, 1.27 (Semple, 1992). To detect C. chinensis in dried peas by flotation, Hurlock (1963) used a sodium silicate solution and observed varying results when the specific gravity of the solution varied from 1.27 to 1.37. However, Somerfield (1989) detected infestation of Bruchus pisorum in dried peas with flotation even at a lower level of 0.5–1.0%. The specific gravity method is a qualitative test and does not indicate the species or the specific life stage present inside the grains; it is not suitable for hulled seeds such as barley, oats, and paddy and for large-seeded grains like corn (Pedersen, 1992). The method is simple and quick and requires minimum laboratory facilities. Because of low weight, defective grains such as shriveled ones will also float with the infested grains during the test. Hence, there is a need to confirm by dissecting the floating grains and check for the presence of insects. Grains containing only eggs or early larval stages cannot be detected because there will be very little diVerences in the density of grains infested with these stages to make them float. Furthermore, the earliest date of pest detection in food grains will vary because the rate of feeding activity of the larvae developing inside the grain (and thereby the damage caused or cavity created) diVers between species.


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A densitometric separation method employing air instead of a solution has also been used to detect pest infestation (Milner et al., 1953). In addition to infested kernels, grains with exit holes, which are indicative of hidden infestation, are also separated. 2. Cracking-flotation method The cracking-floatation procedure, an oYcial method used in the United States (Association of OYcial Analytical Chemists [AOAC], 1997), involves grinding of whole grain such as wheat to a particle size of about 1.5 mm so that the internal insect stages are exposed; the coarsely ground grain is then mixed with an alcohol solution and light mineral oil to enable the insect stages and parts comprising whole adults, larvae or pupae, adult heads, larval head capsules, and cast skins from larvae, pupae, or adults to float to the surface. The latter are then collected on a filter paper and examined under a microscope (Harris et al., 1952). In this method, in addition to the actual number of insects, diVerent life stages present in the sample are observed. A level of detection of larvae of 95–97% has been reported. Thind and GriYths (1979) developed a flotation technique for the detection and determination of mites in animal feeds. The method was later modified for the determination of mite and insect contaminants in food and foodstuVs with improved sensitivity (Thind, 2000). When a mixture of industrial methylated spirit (46% by volume) and glycerol (54%) was used as flotation medium, the recovery of mites and contaminants including insect (T. castaneum) fragments in diVerent types of foodstuVs (dried fruits and nuts, wheat, barley, dried milk, various types of flour, and wheat germ) was noted to be 83% and 89%, respectively. The cracking-flotation method has been compared with other analytical techniques. In his comparative study of four analytical methods for detecting S. granarius in wheat at three levels of infestation over a period of 48 days, Russell (1988) noticed that the results of x-ray and cracking-flotation methods were comparable. Brader et al. (2002) conducted a collaborative study of the detection of insect contamination in wheat by x-ray analysis, cracking-flotation, insect fragment test, and the ELISA technique in three laboratories. Large variations in the results of the cracking-flotation method between laboratories were observed; moreover, false negatives (i.e., zero results with 10 infested kernels) have also been encountered. Xingwei et al. (1999) compared the flotation method with a rearing method, the ninhydrin method, the x-ray method, and the CO2 method for the detection of hidden infestation of S. zeamais, R. dominica, and S. cerealella in wheat and rice and C. chinensis in mung bean. The flotation method was the least accurate in determining the insect population in the commodities.

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S. RAJENDRAN D. FRAGMENT COUNT OR ACID HYDROLYSIS METHOD

Pest infestation in food commodities can also be detected by the presence of fragments of insects such as elytra, head capsules, mandibles, and other sclerotized parts. The fragment count method basically involves a flotation technique but in a modified way. In the flotation method as applied for whole grains, simple salt solutions are used as a floating medium, whereas in the fragment count method, a mixture of oil and aqueous phase (besides surfactants) is used and the interfering food materials are digested with acid before allowing the insect fragments to float. The filth test (AOAC, 1997) for the determination of extraneous matter like insect fragments and rat hairs in diVerent processed foods is based on the principle of fragment count method only. The test involves digestion of a sample with an acid, wet sieving, or a defatting treatment using a detergent or solvent. Then the insect fragments and rat hairs, which are oleophilic, are separated from food particles by the attraction of the oil phase (light mineral oil in an oil–aqueous mixture). The floating fragments are trapped or filtered and examined under a microscope (Dent and Brickey, 1984). Infestation detection by the fragment count method is applicable for various types of processed foods such as flour, chocolate, and powdered spices. However, it requires a trained person to carry out the analysis (Bair and Kitto, 1992; Gentry et al., 2001). In developed countries, there are tolerance limits for insect fragments in processed foods. In the United States, the Food and Drug Administration (FDA) allows a tolerance limit (Defect Action Level) of 75 insect fragments per 50 g of wheat flour. However, the type or size of the insect fragments is not taken into consideration in the total count. Hence, in advanced countries the fragment count method is very important for the food industries and millers. The number of insect fragments present in a processed food is influenced by the method of grinding or processing that the material has undergone. In a study of insect contamination of wheat and wheat flour in 16 mills in the United States, over a period of 12 months, Harris et al. (1952) observed that the ratio of insect fragments in flour to whole insects in the wheat from which it was made varied from mill to mill (13.7 to 1.0). In a comparative study on the determination of insect contamination in grains, Brader et al. (2002) observed wide variation in results of the fragment count test as reported by three laboratories when supplied with wheat samples containing a known number of grains infested with S. granarius. In addition, false positives have been noticed; in the absence of infested kernels, the three laboratories observed 5–33 fragments. In the extraction of insect fragments, the surfactants and the mixture of oil–aqueous phases vary depending on the type of foodstuVs analyzed. For certain food commodities, acid digestion followed by washing with suitable


181

surfactants and sieving, the fragments could be extracted. Accordingly, for the extraction of light filth comprising insect fragments and rodent hairs in diVerent types of cheeses, Nakashima (1994) used Tergitol Anionic 4 and 1% sodium lauryl sulfate solution as surfactants for washing after digesting the sample with 5.7% HCl containing the emulsifiers Igepal CO-730 and Igepal DM-710. The recovery of added insect fragments (elytra of T. castaneum) was about 96% 0.95 in the collaborative study. Similarly, insect fragments in oriental fish products containing spice were isolated by acid digestion, sieving after washing with surfactants, and extraction. About 84–89% recovery of added T. castaneum fragments was reported (Glaze, 1993). To detect insect fragments in grain products such as whole-wheat flour, the sample was digested in a 3% HCl solution, the residue was defatted by boiling in isopropanol and the mixture was run through a No. 230 sieve. The lighter filth containing insect fragments and rat hairs was trapped with mineral oil in a mixture of tween 80 and Na4 EDTA in 40% isopropanol. In flour samples that had been added with insect fragments (elytra of T. castaneum) at the levels of 5, 15, and 30 fragments 50 g 1, the recoveries of insect fragments in the tests ranged from 86.2% to 94.0% (Glaze and Bryce, 1994). The oil–aqueous phase system has the advantage of enhancing the lifting power of the oil globules that attract and concentrate the insect fragments (and rodent hairs). The size and shape of the oil globules can influence the extraction time and the type of fragments extracted. The oil globules are typically big when a simple mineral oil–water mixture is used for trapping the fragments. When ethanol or isopropanol is added to the aqueous system, the oil globules become smaller and the extraction time becomes longer, but the numbers of smaller fragments trapped are increased (Dent and Brickey, 1984). E. STAINING TECHNIQUES

