Unconventional Sources of Dietary Fiber. Physiological and in Vitro Functional Properties 9780841207684, 9780841210295, 0-8412-0768-2

Table of contents :
Title Page......Page 1
Half Title Page......Page 3
Copyright......Page 4
ACS Symposium Series......Page 5
FOREWORD......Page 6
PdftkEmptyString......Page 0
PREFACE......Page 7
1 Effect of Conventional and Unconventional Dietary Fibers in Colon Carcinogenesis......Page 9
Correlation and Case-Control Studies......Page 10
Possible Mechanism of Protective Effect of Dietary Fiber In Colon Carcinogenesis......Page 11
Animal Model Studies in Colon Carcinogenesis: Effect of Conventional and Unconventional Fibers......Page 14
Conclusions......Page 21
Literature Cited......Page 22
2 Leguminous Seed Fiber Uses in Disease States and Effects on Carbohydrate Digestion in Vitro and Absorption in Vivo......Page 25
Effect of Leguminous Fiber on Gut Physiology......Page 26
Therapeutic use of Fiber in Diabetes: Need for Fiber Supplemented Foods......Page 29
The Glycemic Index......Page 31
High Fiber Foods and Diabetic Treatment......Page 33
Conclusion......Page 36
Literature Cited......Page 37
3 Structural Chemistry of Some Nonstarchy Polysaccharides of Carrots and Apples......Page 40
Results and Discussion......Page 41
Experimental......Page 51
Acknowledgments......Page 54
Literature Cited......Page 55
4 Legumes and Their Soluble Fiber: Effect on Cholesterol-Rich Lipoproteins......Page 56
METHODS......Page 58
RESULTS......Page 60
DISCUSSION......Page 63
Literature Cited......Page 65
5 Purified Psyllium Seed Fiber, Human Gastrointestinal Tract Function, and Nutritional Status of Humans......Page 67
Effect on Proximal Composition of Feces......Page 69
Effect on Fecal Fiber Composition......Page 72
Effect on Blood Serum Lipid Patterns......Page 73
Acknowledgments......Page 74
Literature Cited......Page 75
6 Locust Bean Gum in Food Products Fed to Familial Hypercholesterolemic Families and a Type IIB Patient......Page 77
Results......Page 83
Discussion......Page 92
Literature Cited......Page 95
7 Pectins and Guar Gum: Effect on Plasma Lipoproteins and Tissue Lipoprotein Lipase Activity in Rats......Page 99
Materials and Methods......Page 101
Results and Discussion......Page 102
General Discussion......Page 106
Acknowledgments......Page 108
Literature cited......Page 109
8 Aminopolysaccharides—Their Potential as Dietary Fiber......Page 111
General Properties......Page 112
Potential Applications in Foods......Page 114
Physiological Activity......Page 115
Possible Mechanism of Hypocholesterolemic and Hypolipidemic Activity of Chitosan......Page 118
Literature Cited......Page 127
9 Catabolism of Mucopolysaccharides, Plant Gums, and Maillard Products by Human Colonic Bacteroides......Page 129
Polysaccharide Utilization by Colon Bacteria......Page 131
Strategies for Polysaccharide Metabolism......Page 133
Utilization of Maillard Products......Page 134
Literature cited......Page 139
10 Some in Vitro and in Vivo Properties of Dietary Fibers from Noncereal Sources......Page 141
Fiber Sources......Page 142
Fermentability and Water Holding Capacity......Page 144
Summary......Page 146
Literature Cited......Page 147
11 Dietary Pectin: Effect on Metabolic Processes in Rats......Page 148
Literature Cited......Page 158
12 Chitin and Chitosan: Influence on Element Absorption in Rats......Page 160
Materials and Methods......Page 163
Results......Page 166
Discussion......Page 182
Literature Cited......Page 187
13 Citrus Pectic Polysaccharides—Their in Vitro Interaction with Low Density Serum Lipoproteins......Page 190
Literature Cited......Page 194
14 Washed Orange Pulp: Characterization and Properties......Page 196
Methods and Materials......Page 197
Results and Discussion......Page 198
Literature Cited......Page 208
15 Dietary Fiber from Citrus Wastes: Characterization......Page 210
Analyzing Citrus Residue......Page 213
Composition of Citrus Processing Residue......Page 217
Conclusion......Page 220
Literature Cited......Page 223
16 Cellulose, Xylan, Corn Bran, and Pectin in the Human Digestive Process......Page 225
Experimental......Page 227
Results And Discussion......Page 229
Literature Cited......Page 242
17 Properties of Wood Lignin......Page 244
Effect of Cations and Complexing Ligands on the Adsorption of Nitrosamines on Lignin......Page 245
Binding of Heavy Metals on Lignin......Page 247
Experimental......Page 250
Literature Cited......Page 253
18 Chemical and Physical Properties of Tobacco Fiber......Page 254
Materials and Methods......Page 255
Results......Page 257
Discussion......Page 262
Literature Cited......Page 267
19 Products of Wheat Straw Biodegradation by Cyathus stercoreus......Page 269
Materials and Methods......Page 271
Results and Discussion......Page 274
Literature Cited......Page 285
20 Biphenyl Hydroxylase, Arylhydrocarbon Hydroxylase, and Epoxide Hydrolase Activities in Intestinal and Liver Microsomes of Rats Fed Selected Types of Dietary Fibers......Page 287
Previous Work Relating to Selection of Substrates......Page 288
Methods and Materials......Page 290
Results and Discussion......Page 291
Literature Cited......Page 303
C......Page 307
D......Page 309
G......Page 310
L......Page 311
Ρ......Page 312
Τ......Page 313
W......Page 314
Z......Page 315

Citation preview

Unconventional Sources of Dietary Fiber

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

Unconventional Sources of Dietary Fiber Physiological and in Vitro Functional Properties Iva General Mills, Inc. Based on a symposium sponsored by the ACS Division of Agricultural and Food Chemistry at the 183rd Meeting of the American Chemical Society, Las Vegas, Nevada, March 28-April 2, 1982

214

ACS SYMPOSIUM SERIES

AMERICAN

CHEMICAL

SOCIETY

WASHINGTON, D.C. 1983

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

Library of Congress Cataloging in Publication Data Unconventional sources of dietary fiber. (ACS symposium series, ISSN 0097-6156; 214) "Based on a symposium sponsored by the ACS Division of Agricultural and Food Chemistry at the 183rd Meeting of the America Las Vegas, Nevada, March 28-Apri Includes bibliographies and 1. Food-Fiber content—Addresses, essays, lectures. I. Furda, Ivan, 1938. II. American Chemical Society. Division of Agricultural and Food Chemistry. III. American Chemical Society. National Meeting (183rd: 1982: Las Vegas, Nev.) IV. Series. TX553.F53U53 1983 ISBN 0-8412-0768-2

613.2'8

83-2691

Copyright © 1983 American Chemical Society All Rights Reserved. The appearance of the code at the bottom of the first page of each article in this volume indicates the copyright owner's consent that reprographic copies of the article may be made for personal or internal use or for the 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. for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to copying or transmission by any means—graphic or electronic—for any other purpose, such as for general distribution, for advertising or promotional purposes, for creating new collective work, for resale, or for information storage and retrieval systems. The copying fee for each chapter is indicated in the code at the bottom of the first page of the chapter. The citation of trade names and/or names of manufacturers in this publication is not to be construed as an endorsement or as approval by ACS of the commercial products or services referenced herein; nor should the mere reference herein to any drawing, specification, chemical process, or other data be regarded as a license or as a conveyance of any right or permission, to the holder, reader, or any other person or corporation, to manufacture, reproduce, use, or sell any patented invention or copyrighted work that may in any way be related thereto. PRINTED IN THE UNITED STATES

AMERICA

Society Library 1155 16th St. N. W. In Unconventional Sources of Dietary Washington, D. C.Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

20036

ACS Symposium Series M. Joa

Advisory Board David L. Allara

Robert Ory

Robert Baker

Geoffrey D. Parfitt

Donald D. Dollberg

Theodore Provder

Brian M . Harney

Charles N. Satterfield

W. Jeffrey Howe

Dennis Schuetzle

Herbert D. Kaesz

Davis L. Temple, Jr.