Staining secretions (egg plugs) or body fluids of insects (hemolymph) and entry holes as a means of detecting hidden insect infestation in food commodities were considered as early as the 1950s. Staining, a chemical indicator technique, is a direct method of establishing hidden living infestation in a commodity. Staining techniques are of three types (Table V). In the first method, mucilaginous secretions of weevils are stained with a suitable chemical compound. Weevils including S. oryzae, S. granarius, and S. zeamais attacking stored cereals deposit their eggs inside the grains and plug the holes or egg cavities with saliva. Using suitable coloring agents, the egg plugs in grains can be stained and identified. The extent of infestation in a grain sample is estimated by the number of egg plugs observed. The egg plugs are likely to be dislodged during grain handling, so there are chances of

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TABLE V DETECTION OF INSECT INFESTATION BY STAINING TECHNIQUES

Chemicals used

Color spot

Reference

Specific for weevil egg plugs in grains Acid fuchsin Gentian violet Berberine sulfate

Cherry red Purple Greenish yellow

Frankenfeld (1948) Goossens (1949) Milner et al. (1950a)

Specific for weevil entry holes in pulses Iodine-potassium iodide

Black

Frankenfeld (1948)

General infestation in whole grains Ninhydrin

Purple

Ashman et al. (1970); Dennis and Decker (1962)

recording less than the actual infestation level (Potter et al., 1952). Moreover, this technique is not applicable for other internal infesters such as R. dominica and S. cerealella that oviposit outside the grain. When 0.5% acid fuchsin or 1% gentian violet is used for detecting insect egg plugs in cereals, it was noted that both grains damaged by insect feeding activity and those damaged by handling and mechanical operations that exposed endosperm also are stained by the compounds, resulting in false positives; only after careful examination can one diVerentiate damaged grains that are colored only at the periphery from that of egg plugs, which are stained completely (Fleurat-Lessard, 1988). To overcome this problem, Milner et al. (1950a) used 20% berberine sulfate, an alkaloid, as the coloring agent where the egg plugs are stained fluorescent yellow when observed under ultraviolet (UV) light of 366-nm wavelength. Reed and Harris (1953) compared the staining methods using berberine sulfate and acid fuchsin in the detection of egg plugs in wheat and corn; the acid fuchsin test proved more sensitive than the berberine sulfate test. The second method involves staining larval entry holes in pulse seeds. In the third method, ninhydrin (triketohydrindene hydrate) has been used to react with the body fluids of insects developing inside grains. A purple spot develops when ninhydrin reacts with the free amino acids (and keto acids) present in the body fluids of insects (Dennis and Decker, 1962). Ninhydrin at a 0.3% level in acetone is impregnated into filter paper. When infested grains are crushed between the folds of ninhydrin-treated paper, the body fluids from the crushed insect bodies react with ninhydrin in the paper. Purple spots develop in the paper in 20–30 minutes at room temperature (20–25 8C), the number of spots indicating the number of insects present.


183

The development of color spots can be hastened if the paper is heated to 50 8C for 5–10 minutes. The intensity of the color spots developed depends on the life stage or size of the insect. The infestation level in the sample is expressed as the number of hidden insects (color spots developed) per kilogram of grain. An automated machine with a flow rate of 300 kernels/ min and with the ninhydrin treated paper preheated to 120 8C for rapid color spot development was developed in the United States to detect hidden infestation in grains (Dennis and Decker, 1962). Subsequently in the United Kingdom, a smaller and portable machine known as the ‘‘Ashman-Simon infestation detector’’ has been developed (Ashman et al., 1970); the latter has been claimed to detect 5–10% of eggs and early larvae, 40–60% of middle age larvae, and 80–90% of mature larvae in cereals. The ninhydrin staining method is simple and it needs very little training to conduct the test. The drawbacks of this technique include the following: (1) Eggs and early larvae are not detected, (2) grain having higher moisture content of 15% or more or having fungi gives a color reaction even in the absence of insect infestation, and (3) the ninhydrin-impregnated paper develops purple spots when touched by hand because of the contact of amino acids in perspiration on the fingers. In a comparative study on detecting hidden infestations of S. zeamais, R. dominica, S. cerealella, and C. chinensis in cereals and pulses by five detection methods (rearing method, CO2 method, flotation method, ninhydrin method, and x-ray method), Xingwei et al. (1999) observed the following order (from high to low) in terms of accuracy in detection: rearing method > ninhydrin method > x-ray method > flotation method, and the CO2 method was rapid but not quantitative. However, when comparing different detection methods for dried peas infested with C. chinensis, Hurlock (1963) reported that the staining methods are unsuitable. With 0.002% berberine solutions, he noted that there was no yellow fluorescence of the bruchid eggs when observed under UV light. When 1% iodine in potassium iodide solution was used for staining, in addition to insect eggs, the exit holes and other damaged parts in the sample were stained black, making it diYcult to diVerentiate. A similar problem was encountered when acid fuchsin was used as the staining medium. F. CO2 ANALYSIS

Howe and Oxley (1944) proposed the use of carbon dioxide (CO2) produced in food grains and grain products as an indicator of insect infestation, particularly hidden infestation. The intergranular air in normal grain, which is free from insect infestation, contains about 0.03% CO2. This level varies depending on the moisture content of the grain because of the metabolism

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of the grain itself; the higher the moisture content, the greater the CO2 level. But in the presence of pest activity, the CO2 level increases more rapidly. The pests responsible for CO2 output include insects, microorganisms, and mites. The CO2 or respirometric method involves incubation of the grain or its product in an airtight enclosure for 24 or 48 hours at controlled temperatures (258 or 35 8C), and then estimating the intergranular CO2 content with a suitable instrument. When the grain moisture content is less than 15% and is free from insect infestation, the production of CO2 by 1 kg of grain in 24 hours at 35 8C will be less than 0.25%. However, when there is insect or any other pest infestation, the CO2 level increases proportionately. A CO2 concentration of about 0.5% or more is indicative of high-level pest activity in the sample. The CO2 content of the intergranular air can be measured by a gasometric method or an infrared gas analyzer. The infrared gas analyzer is relatively sensitive and can measure low-level infestation (Bruce and Street, 1974; ISO, 1987). The interpretation of the results based on CO2 concentrations depends on whether a gasometric or infrared gas analysis is used (Table VI). When an infrared gas analyzer is used, the incubation time for CO2 measurement can be reduced to less than 24 hours (Fleurat-Lessard, 1988). In a laboratory study, Street and Bruce (1976) showed that the presence of a single larva of E. cautella in stored dates could be detected by measuring CO2 concentration with a Luft type of infrared gas analyzer. The CO2 evolved can also be determined by a gas chromatograph with a TCD detector or interference refractometer. Among the instrumental methods of analysis

TABLE VI PEST INFESTATION LEVELS IN FOOD GRAINS AS INTERPRETED ACCORDING TO THE METHOD OF INSTRUMENTAL ANALYSIS OF CO2 IN 1 KG OF GRAIN AFTER INCUBATION FOR 24 HOURSa

Method Gasometric (% CO2 v/v)b

Infrared (ml of CO2/min)

Level of infestation

1.0

6.0

Nil or negligible Low level Light to moderate Moderate to heavy Heavy

a

From ISO (1987). Applicable for wheat, peas, polished rice, small yellow maize, and similar small huskless hard grains; for others, the CO2 concentration is multiplied by correction factors (e.g., large white maize, 1.18; barley, 1.25; and oats, 1.39).