Marvin Margoshes

Charles S. Tuesday

Donald E. Moreland

C. Grant Willson

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

FOREWORD The ACS SYMPOSIUM SERIES was founded in 1974 to provide

a medium for publishing symposia quickly in book form. The format of the Series parallels that of the continuing ADVANCES IN CHEMISTRY SERIES except that in order to save time the papers are not typeset but are reproduced as they are submitted by the authors in camera-ready form. Papers are reviewed under the supervision of the Editors with the assistance of the Series Advisory Board and are selected to maintain the integrity of the symposia; however, verbatim reproductions of previously published papers are not accepted. Both reviews and reports of research are acceptable since symposia may embrace both types of presentation.

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

PREFACE

ΤΓ

H E IDEA OF ORGANIZING THIS SYMPOSIUM was born at the 179th Annual ACS meeting in Houston in 1980, from discussions with the previous chair­ man of the Agricultural an was felt that the subject spectrum that should not cover excessively the current common, almost classical types, offibersuch as wheat and corn bran or cellulose. It should also encompass less conventional sources of dietaryfiberwhich would be the main topic of this symposium. Because soluble nondigestible plant polysaccharides are now included in the definition of dietary fiber, and because numerous nondigestible polymers of nonplant origin exist, greater attention should be paid to these alternatives. These polymers have a variety of unique physiological, chemical, and functional properties that need to be better understood and utilized. This type of understanding is frequently achieved when different points of view are presented and a variety of scientists engage in discussion. Presentations in this volume come from chemists, medical researchers, and microbiologists, as well as nutritionists and food scientists. In a few cases, rather exoticfibertypes such as tobaccofiber,wheat straw lignin, or shellfish aminopolysaccharides are discussed. Other sources include psyl­ lium, different legumes, and vegetable and fruitfibers.There is no doubt that additional unconventionalfibersources will be continuously identified and increasingly used. It is my hope and belief that this publication will help in the expan­ sion and utilization offibersources in foods, as well as in pharmaceutical and medical preparations. After all, we should recognize that the chance to accomplish this is quite favorable because "complex carbohydrates," which are the main constituents of dietaryfiber,represent the only group of major food components for which increased daily intake is repeatedly being recommended. I would like to express my appreciation and thanks to all participants of this symposium and to the writers of the individual chapters for their efforts and contributions. Without them, this undertaking and publication

ix In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

would not be possible. Finally, I am grateful to Akiva Pour-El for his administrative help, and to General Mills and The Quaker Oats Company for their generousfinancialsupport. IVAN F U R D A

General Mills, Inc. Minneapolis, MN 55427 November 1982

χ

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

1 Effect of Conventional and Unconventional Dietary Fibers in Colon Carcinogenesis BANDARU S. REDDY, KENSHI

1

WATANABE,

and HIDEKI

2

MORI

American Health Foundation, Naylor Dana Institute for Disease Prevention, Valhalla, NY 10595

Epidemiologi gest a p r o t e c t i v against colon cancer. This p r o t e c t i o n by the d i e t a r y f i b e r s may be mediated through (a) the di­ lution of tumorigenic compounds i n the gut,(b) the binding of tumorigenic compounds to the f i b e r i n the gut, and (c) an i n d i r e c t e f f e c t on the meta­ bolism of carcinogens. Experiments were conducted i n animal models to study the e f f e c t of c e r t a i n non-conventional d i e t a r y f i b e r s on colon c a r c i n o ­ genesis. Rats were fed the d i e t s c o n t a i n i n g al­ falfa, c i t r u s pulp, p e c t i n , wheat bran, undegraded carrageenan or cholestyramine and t r e a t e d with azoxymethane (AOM), 3,2'-dimethyl-4-aminobiphenyl (DMAB), or methylnitrosourea (MNU) to induce tumors. Animals fed the wheat bran or c i t r u s pulp and t r e a t e d with AOM or DMAB had a lower colon tumor incidence than d i d those fed the c o n t r o l d i e t and treated with r e s p e c t i v e carcinogens. Al­ falfa d i e t had no e f f e c t on AOM- or MNU-induced colon tumors, whereas the p e c t i n d i e t i n h i b i t e d ΑΟΜ-induced but not MNU-induced colon tumors. Animals fed the d i e t s c o n t a i n i n g undegraded c a r r a ­ geenan or cholestyramine developed more AOM and/or MNU-induced colon tumors than d i d the r a t s fed the c o n t r o l d i e t . Thus, the p r o t e c t i v e e f f e c t of var­ ious d i e t a r y f i b e r s i n colon carcinogenesis de­ pends on the type of f i b e r fed, as w e l l as the type of carcinogen used to induce tumors.

1 2

Current address: Kagoshima University School of Medicine, Kagoshima, Japan Current address: Gifu University School of Medicine, Gifu City, Japan

0097-6156/83/0214-0001$06.00/0 © 1983 American Chemical Society

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

2

UNCONVENTIONAL SOURCES OF DIETARY FIBER Rapid progress has been made i n basic concepts concerning carcinogenesis (_1>2^· There are agents which are genotoxic, which by d e f i n i t i o n i n t e r a c t with the gene to y i e l d an abnormal genetic m a t e r i a l and would be considered i n i t i a t i n g agents (3)· The second broad c l a s s of agents act by e p i g e n e t i c mechanisms and tend to i n c r e a s e the development of l e s i o n s i n i t i a t e d by genotoxic carcinogens. Thus, t h i s l i s t includes co-carcinogens which operate at the same time as genotoxic carcinogens and can a l t e r the metabolism of a genotoxic agent with an increased r a t i o of a c t i v a t i o n / d e t o x i f i c a t i o n m e t a b o l i t e s . Such agents can a l s o act as more c l a s s i c tumor promoters which e x h i b i t t h e i r e f f e c t a f t e r the a c t i o n of a genotoxic carcinogen. Current concepts of colon cancer e t i o l o g y stem from m u l t i d i s c i p l i n a r y research e f f o r t s based on three major approaches, namely: (1) the v a r i a t i o n i n incidence of colon cancer as a f u n c t i o n of area of residence population; (2) the change and (3) d e t a i l e d l a b o r a t o r y s t u d i e s i n humans, i n animal models, and through i n v i t r o systems* The consistency of these f i n d i n g s suggests that environmental f a c t o r s i n g e n e r a l , and dietary f a c t o r s i n p a r t i c u l a r , play a dominant r o l e i n the development of colon cancer i n humans. Epidemiologic studies suggest that d i e t s p a r t i c u l a r l y high i n t o t a l f a t and low i n f i b e r and i n c e r t a i n vegetables as w e l l as high intake of beef are g e n e r a l l y a s s o c i a t e d with an i n c r e a s ed incidence of l a r g e bowel cancer i n man (4-10). Dietary f a t may be a r i s k f a c t o r i n the absence of f a c t o r s that are p r o t e c t i v e , such as use of high f i b r o u s foods and f i b e r (11,12). As an example, i n F i n l a n d , where the d i e t a r y intake of f a t i s s i m i l a r to many of the Western c o u n t r i e s and the f i b e r intake i s higher, the incidence of colon cancer i s lower than a l l of the Western countries. This b r i e f review evaluates current research on the r e l a t i o n between d i e t a r y f i b e r and large bowel cancer i n humans, i n c l u d i n g the use of animal models. This b r i e f review also presents an e v a l u a t i o n of the mechanism whereby c e r t a i n d i e t a r y f i b e r s i n c l u d i n g conventional and unconventional f i b e r s modify the r i s k f o r the development of colon cancer. C o r r e l a t i o n and Case-Control Studies C r o s s - n a t i o n a l c o r r e l a t i o n s between the incidence of colon cancer and d i e t a r y h a b i t s have been used to s e l e c t hypotheses f o r t e s t i n g i n c a s e - c o n t r o l and cohort s t u d i e s . These s t u d i e s have shown that c e r t a i n food preferences appear to be a s s o c i a t e d with e i t h e r a h i g h - or a l o w - r i s k f o r colon cancer. When such c o r r e l a t i o n s are supported by experimental evidence from animal s t u d i e s , the hypothesis could be a t t r a c t i v e . B u r k i t t (10,13) f i r s t observed the r a r i t y of l a r g e bowel cancer i n most A f r i c a n populations and suggested that populations consuming a d i e t r i c h i n f i b e r have a lower incidence of t h i s type of cancer, while those e a t i n g r e f i n e d carbohydrates