b


185

for CO2 in the grain samples, the infrared gas analyzer has proven to be relatively sensitive, the response is quicker, and it is easy to operate (Zisman and Calderon, 1991). CO2 measurement is an indirect method of detecting an existing insect infestation. The respiratory rate of insect eggs or early larval stages is negligible, so the CO2 method is not applicable for grains having only those life stages. Also, it cannot be used for grains with moisture content exceeding 15%, because at higher moisture levels, grain alone evolves more CO2 (Semple, 1992). The intergranular CO2 level in an incubated sample is also aVected by the absorption of the gas by the grain itself (water solubility of CO2 is 0.76% v/v). Furthermore, the CO2 concentration inside the enclosure/container is also dependent on its air tightness, as CO2 has a high vapor pressure (71 atm; i.e., 54,000 mm Hg, at 30 8C), and hence, it will tend to leak. Infestation detection by CO2 analysis is only a qualitative test and does not diVerentiate between CO2 production by insects, microorganisms, or mites individually. Even among insects, the rate of production of CO2 varies among species and between life stages of single species. In a laboratory study on wheat infested with 12 adults of T. castaneum and C. ferrugineus, separately and in mixed population in individual jars at 27.58 and 33 8C, Sinha et al. (1986a) observed that T. castaneum produced more CO2 than C. ferrugineus during the experimental period. Generally, the older immature stages of insects produced more CO2 than the adults. In S. oryzae, fourth instar larvae produced 0.25 ml of CO2/min 1, whereas the adults produced 0.01 ml of CO2/min 1 only. Hence, the minimum number of insects required to produce a measurable amount of CO2 or the detection threshold in grains varies (Howe and Oxley, 1952). In a laboratory study in Israel, Zisman and Calderon (1991) noted 0.15–0.30% CO2 in 48 hours in 1 kg of wheat (11.5% moisture content) samples having one or two insects of the species T. castaneum, S. oryzae, and R. dominica at 26 8C. In a 24-hour test at 35 8C with dried peas infested with C. chinensis, Hurlock (1963) estimated about 4.9% of CO2. Xingwei et al. (1999) determined hidden infestation in cereals and pulses using five methods and concluded that the rearing method followed by CO2 measurement at 25 8C for 24 hours is the best. The CO2 analysis method is also applicable for monitoring infestation in farm grain storage bins. Detection of pests and grain quality deterioration in wheat, barley, and maize stored in airtight steel bins in Canada and the United States was accomplished by checking CO2 levels inside bins. The insects present in the bin included Cryptolestes spp., T. castaneum, and Plodia interpunctella. CO2 concentrations up to 2% were recorded in infested bins (Sinha et al., 1986b); in uninfested bins, the CO2 level was only 0.03%. CO2 measurement is particularly suitable for the detection of the insects in food grains in transit and for grains intended for binning/loading in silos,

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and it requires minimum apparatus/facilities. Furthermore, a large number of samples can be analyzed for CO2 content simultaneously. G. URIC ACID DETERMINATION

The excreta of stored product insects are composed primarily of uric acid. However, in some species, the presence of other purines in lesser proportions has been reported. Ammonia is found in the excreta of larvae of Ephestia kuehniella, urea and allantoin in Tenebrio molitor, and both urea and xanthin in larval excreta of C. cephalonica (Bursell, 1967). Uric acid has been used as an indicator of insect infestation in cereals and cereal products since the 1950s (Subrahmanyan et al., 1955; Venkatrao et al., 1957). Subsequently, several methods have been described to determine uric acid levels in infested foodstuVs (Table VII). Most of these methods are modifications of tests originally developed for analysis in clinical samples. Pachla et al. (1987) have reviewed the methods of uric acid determination in foodstuVs and biological fluids. In the earliest colorimetric methods, uric acid was determined using Benedict’s reagent (Benedict and Franke, 1922). In the reaction, either sodium cyanide or molybdate and arseno-tungstate reagents were used or the detection limit in these methods was low at 8 mg/g. It was then realized that in the colorimetric method, some of the uric acid–like substances also react with the color reagent, giving exaggerated values for uric acid. This was proven by the reaction of extract from the foodstuVs with and without uricase enzyme. The enzyme uricase specifically breaks down uric acid and the values obtained for uric acid–free sample are known as apparent uric acid. The ‘‘apparent’’ uric acid content was more in infested pulses than in cereals (Venkatrao et al., 1959). The apparent uric acid was deducted from the values obtained for the samples without any uricase enzyme activity (‘‘total’’ uric acid), to arrive at the ‘‘actual’’ uric acid (Pillai et al., 1975). The ‘‘total’’ uric acid indicates excretory substances produced by insects and the metabolites released by fungi. Thus, the total uric acid content in a product depends on its insect population, mold count, and moisture damage to the food grain. In the colorimetric method, which involves the reaction between phosphomolybdic acid and uric acid, the intensity of color developed for measurement was relatively less. However, the sensitivity was better (detection limit 8 mg/g) than when sodium cyanide and arseno-tungstic acid were used for colorimetry (Majumdar and Agarwal, 1991). In addition to the low sensitivity of the colorimetric methods, there are problems such as risk when using sodium cyanide or arseno-tungstate as one of the reagents and interference due to turbidity of the extract; also, they are not useful for highly colored spices. Joshi et al. (1985) improved the oYcial method of infestation detection (Bureau of Indian Standards [BIS], 1970) in food


187

TABLE VII METHODS FOR DETERMINING URIC ACID IN FOODSTUFFS

Method

Commodity

I. Colorimetry

Detection limit (mg/g)

Reference

Cereals

8.0

Majumdar and Agarwal (1991); Subramanyan et al. (1955); Venkatrao et al. (1959)

Cereal Cereal Cereal Cereal

product product product product

100 0.5 32.0 0.6

Farn and Smith (1963a) Sen (1968) Sen and Smith (1966) Sen and Vazquez (1969)

Spices

1.0

Brown et al. (1982)

Cereal product Cereals

20.0 0.1

Holmes (1980) Lamkin et al. (1991)

Cereal and cereal products Cereal products

1.0

Wehling and Wetzel (1983) Pachla and Kissinger (1977)

Spices

5.0

Sengupta et al. (1972)

Cereal product

100

Venkatrao et al. (1960a)

Cereal product

1.0

Wehling et al. (1984)

II. Enzymatic A. Direct method a. Using UV b. Colorimetric B. Indirect method III. Fluorometry IV. HPLC A. UV detection B. Thin layer amperometric detection V. Thin-layer chromatography VI. Paper chromatography VII. Liquid chromatography

2.0

grains in India for use with floury materials and pulses. The modifications to this colorimetric method include change in wavelength for measurement at 510 nm instead of 715 nm and use of higher quantities of sodium tungstate and sulfuric acid for analysis of pulses, which are highly proteinaceous. In dried mushrooms, the uric acid is separated and extracted with an ammonium carbonate solution, purified with Sephadex G-10 and determined by spectrophotometry after adding Benedict’s reagent (Mlodecki et al., 1972). Manual or automated methods involving uricase enzyme were subsequently developed for use with cereal products (Farn and Smith, 1963b; Sen and Smith, 1966). Laessig et al. (1972) used an autoanalyzer, an instrument deployed for determining uric acid in blood, for uric acid analysis in