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

1.

REDDY ET AL.

Dietary Fibers in Colon Carcinogenesis

and l i t t l e f i b e r have a higher i n c i d e n c e . A recent study comparing low-risk populations i n Kuopio, F i n l a n d , with those at high r i s k i n Copenhagen i n d i c a t e d that d i e t a r y f i b e r intake i s higher i n F i n l a n d compared with Copenhagen (14). The F i n n i s h population i s unique i n t h i s respect because i n t h i s country the t o t a l d i e t a r y f a t i s s i m i l a r to countries with high rate of colon cancer. The c r u c i a l d i f f e r e n c e i n d i e t a r y intake between F i n l a n d and Denmark and other Western countries may r e l a t e to d i e t a r y f i b e r and meat. Our data a l s o suggest that one of the f a c t o r s c o n t r i b u t i n g to the low r i s k of large bowel cancer i n Kuopio appears to be that a high intake of d i e t a r y f i b e r (mainly c e r e a l f i b e r ) leads to increased s t o o l bulk, i n e f f e c t d i l u t i n g tumorigenic compounds i n the colon (12)· The r e s u l t s are cons i s t e n t with a p o s s i b l e r o l e f o r d i e t a r y f i b e r i n the prevention of colon cancer i n humans Case c o n t r o l studie i b l e r e l a t i o n of d i e t a r Dales ejt a l . (15) found that among American blacks s i g n i f i c a n t l y more colon cancer p a t i e n t s than c o n t r o l s reported that t h e i r d i e t was high i n saturated f a t and low i n f i b r o u s foods. Inv e s t i g a t i n g many d i e t a r y c o n s t i t u e n t s , Modan et a l . (16) d i s covered that those c o n t r i b u t i n g l e s s to the d i e t s of p a t i e n t s with colon cancer than to the d i e t s of c o n t r o l s were those containing fiber. Bjelke (17) who interviewed hospitalized p a t i e n t s and c o n t r o l s i n Minnesota and i n Norway, learned that c o l o r e c t a l cancer p a t i e n t s l e s s f r e q u e n t l y ate vegetables, in p a r t i c u l a r the Minnesota p a t i e n t s ate l e s s cabbage. S i m i l a r l y , Graham et_ a l . (18) found that i n d i v i d u a l s who ate vegetables such as cabbage, b r o c c o l i and Brussels sprouts had a lower r i s k of colon cancer. These studies i n d i c a t e that d i e t s with a high intake of t o t a l f a t and a low intake of c e r t a i n f i b e r s and c e r t a i n vegetables are g e n e r a l l y a s s o c i a t e d with an increased incidence of colon cancer i n humans. Even i n populations consuming high amount of f a t , high d i e t a r y f i b e r acts as a p r o t e c t i v e f a c t o r i n colon c a r c i n o g e n e s i s . P o s s i b l e Mechanism of P r o t e c t i v e E f f e c t of D i e t a r y F i b e r In Colon Carcinogenesis Although the concept of f i b e r involvement i n colon c a r c i n o genesis i s a t t r a c t i v e , the data o f t e n appear c o n t r a d i c t o r y and confusing. Discrepancies may have a r i s e n from the general misuse of f i b e r terminology. As w e l l , experimental design has f a i l e d to account f o r the p o s s i b l e subtle e f f e c t of i n h i b i t o r s , e s p e c i a l l y i n r e l a t i o n to the promoting process. Evaluations of the b i o l o g i c f u n c t i o n of d i e t a r y f i b e r have often lacked comp l e t e information on the nature of the f i b e r . Dietary f i b e r comprises a heterogenous group of carbohydrates, i n c l u d i n g c e l l u l o s e , hemicellulose and p e c t i n , and a noncarbohydrate substance, l i g n i n (19). According to Van Soest (20), f i b e r s can be c l a s s i f i e d i n t o three groups: vegetable

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

3

4

UNCONVENTIONAL

SOURCES OF DIETARY

FIBER

f i b e r s , which are h i g h l y fermentable and have l i t t l e i n d i g e s t i b l e residue; brans, which are l e s s fermentable; and chemically p u r i f i e d f i b e r s , such as c e l l u l o s e , which are r e l a t i v e l y nonfermentable. Pectins and gums, s o l u b l e substances that are not true f i b e r s , are considered part of the d i e t a r y f i b e r complex because of the s i m i l a r e f f e c t s they can e l i c i t i n the d i e t . Wheat bran and vegetable and f r u i t f i b e r s have d i f f e r e n t percentages of c e l l u l o s e , hemicellulose and l i g n i n . Carrageenan, a broad generic c l a s s of s u l f a t e d polysaccharides derived from a wide range of seaweed species, can be c l a s s i f i e d as non-conventional f i b e r and i s used i n food as an e m u l s i f i e r , s t a b i l i z e r , thickener and g e l l i n g agent. The p r o t e c t i v e e f f e c t of d i e t a r y f i b e r may be due to ads o r p t i o n , d i l u t i o n or metabolism of cocarcinogens, promoters and y e t - t o - b e - i d e n t i f i e d carcinogens by the components of the f i b e r (12,21,22). There i some other f i b e r s can bin v i t r o (23). This i n d i c a t e s that the d i f f e r e n t types of nonn u t r i t i v e f i b e r s possess s p e c i f i c binding p r o p e r t i e s . Dietary f i b e r could a l s o a f f e c t the enterohepatic c i r c u l a t i o n of b i l e s a l t s (24) which act as colon tumor promoters (6)· F i b e r not only i n f l u e n c e s b i l e a c i d metabolism (12,22) thereby reducing the formation of p o t e n t i a l tumor promoters i n the colon, but a l s o exerts a s o l v e n t - l i k e e f f e c t i n that i t d i l u t e s p o t e n t i a l carcinogens and cocarcinogens by i t s bulking e f f e c t (12) and i s able to bind b i l e acids and c e r t a i n carcinogenic compounds (23,25,26). On the other hand, d i e t a r y undegraded carrageenan markedly enhances the b i l e a c i d content i n the colon, thereby i n c r e a s i n g the p o t e n t i a l colon tumor promoters i n the gut ( 6 ) . Smith-Barbaro et a l . (26) i n our l a b o r a t o r y determined the c a p a c i t y of various f i b e r s to bind the colon carcinogen 1.2-dimethylhydrazine (DMH) in vitro. The percent of DMH bound to wheat bran, corn bran, a l f a l f a f i b e r and dehydrated c i t r u s pulp was dependent on pH of the medium as w e l l as the type of f i b e r examined. Results from t h i s study show that at c o l o n i c pH, a greater percent of DMH was bound by wheat bran than by c i t r u s pulp. Therefore, i t i s p o s s i b l e that c e r t a i n f i b e r s bind c a r cinogen at c o l o n i c pH, thus making i t u n a v a i l a b l e f o r contact with the c o l o n i c mucosa. Other f i b e r s such as p e c t i n ( s o l u b l e f i b e r ) do not bind DMH at c o l o n i c pH, but may modify the metabolism of carcinogen v i a a c t i v a t i o n / d e a c t i v a t i o n steps e i t h e r i n the l i v e r and/or i n the c o l o n i c mucosa. I n v e s t i g a t i o n s have been c a r r i e d out i n s e v e r a l l a b o r a t o r i e s to determine whether there are d i f f e r e n c e s i n f e c a l c o n s t i t uents between populations at high and low r i s k of colon cancer, and whether changes i n the f i b e r content of the d i e t would a l t e r the concentration of f e c a l b i l e acids that act as colon tumor promoters and the a c t i v i t y of f e c a l m i c r o f l o r a . Recently, we studied healthy i n d i v i d u a l s i n Kuopio, F i n l a n d , an area of low r i s k f o r the development of colon cancer (12). Dietary

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

1.

REDDY ET A L .

Dietary Fibers in Colon Carcinogenesis

5

h i s t o r i e s i n d i c a t e d that the t o t a l f a t consumption i s s i m i l a r to that i n the United States but the intake of c e r e a l f i b e r i n F i n land i s higher and the d a i l y output of feces three times higher than that of healthy i n d i v i d u a l s i n the United S t a t e s . The conc e n t r a t i o n of f e c a l secondary b i l e a c i d s , mainly deoxycholic a c i d and l i t h o c h o l i c a c i d i s l e s s i n Kuopio than i n the United States, but the t o t a l d a i l y output i s the same i n the two popul a t i o n s because of the t h r e e f o l d greater d a i l y output of feces i n Kuopio. This suggests that increased f e c a l bulk d i l u t e s suspected carcinogens and promoters that may be i n d i r e c t contact with the large bowel mucosa. Cummings (27) demonstrated that f i b e r from c a r r o t , cabbage, apple, bran and guar gum produces d i f f e r e n t responses i n f e c a l weight i n humans r e l a t e d to the i n take of pentose-containing polysaccharides i n the f i b e r . The f e c a l weight increased by 127% when bran was added to the d i e t and 20% when guar gum duced intermediate changes In another study, Cummings (28) reported that an increase i n c e r e a l f i b e r intake from 17 to 45 g/d increased the f e c a l weight from 79 to 228 g/d and d i l u t e d the f e c a l b i l e a c i d s . Kay and Truswell (29) showed that adding wheat f i b e r to the d i e t decreased the concentration of f e c a l b i l e acids and n e u t r a l s t e r oids because of the bulking e f f e c t of f i b e r , whereas the a d d i t i o n of p e c t i n to the d i e t increased the f e c a l s t e r o i d and b i l e a c i d output. These r e s u l t s suggest that the e f f e c t on f e c a l b i l e a c i d e x c r e t i o n may depend on the type of f i b e r consumed. The e f f e c t of d i e t a r y wheat bran and a l f a l f a at 15% l e v e l on the composition of f e c a l b i l e acids was studied i n r a t s fed a s e m i p u r i f i e d d i e t (30). Diets containing wheat bran and a l f a l f a caused a s i g n i f i c a n t increase i n s t o o l weight. The concentrat i o n of f e c a l b i l e a c i d s , p a r t i c u l a r l y hyodeoxycholic acid, p-muricholic a c i d , deoxycholic a c i d and l i t h o c h o l i c a c i d was lower i n r a t s f e d wheat bran, compared to those f e d a c o n t r o l d i e t , b u t the d a i l y output of these b i l e acids was the same i n both groups. A l f a l f a had no e f f e c t on the concentration of f e c a l b i l e a c i d s , but the d a i l y e x c r e t i o n of deoxycholic a c i d , l i t h o c h o l i c a c i d and 1 2 - k e t o l i t h o c h o l i c a c i d was increased compared to the c o n t r o l d i e t . I t i s apparent from t h i s study that the f e c a l e x c r e t i o n of b i l e acids v a r i e s with the type and amount of d i e t a r y f i b e r . U n t i l r e c e n t l y , the nature of the carcinogens responsible f o r c o l o n cancer not only was obscure, but there were no r e a l leads. Because of p o t e n t i a l importance of f e c a l mutagens i n the genesis of l a r g e bowel cancer and of p o s s i b l e r o l e of d i e t a r y ^ f a c t o r s i n the i n d u c t i o n of colon cancer, the f e c a l mutagenic a c t i v i t y of various population groups with d i s t i n c t d i e t a r y habits and v a r i e d colon cancer incidences was determined by several investigators. E h r i c h jet a l . (31) have demonstrated that the s t o o l s of South A f r i c a n urban whites who consume a h i g h - f a t , l o w - f i b e r d i e t and who are at high r i s k f o r colon

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

6

UNCONVENTIONAL SOURCES OF DIETARY FIBER cancer development were higher (17% of the i n d i v i d u a l s ) i n mutagenic a c t i v i t y with Salmonella typhimurium s t r a i n s TA98 and TA100 without microsomal a c t i v a t i o n compared to South A f r i c a n urban and r u r a l blacks (0-5% of the i n d i v i d u a l s ) who consume a low-fat, h i g h - f i b e r d i e t and who are at l o w - r i s k . Bruce et a l . (32) were the f i r s t to show that the feces of some normal humans consuming a h i g h - f a t , l o w - f i b e r d i e t contained compounds that caused d i r e c t mutagenesis of TA98 and TA100 i n the Ames assay. They have a l s o demonstrated that increased d i e t a r y fiber, -tocopherol or v i t a m i n C reduced f e c a l mutagens. Kuhnlein et a l . (33) compared f e c a l mutagens (water e x t r a c t s ) from a group of vegetarians consuming h i g h - f i b e r d i e t s with those from persons on t y p i c a l North American d i e t s c o n t a i n i n g meat. On TA100 and TA98, ovo-lactovegetarians and s t r i c t vegetarians had lower l e v e l s of f e c a l mutagens than non-vegetarians. Correlation studies between the p i n d i c a t e the presence o Recently, we have s t u d i e d f e c a l mutagens of 3 populations with d i s t i n c t r i s k f o r the development of colon cancer, a highr i s k population i n New York (non-SDA) consuming a h i g h - f a t , lowf i b e r , mixed-Western d i e t , a low-risk v e g e t a r i a n SDA (SeventhDay A d v e n t i s t s ) and a l o w - r i s k population i n Kuopio, F i n l a n d consuming a h i g h - f a t , h i g h - f i b e r d i e t (34). F e c a l samples of non-SDA were h i g h l y mutagenic i n TA98 without microsomal a c t i v a t i o n , followed by TA100 without a c t i v a t i o n and TA100 with a c t i vation. None of the samples of SDA tested showed mutagenic a c t i v i t y i n any of the t e s t e r systems, whereas Kuopio samples e x h i b i t e d a c t i v i t y only i n TA98 with microsomal a c t i v a t i o n . Animal Model Studies i n Colon Carcinogenesis; E f f e c t of Conventional and Unconventional F i b e r s Research on the mechanisms of cancer causation i n the large bowel has been a s s i s t e d by the discovery of s e v e r a l animal models that m i r r o r the type of l e s i o n s seen i n man. These models i n c l u d e (a) i n d u c t i o n of l a r g e bowel cancer i n r a t s through chemicals such as 3-methyl-4-aminobiphenyl, or 3-methyl2-naphthylamine; (b) d e r i v a t i v e s and analogs of c y c a s i n and methylazoxymethanol (MAM) such as azoxymethane (AOM and 1,2-dimethylhydrazine (DMH), which work w e l l i n r a t s and mice of s e l e c t e d s t r a i n s ; and (c) i n t r a r e c t a l a d m i n i s t r a t i o n of d i r e c t a c t i n g carcinogens of the type of a l k y l n i t r o s o u r e a s , such as methylnitrosourea (MNU) or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) which lead to cancer of the descending colon i n every species tested so f a r . The r e l a t i o n between d i e t a r y f i b e r consumption and c o l o n cancer has been s t u d i e d i n experimental animals. Wilson et_ a l . (35) found that Sprague-Dawley r a t s fed a d i e t c o n t a i n i n g 20% corn o i l or beef f a t and 20% wheat bran had fewer benign colon tumors induced by DMH given by g a s t r i c i n t u b a t i o n than rats fed a c o n t r o l d i e t c o n t a i n i n g 20% f a t and no bran. It i s possible that with a d d i t i o n a l time, a number of tumors that were c l a s s i -

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

1.