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food samples like flour and other cereal products and milk powder. With this method, the turbidity problem was eliminated by dialysis of the extract to separate uric acid from interfering substances. The detection limit of uric acid was 40 mg/g. A similar method with improved sensitivity (5 mg/g) that used immobilized uricase for wheat flour samples was reported by Roy and Kvenberg (1981). Brown et al. (1982) developed a modified enzymatic method using a glucose analyzer for highly colored whole spices. With this method, the degradation of uric acid by the enzyme uricase is monitored with an oxygen-sensitive electrode. In the enzymatic method, either UV detection or colorimetry was used. The enzymatic UV method is considered relatively simpler than the enzymatic colorimetric method (Farn and Smith, 1963a). The thin-layer chromatography (TLC) method is also specific for actual uric acid present in a sample (Thrasher and Abadie, 1978). The technique has been claimed to be particularly suitable for spices with high color content (Sengupta et al., 1972). An advanced method of analysis involving high-performance liquid chromatography (HPLC) either with UV detection or with thin-layer amperometric detection was then developed with sensitivity ranging from 0.03 to 2.0 mg/g of cereal and cereal products. In the HPLC method involving paired ion chromatography, Cohen (1983) could estimate individual purines (at low level, i.e., 500 pg) including uric acid, hypoxanthine, and xanthine. Other methods, mainly useful for qualitative or confirmatory tests, include TLC (Sengupta et al., 1972; Thrasher and Abadie, 1978) and paper chromatography (Venkatrao et al., 1960a). Insect detection by uric acid analysis has been compared with other detection methods for sensitivity and reliability. Wehling et al. (1984) observed positive correlation between number of insects present (S. granarius, S. oryzae, and R. dominica) and level of uric acid determined by liquid chromatography. Sen and Vazquez (1969) observed good correlation between the insect fragment count method and uric acid determination in infested flour. A good correlation between kernel damage due to insect feeding, insect fragment level, and uric acid content in wheat infested with S. oryzae, R. dominica, and T. granarium was also observed (Subramanyan et al., 1955). Galacci (1983) improved the semiautomated colorimetric method of Roy and Kvenberg (1981) and reported that the uric acid determination and fragment count data were comparable; it was observed that uric acid value of 600 mg/50 g of flour (12 mg/g) and 50 insect fragments per 50 g of flour sample (earlier FDA Defect Detection Level) were related. Wehling and Wetzel (1983) and Pachla et al. (1987) discussed the merits and drawbacks of manual colorimetry, enzymatic, and fluorometric methods of uric acid analysis in foodstuVs and favored HPLC because it can detect uric acid at the desired commercial level.


189

Infestation detection by uric acid determination has been considered relatively less sensitive because it requires a substantial insect population to produce a measurable amount of uric acid and it lacks reproducibility. However, sensitivity is greatly improved with the HPLC methods (Pixton, 1965; Wehling and Wetzel, 1983). Furthermore, the uric acid level in a particular sample indicates not only the existing but also the past infestation. Nevertheless, uric acid measurement as an indicator of the level of insect infestation is still followed in developing countries where infestation level is relatively higher (Joshi et al., 1985). The amount of uric acid excreted insect/day has been noted to vary depending on the life stage, insect density, and their nutritional status (Farn and Smith, 1963b). In T. castaneum, among the life stages the rate of excretion of uric acid was highest in the larvae (18.0 5.5 mg/mg of body weight/day), whereas in the adults it was low at 5.9 1.5 mg/mg of body weight/day (Sen, 1968). The amount of uric acid produced by three stored product species developing part of their life cycle inside wheat was compared by Wehling et al. (1984). The highest amount of uric acid was produced by S. granarius (1.27 mg/g of uric acid–infested kernel/100 g of grain) followed by S. oryzae (0.48 mg/g infested kernel/100 g) and R. dominica (0.33 mg/g infested kernel/100 g). Callosobruchus spp. infesting pulses such as black gram and field bean excreted relatively higher quantities of uric acid (3100 and 4875 mg/100 g, respectively), whereas in sorghum infested with S. oryzae and wheat flour by T. castaneum, it was low at 700 and 160 mg/100 g, respectively, during a storage period of 5 months (Venkatrao et al., 1959). This was due to the higher protein/purine content of the pulses and the higher multiplication rate of Callosobruchus than the other insects. With some species (e.g., R. dominica), the amount of uric acid present in a particular grain sample also depends on handling of the grain such as cleaning or aspiration. This is because uric acid is accumulated more in the dust materials (Wehling et al., 1984). The proportion of distribution of uric acid either in frass or in the grains depends on the insect species (Pillai et al., 1975).

H. IMAGING TECHNIQUES

Internal infestation in food grains and other agricultural produce can be detected without damaging the product by employing diVerent types of imaging techniques based on x-rays, nuclear magnetic resonance (NMR), near-infrared (NIR), and computed tomography (CT) scanning. There has been more focus and advancement on these techniques in developed countries.

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S. RAJENDRAN

1. X-ray technique X-ray or radiographic techniques have various applications including the detection of internal damage due to ‘‘hollow heart’’ in potatoes, ‘‘watercore’’ in apples, and insect infestation in fruits and food grains (Tollner, 1993). The method had also been used for the detection of the seed weevil Cryptorynchus mangiferae in mango fruits (Thomas et al., 1995). The x-ray absorbance of a test region is known to be aVected by tissue density, water content, presence of insects, or other foreign materials and eVective atomic number. Any changes in these parameters in the test region exceeding a volume of 1 mm3 are detected by the x-ray system (Tollner, 1993). The required exposure time and voltage of x-rays vary according to commodity, the degree of penetration, and the contrast required; grains having higher moisture need a higher voltage for the penetration of x-rays (Semple, 1992). Also, the denser the matter, the higher its atomic number and the greater the x-ray absorption. For grains, the x-ray exposure varies from several seconds to a few minutes. The x-ray method for the detection of hidden infestation in food grains was developed by Milner et al. (1950b) and is an oYcial method in the United States (American Association of Cereal Chemists [AACC], 2000). It detects both living and dead insects; however, egg and early larval stages are not detected. In addition to the need for an expensive machine to generate x-rays and films for exposure, it requires an experienced person to operate the equipment and to interpret the radiographs. To radiograph a grain sample, about 100 g of grain is spread as a flat single layer in a nonabsorbent tray and exposed to x-rays. The x-ray technique is particularly useful for screening packed foods. In food processing facilities, it has an important role because it can check a series of samples to meet quality-control standards. Available machines, which produce soft x-rays having relatively long wavelengths and weak transmission power, are provided with a microprocessor image analyzer, which provides a clear picture of the internal infestation in individual kernels. Unlike hard x-rays, the soft x-ray system is rapid, does not involve elaborate preparation of the samples, and can vividly distinguish living and dead insects. Soft x-ray–based detection of internal infestation of diVerent stored grain insects in wheat, rice, and mung bean has been reported (Xingwei et al., 1999; Karunakaran, 2002). Brader et al. (2002) examined the accuracy of determining insect contamination in wheat by x-ray analysis. It was observed that the x-ray analysis technique is one of the most accurate methods next to ELISA in detecting late instar larvae of S. granarius in wheat. Hurlock (1963) compared the radiographic method with that of staining techniques, flotation method, and CO2 analysis to detect hidden infestation of C. chinensis in dried whole green


191

peas. He observed that the results with the x-ray method were consistent and the accuracy was better than that of other methods. Schatzki and Fine (1988) examined radiograms of wheat kernels infested with S. zeamais, S. oryzae, R. dominica, and S. cerealella and observed that the infestation of the respective species could be detected with 80% accuracy at 8, 7, 27, and 15 days after oviposition, respectively. False positives due to damaged kernels and the germ portion were recognized at 0.08%. To overcome the problem of expensive x-ray film radiography and time-consuming human visual inspection, an automated inspection system of machine recognition of insect damage in food grains has been developed (Keagy and Schatzki, 1993). However, the latter requires suitable software for the analysis and interpretation of the results. Recognition of hidden weevil infestation of fourth instar larvae (26–28 days after oviposition) of S. granarius with an x-ray and image-processing technique in wheat has been reported. The image-processing algorithm recognized about 50% of infested kernels and the false positive was limited to 0.5% only (Keagy and Schatzki, 1993). Machine recognition generally increased with successive larval stage, from second instar to fourth instar. Unlike human visual detection, the machine recognition decreased at prepupa stage for S. zeamais and pupal stage in S. granarius and then increased again with later stages. Kim and Schatzki (2001) reported the application of x-ray imaging and single processing techniques for the detection of ‘‘pinholes’’ in almonds due to insect damage. Navel orange worm larvae (Amyelois transitella) when feeding on almonds make narrow holes of less than 1 mm in diameter. This damage, known as pinholes, is diYcult to observe manually but is detected by x-ray imaging. In this context, an automatic method of detecting insect produced pinholes in the nuts was developed. The pinhole-damaged region appeared slightly darker than the undamaged region (in almond nuts) on x-ray film or line-scanned images and was recognized by a machine-recognition algorithm. Casasent et al. (2001) studied new morphological image-processing operations for inspection of pistachio nuts for insect damage and other defects. In the new system consisting of a blobcoloring algorithm, filters and watershed transforms, images of the nuts were produced irrespective of their orientation (touching and nontouching nuts). 2. Near-infrared reflectance spectroscopy Near-infrared reflectance spectroscopy (NIRS) has been used in food industries for the determination of water, proteins, and oil content in food grains and other commodities (Williams and Norris, 2001). NIRS involves measurement of absorption spectra of a test material in the infrared region of 700–2500 nm. Compositional changes in the commodity are then correlated

192

S. RAJENDRAN

with spectral changes by suitable statistical techniques such as partial least squares, Fourier transformations, or neural networks. The insects present in food commodities are detected by NIRS due to their hemolymph, lipids, or chitin content. Chitin is also present in fungi. Hence, interference may be encountered due to fungi during the detection of insects in stored grains and this has to be examined in detail. In fact, NIRS has also been deployed for detecting fungal contaminants in other agricultural produce. Wilkin et al. (1986) showed that the presence of the flour mite, Acarus siro, in animal feed can be detected at a level of 105 mites/kg using NIRS. The authors considered that mite hemolymph was responsible for a change in the absorbance of water, resulting in a shift from 1934 nm to 1928 nm. In a preliminary investigation, Chambers et al. (1992a) demonstrated that NIRS could detect adults of S. granarius, O. surinamensis, T. castaneum, and A. advena in wheat. Samples of infested and uninfested kernels were scanned from 1100 to 2500 nm at 2-nm intervals and the reflectance spectra were recorded as log 1/R. The log 1/R spectra revealed increased absorption at the wavelengths of 1450 and 1940 nm, possibly due to water. Subsequently, it was reported that there were clear diVerences in the spectra of live and dead insects (adults of O. surinamensis). Furthermore, the detection of insects by NIRS was noted to be independent of their orientation during scanning (Chambers et al., 1992b). Internal infestation consisting of larvae or pupae of S. granarius and external infestation comprising O. surinamensis adults in wheat was detected by NIRS (Ridgway and Chambers, 1996). The sensitivity of detecting external infestation was, however, low at 270 insects/kg of grain or more, probably because of the position of the insects within the grain sample. Chambers and Ridgway (1996), while checking internal infestation (e.g., pupae of S. granarius) in single wheat kernels by NIRS, observed a decrease in absorbance (increase in reflectance) during scanning of kernels at 400– 2500 nm. However, it was noted that measurement at two wavelengths (i.e., 1194 nm and 1304 nm) is adequate for detection instead of the full spectrum. Ghaedian and Wehling (1997) demonstrated that wheat kernels containing late-instar larvae of S. granarius could be detected by NIRS by the analysis of reflectance spectra at 1100–2500 nm; the infested kernels are distinguished based on calibration models using principal component analysis (PCA) of NIR spectra of the kernels and by calculating Mahalanobis distances. To reduce the cost of the NIRS system, the authors suggested using less expensive filter-based instruments rather than scanning monochromator instruments. Ridgway et al. (1999) reported the detection of S. granarius larvae and pupae in individual wheat kernels by NIRS in the very near-infrared region of 700–1100 nm using two wavelength models, log 1/R (982 nm) log 1/R (1014 nm) or log 1/R (972 nm) log 1/R (1032 nm), with 96% accuracy


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in pest detection. They opined that the spectral diVerences between sound and infested kernels were either due to changes in starch content (decreased) for log 1/R (982 nm) log 1/R (1014 nm) or changes in moisture content (increased) for log 1/R (972 nm) log 1/R (1032 nm) with infestation; the loss in starch was due to insect feeding. They also expected that the cost of analysis by NIRS imaging techniques could be reduced if silicon detector–based charge coupled device (CCD) camera is used. An automated NIRS system capable of scanning individual grains containing late-instar larvae of S. oryzae, R. dominica, or S. cerealella at the rate of 15 kernels/min has been developed in the United Kingdom (Chambers et al., 1998). The system was eVective and could detect the infestation irrespective of the type/class of wheat, its protein content (range 11.32– 16.2%) and moisture content (range 10.0–13.2%). The minimum detectable size of the insects by NIRS varied between species. As identified by x-ray analysis, the NIRS system has been shown to detect R. dominica as small as 1.1 mm2 with 95% level confidence, whereas for S. oryzae it was 2.0 mm2, and for S. cerealella 2.7 mm2. For a particular insect species, the accuracy of detection increases as insect development proceeds. Accordingly, in S. oryzae the accuracy of detection of first instar larvae was 10%, second instar larvae 24%, third instar larvae 82%, fourth instar larvae 95%, and the accuracy was 100% for pupae and adults. The detection of internal infestation of R. dominica, S. oryzae, and S. cerealella in wheat kernels by an automated NIRS system was reported by Dowell et al. (1998). Single kernels were scanned at the rate of 1/4 sec and the spectral data at wavelengths of 1000–1350 nm and 1500–1680 nm were recorded. The detection has been attributed to chitin in insects. Dowell et al. (1999) showed that NIRS could be used for identifying 11 species of stored grain insects due to the characteristic chemical composition of individual species or due to the diVerences in the absorption characteristics of cuticular lipids of the insects. However, the accuracy of detection varies with the species; R. dominica and P. truncatus could be diVerentiated with 100% accuracy; Cryptolestes spp., with 90% accuracy; Sitophilus spp., with 83% accuracy; and Tribolium spp., with 85% accuracy. Perez-Mendoza et al. (2003) demonstrated detection of S. oryzae insect fragments in flour by NIRS using a DA 7000 spectrometer. They noted that the accuracy of detection increased with the number of fragments in a sample (i.e., >90% accuracy at a level of 130 fragments/50 g of flour, 90% at >75 fragments/50 g and 1) is the measure of the curve’s upward concavity at higher deformations and has been related to the last compression mechanism stage in which primary particle rearrangement, deformation, fracture, and densification occur. This four-parameter model has proven successful in describing the sigmoid-shaped force deformation (Figure 17) over full compression ranges in instant agglomerated coffee, instant agglomerated low-fat (2%) milk, and instant agglomerated nonfat milk after confined uniaxial compression tests (Yan and Barbosa-Cańovas, 1997). Figure 17 describes a typical forcedeformation curve obtained in the experiments and different strain zones

FIG. 17 Force-deformation relationship and description of Equation 16, shown in a four-parameter compression model (adapted from Yan and Barbosa-Cańovas, 1997).