REDDY ET AL.

Dietary Fibers in Colon Carcinogenesis

f i e d as benign might have developed a more i n v a s i v e c h a r a c t e r . There was no d i f f e r e n c e i n the incidence of colon cancer between the r a t s fed corn o i l and those fed beef f a t . In another study, Clapp et^ a l . (36) found that DMH-induced colon tumors were i n creased i n BALB/C mice fed a semisynthetic d i e t c o n t a i n i n g 5% f a t and 20% W/W corn bran, soybean bran and s o f t winter wheat bran. DMH-induced colon tumor incidence i n mice fed the c o n t r o l d i e t was s u r p r i s i n g l y low, about 12%. In these s t u d i e s , the experimental d i e t s c o n t a i n i n g high-fat/no bran and h i g h - f a t / h i g h bran, (35) or low-fat/no-bran and low-fat/high bran (36) d i f f e r ed s u b s t a n t i a l l y i n c a l o r i c d e n s i t y . Animals on high bran d i e t s might have consumed q u a n t i t a t i v e l y d i f f e r e n t l e v e l s of vitamins and minerals compared to those fed high fat-no bran d i e t or low fat-no bran d i e t s because the r a t s adjust t h e i r food intake to compensate f o r n o n - d i g e s t i b l e f i b e r i n high bran d i e t . It i s a l s o p o s s i b l e that c e r t a i and damage to c o l o n i c a p a c i t y of the i n t e s t i n a l t r a c t as w e l l as b i l e a c i d - b i n d i n g c a p a c i t y (37,38) which have bearing on colon c a r c i n o g e n e s i s . A recent study by F l e i s z e r et_ a l . (39) i n d i c a t e d that the incidence of colon tumors induced by DMH i n r a t s decreases as the d i e t a r y intake of f i b e r i n c r e a s e s . The number of animals used i n t h i s study was small. The d i e t s i n that study, namely high bran d i e t (28% f i b e r ) , s p e c i a l chow (15% f i b e r ) , r a t chow (5% f i b e r ) F l e x i c a l d i e t (0% f i b e r ) d i f f e r e d not only i n c o n s i s tency (that i s , s o l i d or l i q u i d ) but a l s o i n the proportions of p r o t e i n and f a t s , which have been shown to have an independent e f f e c t on colon carcinogenesis induced by DMH. However, the col o n cancer incidence i n f i b e r - f r e e ( F l e x i c a l ) group was lower than i n r a t chow group. Some r e d u c t i o n i n tumor incidence i n the r a t s i n g e s t i n g a h i g h - f i b e r d i e t might be expected on the b a s i s of reduced energy i n t a k e . Although the study's f i n d i n g s suggest that reduced intake alone cannot account f o r the s i g n i f i c a n t p r o t e c t i v e e f f e c t of d i e t a r y bran, a b e t t e r experimental design might have strengthened the r e s u l t s . In another study, Cruse e_t a l . (40) found that a d i e t cont a i n i n g 20% wheat bran had no e f f e c t on colon carcinogenesis i n duced by DMH i n r a t s . Not only were the number of animals used i n t h i s study small (10 r a t s / g r o u p ) , but a l s o the doses of the chemical i n t h e i r experiment were so high (40 mg/kg body weight/week f o r 13 weeks) that any p r o t e c t i v e e f f e c t of bran might have been unobservable. In a study of the e f f e c t of d i e t on chemical c a r c i n o g e n e s i s , i t i s important to avoid exposing the animal to an excessive l e v e l of carcinogen f o r a long p e r i o d , as t h i s may obscure more subtle changes induced by c e r t a i n d i e t a r y modifications. In a d d i t i o n , d i f f e r e n c e s i n c a l o r i c density of experimental d i e t s a t t r i b u t a b l e to the d i l u t i o n a l e f f e c t of added f i b e r f u r t h e r complicated the i n t e r p r e t a t i o n of the data. In f a c t , the data presented by Cruse et a l . ( 4 0 ) suggest that a h i g h - f i b e r d i e t reduces the frequency of death due to DMH i n rats.

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

7

8

UNCONVENTIONAL

SOURCES

OF

DIETARY

FIBER

The e f f e c t of a d i e t c o n t a i n i n g 15% a l f a l f a , p e c t i n or wheat bran on c o l o n carcinogenesis by MNU or AOM was studied i n F344 r a t s by Watanabe et a l . (41). In t h i s study, the e x p e r i mental d i e t s were not adjusted i s o c a l o r i c a l l y . The a d d i t i o n of p e c t i n or wheat bran to the d i e t g r e a t l y i n h i b i t e d colon tumor incidence induced by AOM, a carcinogen r e q u i r i n g host-mediated metabolic a c t i v a t i o n (Table I)· However, the incidence of AOMinduced colon tumors was not i n f l u e n c e d by the a d d i t i o n of a l f a l f a to the d i e t . The d i e t s c o n t a i n i n g wheat bran and p e c t i n d i d not protect against MNU-induced colon c a r c i n o g e n e s i s . Table I Colon Tumor Incidence i n Female F344 Rats Fed D i e t s Containing P e c t i n , A l f a l f a or Wheat Bran and Treated with Azoxymethan

% Animals Colon Tumors Diet

Azoxymethane treated 57 10 53

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In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

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In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

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In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

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UNCONVENTIONAL SOURCES OF DIETARY FIBER