COMPRESSION AND COMPACTION

271

that explain the three steps for the agglomerated compression mechanism described earlier.

B. COMPRESSIBILITY BY CONFINED UNIAXIAL COMPRESSION TESTS

The influence of key parameters, such as particle size and moisture content, in food powder development, manufacturing, and control has been widely studied. Other effects provided using cells with different geometries, mixtures of different size particles, or anticaking agents can also be found in various publications. The following sections summarize a literature review of historical contributions and new research on these effects and the use of compression properties for flowability characterization. 1. Effect of particle size Particle size is one of the factors greatly influencing the physical properties of particulate systems, such as bulk density, compressibility, and flowability (Barbosa-Cańovas et al., 1987). Equations 13–15 show the effects of particle size on the compression behavior. Yan and Barbosa-Cańovas (1997, 2000) found that particle size played an important role in affecting compressibility (C2) changes in three agglomerated food powders (low-fat [2%] milk, instant nonfat milk, and instant coffee) and two noninstant powders (ground coffee and cornmeal). For the three agglomerated powders, the compressibility (C2) increased with particle size, given that larger agglomerated particles have a lower bulk density because of larger voids between and within agglomerates. Meanwhile, Rennie et al. (1999) found that the particle size of dairy powders had a markedly decreasing effect on cohesion. In the case of cohesive agglomerated dairy powders, it can be deduced that less cohesive materials with larger particle sizes are more easily compressed (i.e., will show increased compressibility). Molina et al. (1990) obtained a good fit in Equation 15 and used compressibility (A2) to evaluate the particle size influence in ground coffees for two brands. Compressibility values were considerably different for two brands at a given size. Factors such as chemical composition, roasting, and grinding conditions may affect compressibility values as well. However for each brand tested, compressibility did not vary for coarser fractions (500– 2360 mm). The truly fine fraction (180–250 mm) formed a low-density open bed structure, which offered a higher compressibility. Therefore, a combination of high compressibility and low density in fine powders proved to be a good indicator of fine powder cohesiveness.

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´ NOVAS AND P. JULIANO G. V. BARBOSA-CA

Yan and Barbosa-Cańovas (2000) found that ground coffee and cornmeal fit Equation 13 and powder compressibility (C2) was affected by particle size. Finer particles gave higher compressibility, and larger particles had higher loose bulk density, coinciding with the research of Molina et al. (1990). Larger particles mainly underwent the two steps in the fine powder compression process explained earlier. However, for smaller particles, the compression process stopped at the first compression step filling the space voids because of the bulk high porosity. Particle size was also evaluated in encapsulated materials by comparing single- and double-encapsulated butter oil powders in sucrose (0.10

Hollenbach et al. (1982) showed compressibility to be a more reproducible index than parameters such as angle of internal friction or unconfined yield stress obtained from the Jenike shear test (ASTM D 6128). Conversely, Ehlermann and Schubert (1987) sustained that compressibility results from materials of different composition cannot be compared and that flowability characterization through compressibility must be made specifically for each food variety. Moreover, confined uniaxial compression is a ‘‘simple’’ compression test that provides an approximate measure of the flowability of powders. Therefore, it is not suitable for silo design but may prove to be a convenient method for process control in any food laboratory (e.g., to evaluate particle cohesion). Table II offers a range value definition for flowability classification by comparing flow function (ratio between the maximum consolidation stress and unconfined yield stress) with compressibility. Yan and Barbosa-Cańovas (2000) used the criteria defined earlier to characterize powder flowability with compressibility measurements in ground coffee and cornmeal. Coarser ground coffee or cornmeal had a lower compressibility and thus better flowability than the finer grinds. However, the same conclusion cannot be drawn in porous agglomerates because compression mechanisms are different. Compressibility in agglomerated powders such as instant coffee and milk increases with particle size because of increased brittleness. In this case, free-flowing characteristics of agglomerates are attributed to a decrease in stickiness. Thus, the cohesion C is not applicable to flowability in agglomerated brittle powders. C. COMPACTION IN FOOD POWDERS

The purpose of food powder compaction tests is primarily to simulate density changes during handling and transportation. Subjecting a powder to vibration or impact usually results in its compaction. During vibration of

280


a powder column, the density of the compact usually approaches an asymptotic value determined by the vibration amplitude and frequency. This behavior has been explained with indexes such as the Hausner ratio index and other compaction models. Models developed for the study of attrition and segregation are presented in the following sections. 1. Hausner ratio The Hausner ratio (Grey and Bedow, 1969) has been introduced as an index to measure the amount of bulk density change caused by compaction and has been used to indicate the presence of attractive forces and friction. Malave´-Lo´pez et al. (1985) defined the Hausner ratio as the relation between asymptotic and initial bulk density (Equation 7): r ð20Þ HR ¼ 1 ; r0 where HR is the Hausner ratio, r1 is the asymptotic constant density after a certain amount of taps, and r0 is the initial bulk density of the sample. A more direct expression widely used to evaluate flow properties calculates powder volume changes in a graduated cylinder after a certain period of time or number of taps (Hayes, 1987): HR ¼

rn V0 ¼ ; r0 Vn

ð21Þ

where n is the number of taps applied to the sample, rn and r0 are the tapped and loose bulk density, and V0 and Vn are the loose and tapped volume, respectively. HR can be used to evaluate the flowability from a classification constructed by Hayes (1987), similar to one made by Jenike on the flow function. If the Hausner ratio is 1.0–1.1, the powder is classified as free flowing; 1.1–1.25, medium flowing; 1.25–1.4, difficult flowing; and >1.4, very difficult flowing. The advantage of this method is the simplicity of the instrumentation (Sone, 1972) and the test performance. However, the Hausner ratio may not be a reliable flowability index for most food powders (Peleg, 1978). Experimental results may depend on the test procedure (e.g., number of taps) and the particle size of the tested bulk because compaction patterns of food powders, including the asymptotic density, depend on the vibrations/impacts regimen, which might change the Hausner ratio or vary it dramatically. Chang et al. (1998) evaluated cohesion, angle of repose, and the Hausner ratio of different mixtures of a starchy powder (potato starch) and a proteinaceous powder (wheat protein) at different water activities. As the water


281

activity of each mixture increased, the tapped bulk density, Hausner ratio, angle of repose, and cohesion increased. However, loose bulk densities decreased at different water activities. Mixtures with higher protein content had a notable tendency to aggregate, indicating increased cohesion. The authors developed empirical equations, which included the tested bulk properties for relating fundamental properties of a powder with the relative humidity of the environment. 2. Compaction models Sone (1972) proposed the following relation between bulk density changes and number of taps to model compaction by tapping (Barletta et al., 1993b): n Þ; ð22Þ r1 rn ¼ C expð K where r1 is the asymptotic bulk density, rn is the density after n taps, and C and K are constants. Another model relates the volume (or density) reduction fraction Y(n), similar to the one defined in Equation 4 as a function of the number of taps n (Peleg, 1983): Y ðnÞ ¼

V0

Vn Vn

¼

rbn

rb0 rb0

¼ HR

1¼

n ; A þ Bn

ð23Þ

where rb0 and rbn are the respective densities and A and B are constants. The Hausner ratio may be adjusted to parameter B in Equation 23. The Hausner ratio, defined in Equation 20, can be related to a particular B representing the asymptotic value of V(n) [i.e., when n ! 1, V(n) ! 1/B]. H¼

r1 B 1 ¼ r0 B

ð24Þ

A single exponential model proposed by Malave´-Lo´pez et al. (1985) is expressed as n Y ðnÞ ¼ C½1 expð Þ; ð25Þ N where C is a constant, n is the number of taps, stant characteristic of the system. Equation 26 is a model proposed by Barletta et al. (1993b) to describe compaction during tapping: n Þ þ C2 ½1 expð Y ðnÞ ¼ C1 ½1 expð N1

and N is a condouble-exponential coffee agglomerate n Þ; N2

ð26Þ

282


where Cs and Ns are constants. Furthermore, a four-parameter model (Barletta et al., 1993b) where A, B, C, and D are constants is expressed as n n þ : ð27Þ Y ðnÞ ¼ A þ Bn C þ Dn The compaction characteristics of agglomerated coffee during tapping were studied by Barletta et al. (1993b) using some of the models mentioned earlier. They found that the three- and four-parameter models have shown considerable improvement in fitting experimental data, compared to Equations 22 and 23. To describe the voidage reduction phenomenon of agglomerated powders, three- and four-parameter models are more suitable because they include compaction and attrition effects. 3. Comparison of mechanical compression and vibration by tapping Malave´-Lo´pez et al. (1985) studied the compaction characteristics by observing the vibration and mechanical compression of different kinds of powders, such as instant coffee, wheat flour, rye flour, cornstarch, and soy protein powders, all with different cohesion properties. No clear relation was found between the Hausner ratio and the mechanical compressibility, at least under the test conditions reported in this work. However, HR was found to be useful in qualifying the maximum powder compressibility under vibration (Malave´-Lo´pez et al., 1985). Mechanical compressibility in compression tests is, at least partly, a result of the collapse of an initial open bed structure (supported by cohesive interparticle forces or interparticle liquid bridges), which is totally and irreversibly destroyed. In the case of vibrational tapping tests, the ‘‘openness’’ of the bed structure can be recovered at least to some extent. Thus, different mechanisms yield different tendencies in the compressibility measured by tapping and mechanical compressibility. 4. Compaction and segregation Barbosa-Cańovas et al. (1985) studied segregation tendencies in some food mixtures during vertical and horizontal vibrations and showed that segregation occurs not only in free-flowing powder mixtures, but also in some cohesive powder mixtures. Segregation intensity depends not only on the mixture composition, particle size, and mechanical vibration history, but also on whether the mixture is ‘‘ordered.’’ Barbosa-Cańovas et al. (1985) used the segregation index Sindex after the mixture was subjected to vibration or tapping.


Sindex

sP ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi n Wi ðXi X Þ2 i¼1P ; ¼ n i¼1 Wi

283

ð28Þ

where Wi is the weight of powder in the ith ring of a vibrating multisplit cell, Xi is the concentration of a given component, and X is the mean concentration of the component in the mixture. Segregation tendency in a starch– sugar mixture was inhibited by attractive interparticle forces at certain mixture ratios, whereas a sugar and instant coffee mixture did not show a cohesion effect, segregating almost completely. It was also noticed that increasing the vibration frequency intensified the mixture’s segregation. Percolation velocity during compaction is affected by the particle size ratio and shape (Shinohara, 1997). Bridgwater et al. (1969) developed a mathematical model for this kind of radial dispersion, which was proposed by Shinohara (1997). ln

N0 R ¼ ; N0 N 4ER t

ð29Þ

where N0 is the total number of percolated small particles through a packed bed and N is the number of small particles with centers inside the radius R at compaction time t. Segregation during angle of repose measurement in the slope being formed is especially relevant, given the importance of this test in flow characterization. Percolation is the governing mechanism during the formation of a heap, where the fine particles tend to flow easily to the center. When uniformly sized particles of different materials are mixed, each with different angles of repose, the material with the steepest angle will tend to concentrate in the center, while the one with the flatter angle will concentrate in the outside surface of the heap. 5. Attrition kinetics Particle size analysis is useful for assessing attrition because both fragmentation and fine formation yield separate particle populations with different sizes. Production of midsize particles by means of shattering will lower the particle population’s mean size and increase its size spread, as formation of fines through surface erosion will make the overall size distribution bimodal or multimodal. Barletta et al. (1993b) summarizes the different size distribution patterns in attrition resulting from the predominant attrition mechanisms and reviews the different models that fit these distributions. In studying the attrition kinetics of powder compaction through tapping, one of the most convenient ways to organize attrition data is to plot the

284


percentage of fines retaining original size as a function of characteristic time or number of taps (Barletta et al., 1993a; Malave´-Lo´pez et al., 1985). Several kinetic models have been proposed to characterize these curves. For example, a good two-term exponential model was found that describes the attrition kinetics of agglomerated coffee with original particle size larger than 16 mesh or 1180 mm (Malave´-Lo´pez et al., 1985). WR=o ¼ F expð A1 nÞ þ ð1

F Þ expð A2 nÞ;

ð30Þ

where WR/O is the weight ratio between agglomerates retaining original particle size and the total sample, n is the number of taps, and F and (1 F ) are the fractions of material that have undergone attrition at rates A1 and A2, respectively. A nonexponential model was also suggested: n ; ð31Þ WR ¼ 1 B1 þ B2 n where WR is the weight fraction of particles retaining original size after n taps, and B1 and B2 are constants. If n ! 0, 1/B1 can be considered as the initial attrition rate, and n ! 1, 1/B2 as the asymptotic weight fraction of material under attrition. This nonexponential model has some advantages over exponential models. First, it has a residual (asymptotic) fraction of agglomerates that survived the attrition test, whereas exponential models imply that all agglomerates of original size will disappear. Second, it generally fits the experimental data quite well, and its two constants can be easily calculated in regression models (Barletta et al., 1993a). The attrition index AI (Equation 32) proposed by Barletta and BarbosaCańovas (1993a) was found suitable for studying agglomeration and the effects of agglomerate size and water activity on the attrition kinetics of agglomerated coffee and nonfat milk (Yan and Barbosa-Cańovas, 2001a). AI ¼ Cna ;

ð32Þ

where AI is the attrition index, C and a are constants, and n is the number of taps. AI is defined as AI ¼

F ; R

ð33Þ

where F is the weight fraction of fines generated in the attrition test and R is the size stability related to agglomerates retaining original particle size. R can be calculated by the following formula: Pi¼L i¼1 Wi Si jT¼n ; ð34Þ R ¼ Pi¼L i¼1 Wi Si jT¼0


285

where Wi is the amount of material retained on each sieve, Si is the normalized sieve opening size, and T is the number of taps. Yan and Barbosa-Cańovas (2001a) compared the Hausner ratio to the attrition index AI under different test conditions in agglomerated coffee and milk. The Hausner ratio was closely related to the attrition index at high numbers of taps (>5000 taps) due to agglomerate attrition. However, at low numbers of taps, the Hausner ratio only represented a volume reduction due to agglomerate rearrangement in the test cylinder. Therefore, it was suggested that the Hausner ratio could be used as a simple attrition index for agglomerates at high tap numbers.