lysis. This p a r t i a l l y accounted f o r the l a r g e amount of glucose recovered from t h i s d i e t . However, s i m i l a r amounts of f r e e g l u cose and s t a r c h were a l s o found i n f e c a l samples from the corn bran d i e t while smaller amounts were found i n the f e c a l samples from the other d i e t s i n c l u d i n g the f i b e r - f r e e d i e t s . While d i f f i c u l t i e s i n these analyses prevented better quantitation, the r e s u l t s suggest that both A l p h a c e l and corn bran may have i n h i b i t e d s t a r c h d i g e s t i o n . This may be p a r t i a l l y due to b u l k i n g e f f e c t and l e s s e f f e c t i v e substrate/enzyme i n t e r a c t i o n . Small amounts of glucose were produced from c e l l u l o s e prep a r a t i o n s by h y d r o l y s i s with TFA as already noted (Table I I ) and reported by others (19). The amount produced probably depends on the p h y s i c a l form of the c e l l u l o s e since d i f f e r e n t amounts were obtained from d i f f e r e n t preparations. Subtracting the amounts of f r e e glucose and that derived from s t a r c h from the t o t a l amount obtained some of the A l p h a c e l d i e t accounted f o r from unchanged c e l l u l o s e i t s e l f . This suggests that passage through the g a s t r o i n t e s t i n a l t r a c t may have produced changes that made c e l l u l o s e i n recovered feces more s u s c e p t i b l e to TFA h y d r o l y s i s than that which was f e d . Xylan and Corn Bran. The xylan and corn bran f i b e r s were mainly h e m i c e l l u l o s e and contained more complex mixtures of hydrolyzable monosaccharides than the other f i b e r sources. It was of some i n t e r e s t to consider the r e s u l t s from the h y d r o l y s i s of f e c a l samples from these d i e t s i n an a l t e r n a t e manner. The data were r e p l o t t e d to show monosaccharides recovered as percent of the amount f e d f o r each s u b j e c t . ( F i g u r e s 6 and 7 ) . For comp a r i s o n the composition of the o r i g i n a l f i b e r i s shown on the extreme l e f t as percent dry weight of the f i b e r . As already noted the composition of the x y l a n was i n t e r e s t ing because of i t s high mannose and glucose content, suggesting the presence of glucomannan i n the x y l a n . The feces examined f o r hydrolyzable polysaccharides showed that the mannose ( o r mannan) was h i g h l y fermentable s i n c e only 0-3% of i t survived to be excreted. S i m i l a r l y l e s s than 10% of the glucose o r i g i n a l l y present i n the xylan ingested was recoverable by h y d r o l y s i s of f e c a l samples. Over 70% of the xylose i n the xylan a l s o d i s a p peared. Thus, the o r i g i n a l xylan may be composed of at l e a s t two r e a d i l y fermentable p o l y s a c c h a r i d e s , a xylan (31-4 l i n k e d xylose u n i t s ) and a glucomannan (31-4 l i n k e d mannose and g l u cose i n the r a t i o 3:1). Of the two polysaccharides the g l u c o mannan was more r e a d i l y fermentable s i n c e l e s s of i t s u r v i v e d the d i g e s t i v e process. Corn bran has a l a r g e xylose to arabinose r a t i o of 1.8. Recovery of these sugars i n the feces was the highest of any of the f i b e r s f e d . The xylose to arabinose r a t i o was a l s o c l o s e to that i n the bran f e d , ranging from 1.5-1.7. Galactose, while i n q u i t e small amounts, survived i n roughly the same

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

Figure 6.

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In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983. SUBJECT «

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Figure 7. Recovery o/ monosaccharides from corn bran diets from each subject as percent of amount fed. Composition of fiber fed is shown at left.

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OLSON ET AL.

Cellulose,

Xylan,

Corn Bran, and Pectin

235

proportions. The glucose value of 20% f o r the corn bran i n ­ cludes 16% s t a r c h and, by d i f f e r e n c e , 4% of the glucose is probably t i e d up i n h e m i c e l l u l o s e . Then a c a l c u l a t e d value of hydrolyzable glucose i n the feces based only on glucose i n h e m i c e l l u l o s e i n the o r i g i n a l f i b e r would be 4X that shown i n F i g u r e 7. Comparison of these two sources of hemicellulose i n the d i e t s and t h e i r r e c o v e r i e s i n the feces i n d i c a t e d that t h e i r behavior i n the g a s t r o i n t e s t i n a l t r a c t may depend both on t h e i r composition and p a r t i c l e s i z e . The xylan was a f i n e l y d i v i d e d preparation while the corn bran was composed of much l a r g e r particles. Very l i t t l e of the xylan was recovered i n the feces compared to that recovered from the corn bran. F i b e r Input; F e c a l Output F e c a l output on a f i b e r - f r e e d i e t may i n c l u d e b a c t e r i a t i n a l wall c e l l debris " f i b e r - f r e e " d i e t that i s as yet not recognized as such ( e . g . m a t e r i a l added as e m u l s i f i e r s , e t c ) . F e c a l output from a f i b e r supplemented d i e t minus that from the f i b e r - f r e e output should i n d i c a t e the extent of the f i b e r i t s e l f that was excreted plus the i n c r e a s e i n b a c t e r i a l d e t r i t u s , mucus and other m a t e r i a l perhaps rendered l e s s d i g e s t i b l e due to the presence of the fiber. In Figure 8 the amount of each f i b e r consumed i s shown compared to a corrected average (of the f i v e subjects) t o t a l 3-day dry weight of f e c a l output and amounts of TFA hydrolyzable sugars present. The c o r r e c t i o n was made by s u b t r a c t i n g the average f e c a l output and hydrolyzable sugars (shown i n the ex­ treme r i g h t i n F i g u r e 8) obtained from the f i b e r - f r e e d i e t s from the values obtained on the f i b e r d i e t s . Average f e c a l outputs f o r a l l of the f i b e r d i e t s are l a r g e r than those from f i b e r - f r e e d i e t s . Most of the TFA-hydrolyzable sugars from the p e c t i n and xylan f i b e r s are not recovered i n the feces (9, Γ 7 , 18). Both the A l p h a c e l and corn bran d i e t s produced l a r g e increases i n f e c a l dry weights. For corn bran r e l a t i v e l y s i m i l a r proportions of x y l o s e , arabinose and galactose found i n the bran were found i n the f e c e s . The l a r g e bran p a r t i ­ c l e s and the chemical form of the polysaccharides probably l i m i t s the f e r m e n t a b i l i t y of these hemicelluloses (20, 21). I t was pos­ s i b l e to v i s u a l l y i d e n t i f y corn bran p a r t i c l e s i n f e c a l m a t e r i a l . S t a t i s t i c a l A n a l y s i s . Means and 95% confidence intervals were c a l c u l a t e d for the recovered monosaccharides (Table I I I ) . Data was t r e a t e d by a n a l y s i s of variance f i t t i n g d i e t s and sub­ jects. In some cases transformations were needed to s t a b i l i z e the variances between d i e t s . Significant differences i n the composition of feces as a r e s u l t of d i f f e r e n t d i e t s are c l e a r l y i n d i c a t e d and are consistant with the previous d i s c u s s i o n .

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

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FECAL OUTPUT ON FIBER FREE DIET

« a e i a ÀHViaia ao saonnos ivNOiiNaANOONa

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

0.41

0.77

0.43

0.43

0.72

F i b e r Free

Pectin

Alphacel

Xylan

Corn bran

10.9

0.11

0.12

0.09

0.08

5.1 16.7

0.07 0.16

0.07 0.17

0.04 0.14

0.03 0.13

ARABINOSE Mean 95% C.I.

16.2

4.11

1.13

0.06

0.09

Mean

11.7 22.4

2.97 5.68

0.81 1.57

0.05 0.09

0.07 0.13

XYLOSE 95% C.I.

FECAL HYDROLYSIS RESULTS:

*For Grams Excreted/3 Day Pooled C o l l e c t i o n ; Ν = 5.

0.49 0.95

0.20 0.66

0.20 0.66

0.54 1.00

0.18 0.64

RHAMNOSE Mean 95% C.I

DIET

Table I I I .

0.15

0.32

0.61

0.13

0.07

0.10 0.23

0.21 0.49

0.40 0.92

0.08 0.19

0.05 0.11

MANNOSE Mean 95% C.I.

3.41

0.45

0.49

0.80

0.44

1.61 5.20

0.32 0.58

0.36 0.62

0.67 0.93

0.31 0.57

GALACTOSE Mean 95% C.I.

3.58

1.36

10.1

1.50

1.17

2.67 4.70

0.94 1.92

8.06 12.5

1.04 2.10

0.79 1.67

GLUCOSE Mean 95% C.I.

MEANS AND 95% CONFIDENCE INTERVALS*

238

UNCONVENTIONAL

Summary And

SOURCES

OF

DIETARY

FIBER

Conclusions

Marthinsen and Fleming (14) reported that methane e x c r e t i o n was g r e a t e r while consuming the xylan and p e c t i n d i e t s than while consuming the other d i e t s . Ingestion of these two f i b e r s a l s o caused higher f l a t u s volume and hydrogen and carbon dioxide exc r e t i o n . The A l p h a c e l and corn bran d i e t s r e s u l t e d i n breath and f l a t u s gas e x c r e t i o n at l e v e l s equivalent to the f i b e r - f r e e d i e t s . These observations are c o n s i s t e n t with the high recovery of f i b e r from the A l p h a c e l and corn bran and the i m p l i e d fermentation and hence low recovery of the x y l a n and the p e c t i n . The r e s u l t s from t h i s work show that d i e t a r y f i b e r sources produce d i f f e r e n t patterns of monosaccharides on d i r e c t TFA h y d r o l y s i s . The r e s u l t s a l s o show that i t i s p o s s i b l e to o b t a i n s i g n i f i c a n t information on the f a t e of d i e t a r y f i b e r sources by d i r e c t TFA h y d r o l y s i s Different dietary fibe terns of monosaccharides that were r e l a t a b l e to the f i b e r sources ingested. A d d i t i o n a l experiments are needed to r e l a t e not only the monosaccharide composition of the f i b e r s fed to f e c a l output but a l s o the s t r u c t u r e s of those f i b e r s and how they may have been modified i n the d i g e s t i v e t r a c t even i f not fermented. The e f f e c t of f i b e r p a r t i c l e s i z e and pretreatment should be s t u d i e d . Free sugar, s t a r c h , c e l l u l o s e and u r o n i c a c i d measurements should be made i n order to o b t a i n a more complete p i c t u r e of what s u r v i v e s and what i s metabolized. The e f f e c t of d i e t a r y f i b e r s on the d i g e s t i o n and u t i l i z a t i o n of other polysaccharides and other food components should be s t u d i e d . This information, together with fermentation data, i n c l u d i n g gas and VFA production, w i l l provide a b e t t e r understanding of the r o l e and value of d i f f e r e n t d i e t a r y f i b e r s and t h e i r e f f e c t s on n u t r i e n t b i o a v a i l a b i l i t y . Acknowledgment The authors thank Dr. Bruce Mackey s t a t i s t i c a l p o r t i o n of t h i s work.

for his assistance for

the

Reference to a company and/or product named by the U. S. Department of A g r i c u l t u r e i s only f o r purposes of i n f o r m a t i o n and does not imply approval or recommendation of the product to the e x c l u s i o n of others which may a l s o be s u i t a b l e . Literature Cited 1. 2.

T r o w e l l , H. Lancet 1974, 1, 503. T r o w e l l , J . ; Southgate, D.A.T.; Wolever, T.M.S.; Leeds, A.R.L.; G a s s u l l , M.A.; Jenkins, D.A. Lancet 1974, 1, 967.

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

16.

3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.

OLSON ET AL.

Cellulose, Xylan, Corn Bran, and Pectin

239

Southgate, D.A.T. "Current Developments of Importance to Health", Ed. Heaton, K.W. Food and Nutrition Press, 1979; Ρ 9. Cummings, J. H. Br. Med. Bull. 1981, 37, 65. Darvill, Α.; McNeil, M.; Albersheim, P.; Delmer, D.P. "Bio­ chemistry of Plants, Vol. 1 The Plant Cell", Ed. Tolbert, N.E., Academic Press, 1980; p 91. Aspinall, G.O. "Biochemistry of Plants, Vol. 3 Carbohydrate: Structure and Function", Ed. Tolbert, N.E., Academic Press, 1980; p 473. James, W.P.T.; Theander, O. Ed. "The Analysis of Dietary Fiber in Food", Marcel Dekker, Inc., New York, 1981. Theander, O.; Amin, P. "Dietary Fibers: Chemistry and Nu­ trition", Ed. Inglett, G.E.; Falkehag, S. I., Academic Press, 1979; p 215 Salyers, Α.; Palmer Spiller, G.A.; Chernoff, ; , ; , ; Nassar, J . J . ; Shipley, E.A. Am. J. Clin. Nutr. 1980, 33, 754. Ehle, F.R.; Robertson, J.B.; van Soest, P.J. J. Nutr. 1982, 112, 158. Calloway, D. "Handbook of Physiology Vol. 6", Ed. Code, C.F., Amer. Physiol. Soc., 1968; p 2839. Bond, J.H.; Levitt, M.D. Am. J. Clin. Nutr. 1978, 31, 169. Marthinsen, D.; Fleming, S.E. J. Nutr. 1982, 112, 1133. Albershiem, P.; Nevins, D.J.; English, P.D.; Karr, A.; Carb. Res. 1967, 5, 340. Olson, A.C.; Gray, G.M.; Chiu, M.C. 182nd meeting American Chemical Society, New York, 1981, AGFD No. 34. Prynne, C.J.; Southgate, D.A.T. Br. J. Nutr. 1979, 41, 495. Cummings, J.H.; Southgate, D.A.T.; Branch, W.J.; Wiggins, H.S.; Houston, H.; Jenkins, D.J.Α.; Jivraj, T.; Hill, M.J. ibid., p 477. Fengel, D.; Wegener, G. "Hydrolysis of Cellulose: Mecha­ nisms of Enzymatic and Acid Catalysis", Adv. in Chem. Series #181, Am. Chem. Soc., 1979; p 145. Heller, S.N.; Hackler, L.R.; Rivers, J.M.; van Soest, P.J.; Roe, D.A.; Lewis, B.A.; Robertson, J. Am. J. Clin. Nutr. 1980, 33, 1734. Dintzis, F.R.; Legg, L.M.; Deatherage, W.L.; Baker, F.L.; Inglett, G.E.; Jacob, R.A.; Reck, S.J.; Munoz, J.M.; Klevay, L.M.; Sandstead, H.H.; Shuey, W.C. Cereal Chem., 1979, 56, 123.

RECEIVED October 29, 1982

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

17 Properties of Wood Lignin C. A. P A D E N , A. S. F R A N K , J. M. WIEBER, B. A. P E T H I C A , and P. Z U M A N Clarkson College of Technology, Departments of Chemistry and Physics, Potsdam, NY 13676 L. J U R A S E K Pulp and Paper Research Institute of Canada, Pointe Claire, Quebec, Canada H 9 R 3J9

To further the component of th y , three aspects of the properties of enzymatically isolated wood lignin are discussed here: (1) the effect of cations and complexing ligands on adsorption of nitrosamines on lignin; (2) the binding of heavy metals on lignin; and (3) the adsorption of bile acids on lignin, wood and cellulose. L i g n i n i s a h i g h l y branched polymer composed o f phenylpropane u n i t s t h a t a r e b e l i e v e d t o be c h e m i c a l l y bound t o t h e c a r b o h y d r a t e s i n wood. E i t h e r d e g r a d a t i v e p r o c e s s e s , such as t h o s e used i n p r o d u c i n g k r a f t o r Klason l i g n i n s , or e x t r a c t i o n with organic solvents, such as those used i n p r o d u c i n g Brauns l i g n i n , a r e used t o i s o l a t e l i g n i n s from wood. L i g n i n s i s o l a t e d by t h e s e methods a r e u n l i k e l y t o be u s e f u l f o r e l u c i d a t i n g t h e r o l e o f l i g n i n i n d i e t a r y f i b e r because t h e y a r e e i t h e r c h e m i c a l l y o r s t r u c t u r a l l y d i f f e r e n t from n a t u r a l l y o c c u r r i n g l i g n i n (_1) . We have c o n c e n t r a t e d o u r e f f o r t s on l i g n i n s o b t a i n e d from wood by enzymatic d i g e s t i o n (2). L i g n i n s p r e p a r e d by t h e enzymatic removal o f t h e c a r b o h y d r a t e s o f p l a n t m a t e r i a l s bear a c l o s e r resemblance t o t h e substances found i n t h e d i g e s t i v e t r a c t . To f u r t h e r t h e u n d e r s t a n d i n g o f l i g n i n as a component o f d i e t a r y f i b e r , studies o f three aspects o f the propert i e s o f wood l i g n i n a r e d i s c u s s e d h e r e : (1) t h e e f f e c t of c a t i o n s and complexing l i g a n d s on a d s o r p t i o n o f n i t r o s a m i n e s on l i g n i n ; (2) t h e b i n d i n g o f heavy m e t a l s on l i g n i n ; and (3) t h e a d s o r p t i o n o f b i l e a c i d s on l i g n i n , wood and c e l l u l o s e .