III. MICROSTRUCTURAL APPROACH FOR COMPRESSION AND COMPACTION A. SCANNING ELECTRON MICROSCOPY STUDIES IN FOOD POWDERS

Because a correlation exists between the microstructure and physical properties of food powders (Aguilera and Stanley, 1999; Kala´b, 1979), the SEM method is appropriate in studying the surface morphology and the internal structure of agglomerated powders. Careful microscopic examination of the failed pieces can provide much insight on the dominant failure modes (Bika et al., 2001). Current micromechanical modeling has clearly demonstrated the importance of structure (packing and resulting porosity) on the properties of agglomerates. However, those models are highly idealized and not capable of predicting quantitatively the mechanical properties of real agglomerates from the properties of constituent materials (Aguilera and Stanley, 1999). Some agglomerates of different materials have been observed to fail because of internal flaws driven by a number of stresses (e.g., internal tensile stress; cracks in the surface; plastic flow at the surface between the agglomerate and platen; and shear stress within the sphere). For brittle particle agglomerates with significant internal flaws, the tensile strength is small compared to the compressive and shear strength, and failure is likely initiated by the internal tensile stress. In any case, a careful microscopic examination of failed pieces can provide much information on the dominant failure mode (Bika et al., 2001). Ghadiri et al. (1991) used SEM to study the impact damage on NaCl particles developed from different processing routes. Local plastic deformation phenomena leading to cracks, including lateral cracks, and flaw paths are illustrated in Ghadiri et al. (1991). Yan and Barbosa-Cańovas (1997)

286


FIG. 19 Fracture lines in a milk powder agglomerate after compression (from Yan and Barbosa-Cańovas, 2000).

studied the microstructure of spray-dried instant low-fat (5%) and nonfat milk before and after compression tests, to characterize their internal forces and compression mechanisms. Before compression, the primary particles in nonfat milk powder were characterized by wrinkled surfaces with deep dents. The 5% low-fat milk powders particles were spherical with small dents and smooth surfaces, with some rims around small globules emerging from the large particles and some craterlike scars. In both cases, the solid bridge force (lactose recrystallization during the instantizing process (Caric´ and Kala´b, 1987) dominated the overall interparticle forces. Because both had very low moisture contents, the chance for liquid bridge formation was diminished. Very small particles adhering to larger ones could be the result of interparticle attraction forces. In Figure 19, crack lines can be observed in low-fat milk due to the primary force-transmitting routes of the applied compression stress. As compression proceeds, particle fracture occurs along these crack lines, load paths bifurcate, and more particles become load bearing. Figure 20 shows how large forces tend to be transmitted similarly along particle chains, as observed in experiments on the assembly of photoelastic discs. These particle chains are positioned to form enclosed circular boundaries, where loaded particles create concentrated failure lines and propagate major principal stress.


287

FIG. 20 Force transmission chains through a granular material while under compressive stress (from Yan and Barbosa-Cańovas, 2000). Width of lines represents the relative magnitude of local stress; the principal stress is vertical.

B. FRACTAL CHARACTERIZATION FOR COMPACTION PROCESSES

The fractal approach analyzes two-dimensional projections of particles by redefining its contour. A given step length can provide a close polygonal contour at different points of the particle projection that geometrically recreates a self-similar particle silhouette. The procedure is based on estimating the silhouette perimeter by adding all the step lengths plus the exact length of the last side to complete the polygon (if different from the step length). Measurement of the silhouette perimeter is repeated by using several different step lengths, which gives different perimeters in each case. The perimeter LP of the corresponding polygon is expressed as (Peleg and Normand, 1985a): LP ¼ nl;

ð35Þ

where n is the number of the polygon’s sides for a particular selected step length l. A logarithmic plot of the perimeter LP against different step lengths l is called a Richardson plot, which produces a curve with a negative

288


slope a due to perimeter increase through step length decrease. The fractal dimension DF of the profile is expressed as DF ¼ 1 þ tanjaj:

ð36Þ

The fractal dimension provides useful information about the particle shape, openness, and ruggedness in the form of single numbers (Simons, 1996). Thus, fractal characterization can be used as a tool to describe the eroding process in particulate materials during compaction (Barletta and Barbosa-Cańovas, 1993b). In fact, fractal dimension has been used as a sensitive attrition index, based on the fact attrition can cause changes in particle shape and surface, and on the scale that a fractal approach is applicable (Peleg and Normand, 1985a). Computer algorithms are generally applied for fractal determination using digitized particle images with special software (Allen et al., 1995). Particles with different origins and shapes, such as carbon aggregates, minerals, protein, catalysts, and instant coffee, showed fractal dimensions (DF) within the range of 1.05–1.36 (Clark, 1986). Particles with DF close to 2.0 are not likely to exist because of inherent mechanical instability (Normand and Peleg, 1986; Peleg and Normand, 1985b). Barletta and Barbosa-Cańovas (1993b) used fractal analysis to characterize the changes in ruggedness of particles from attrition by tapping in instant coffee and instant skim milk. Fractal dimension changes in ruggedness were detected even for low tap numbers decreasing with increasing number of taps. It was proposed that fractal analysis be used for quality assessment of instant food powders and other particulate materials for attrition characterization in compaction or compression processes.

IV. COMPRESSION AND COMPACTION IN FOOD PROCESSING In powder technology, the general behavior of powders under compressive stress or compaction due to mechanical motion is relevant in several applications. The tendency of a food powder’s physical and chemical properties to change relative to temperature-moisture history is a common feature of all food powders (Peleg, 1978). Intrinsic variables like temperature, moisture, and composition can influence the response of food powders to the stress of deformation from tension, shear, or compression. Apart from the influence of temperature and relative humidity conditions during powder processing, different unit operations can provide unique settings for food powder materials to encounter compression and compaction phenomena. In the following sections, we discuss how compaction and compression events directly or indirectly intervene in different processing operations.


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A. SIZE REDUCTION

Size reduction operations can be related with particle or powder bulk volume reduction. The objective of particle size reduction is to produce smaller particles from larger ones. Furthermore, bulk powder volume reduction through compression or compaction can also be considered a size-reduction operation. Compression characteristics are extremely important for bulk size– reduction operations (Murthy and Battacharya, 1998), especially in military and domestic applications. Compressed powders can be used by armed forces for field rations, by astronauts during space travel, and by hikers and others who must carry their own food supplies. Powders can be compressed by mechanical presses with savings in transportation costs, storage space (Van Heyst, 1983), and packaging material. Webb and Hufnagel (1943) reported a 42% savings in space with compressed dry whole milk (Van Heyst, 1983). Particle size reduction, a comminution process, includes operations such as crushing, grinding, and milling (e.g., milling of cereals, grinding of spices) in which food pieces are deformed until breakage or failure. As mentioned earlier, breakage can occur along cracks or defects in the structure of hard materials during compression. Scanlon and Lamb (1995) showed how different types of fracture operations affected the particle shape differently in dried gelatinized starch comminuted in an impact breakage gun, hammer mill, and blender. Forces commonly used in food processes for particle size reduction are compressive, impact, attrition (or shear), and cutting forces. More than one force usually participates in the comminution operation in industrial sizereduction equipment. In particular, crushing rolls use mainly compressive forces, hammer mills are based on impact, disc mills cause particle attrition through shear force application, and rotary knife cutters use cutting forces. Coarse crushing of hard materials through compressive forces allows a solids reduction to about 3 mm. Impact forces may be associated with coarse, medium, and fine grinding for a variety of food materials (e.g., for nut breakage). Shear or attrition forces are applied for pulverization of powders in the micrometer range (like most food powders). Cutting is a process totally different from comminution, because the operating principles are quite different from those governing the size reduction of hard materials and generally gives a definite particle size and may even produce a definite shape. An ideal size-reduction pattern to achieve a high reduction ratio for hard brittle materials, such as sugar crystals or dry grains, could be obtained by first compressing and then using an impact force, and finally by shearing or rubbing.

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In regards to particle shape and defects, a large piece having many defects can be broken under small stress with very little deformation. On the other hand, smaller pieces have fewer defects and will need a higher breaking strength. Limited by very small particles, purely intermolecular forces must be overcome. This is why grinding is so difficult to achieve in smaller particles. For example, fine grinding of roasted coffee (e.g., to

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