0097-6156/83/0214-0241$06.00/0 © 1983 American Chemical Society

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

242

UNCONVENTIONAL

SOURCES

OF DIETARY

FIBER

E f f e c t o f C a t i o n s and Complexing L i g a n d s on the A d s o r p t i o n o f N i t r o s a m i n e s on L i g n i n The marked d i f f e r e n c e s i n the a d s o r p t i o n o f N - n i t r o s o d i e t h y l a m i n e on d i f f e r e n t l i g n i n p r e p a r a t i o n s e n a b l e d us (.3) t o c o r r e l a t e the a d s o r p t i o n o f t h i s n i t r o s a m i n e w i t h c h e m i c a l s t u d i e s o f the v a r i o u s lignins. The a d s o r p t i o n o f N - n i t r o s o d i e t h y l a m i n e was a l s o used as a d i a g n o s t i c t o o l i n d e f i n i n g optimum c o n d i t i o n s f o r p r e p a r i n g the enzyme l i g n i n . Two f a c t o r s proved t o be o f p a r t i c u l a r i m p o r t a n c e : mechanical p r e t r e a t m e n t o f wood and the c o n d i t i o n s of the enzymatic d i g e s t i o n p r o c e s s ( £ ) . The e f f e c t o f t h e s e f a c t o r s on the m e t a l c o n t e n t i n wood and l i g n i n i s d i s c u s s e d h e r e . The m e c h a n i c a l comminution p r o c e s s can i n t r o d u c e heavy m e t a l s , p a r t i c u l a r l y i r o n i n t o the samples A n a l y s i s by atomic a b s o r p t i o the c a l c i u m , magnesium a f f e c t e d by the comminution method. Samples o f wood p u l v e r i z e d by a H u r r i c a n e P u l v e r i z e r (a hammer m i l l ) , however, had an i r o n c o n t e n t one o r d e r o f magnitude h i g h e r than t h o s e t r e a t e d i n a W i l e y M i l l (a r o t a t i n g steel cutter). In b o t h c a s e s the samples were f u r t h e r b a l l - m i l l e d w i t h ceramic b a l l s . The enzymatic l i g n i n p r e p a r e d from the p u l v e r i z e d and b a l l - m i l l e d wood showed a h i g h i r o n c o n t e n t (Table I ) . The c o n t e n t o f m e t a l i o n s i n the wood samples was reduced by d e i o n i z a t i o n by e x t r a c t i o n w i t h 1 M HC1 and 1 M NaCl and subsequent dialysis. S i m i l a r t r e a t m e n t o f the l i g n i n s showed a d e c r e a s e i n c a l c i u m , magnesium, and z i n c c o n t e n t , w i t h a r e l a t i v e l y low degree o f removal o f i r o n . This i n d i c a t e s t h a t l i g n i n b i n d s i r o n more s t r o n g l y when i s o l a t e d from the wood p o l y s a c c h a r i d e s . Another source o f the metal i o n c o n t e n t of l i g n i n i s from the enzyme s o l u t i o n s . Two t y p e s o f enzymatic p r e p a r a t i o n s were used: a m i x t u r e o f the c e l l u l a s e s produced by c u l t u r e s o f S c h i z o p h y l l u m commune and T r i c h o d e r m a r e e s e i (5) ; and a commercial p r e p a r a t i o n ( C e l l u c a s t , Novo I n d u s t r y , Denmark) which i s a c o n c e n trate of the c e l l u l a s e s produced by T r i c h o d e r m a v i r i d e . Both types o f enzyme p r e p a r a t i o n c o n t a i n m e t a l i o n s d e r i v e d from the c u l t u r e media. N - n i t r o s o d i e t h y l a m i n e i s s t r o n g l y adsorbed onto k r a f t l i g n i n (6) , K l a s o n l i g n i n (7) , and onto f r e s h l y p r e p a r e d o r s t o r e d samples o f enzyme l i g n i n w i t h low r e s i d u a l sugar p r e p a r e d from W i l e y - m i l l e d wood. By c o n t r a s t a l l o f the enzymatic l i g n i n samples p r e p a r e d from H u r r i c a n e - p u l v e r i z e d wood were poor a d s o r b e r s o f nitrosamine. We b e l i e v e t h a t i r o n , i n t r o d u c e d by the H u r r i c a n e p u l v e r i z e r , forms a complex w i t h l i g n i n t h a t a l t e r s i t s adsorptive properties. In one enzyme l i g n i n sample f r e s h l y p r e p a r e d from H u r r i c a n e - p u l v e r i z e d wood, the a d s o r p t i o n o f n i t r o s a m i n e s was enhanced by washing

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

17.

PADEN ET A L .

Properties of Wood

Table Minerals i

Aspe

Wood, H u r r i c a n e pulverized, ball-milled Wood, same,

Lignin, Lignin,

enzymatic same,

0

I Wood

d Lignin

Ca

Mg

Fe

Zn

1600

220

350

50

40

3

90

6

1400

170

30

50

1200

75

370

8

90

9

280

7

deionized

Wood, W i l e y - m i l l e d ball-milled

243

Lignin

deionized^

a

Determined

b

E x t r a c t e d w i t h 1 M HC1 and 1 M N a C l , d i s t i l l e d water.

c

From Hurricane-pulverized,

by atomic

absorption. dialyzed

ball-milled

against

wood.

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

244

UNCONVENTIONAL SOURCES OF DIETARY FIBER

the l i g n i n w i t h a 1 molar s o l u t i o n o f c a l c i u m , sodium or l i t h i u m c h l o r i d e ( F i g u r e 1 ) . The a d s o r p t i o n p r o p e r t i e s of l i g n i n increase with increasing i o n i c radius of the c a t i o n . T h i s i n d i c a t e s t h a t p r o b a b l y an i o n i c s t r e n g t h e f f e c t i s i n v o l v e d , a f f e c t i n g the c o m p o s i t i o n of the l a y e r o f s o l v e n t (water) a t the s u r f a c e o f lignin. However, when the sample o f l i g n i n was s t o r e d i n a semi-wet s t a t e a t 4 ° C f o r a month, t h i s enhancement o f n i t r o s a m i n e a d s o r p t i o n by c o n t a c t w i t h c o n c e n t r a t e d s a l t s o l u t i o n s was l o s t . A n o t h e r sample o f enzymatic l i g n i n f r e s h l y p r e p a r e d from H u r r i c a n e - p u l v e r i z e d wood was suspended i n 0.1 M EDTA and s t o r e d a t 4 ° C f o r one week. This treatment a l s o enhanced the a d s o r p t i o n o f n i t r o s a m i n e (Figure 2). I t t h e r e f o r e appears t h a t i o n exchange o f heavy m e t a l s , n o t a b l EDTA enhances the a d s o r p t i o matic l i g n i n . S y s t e m a t i c c o n t r o l o f the heavy m e t a l ions i n enzymatically prepared l i g n i n s i s a necessary feature of future s t u d i e s . B i n d i n g o f Heavy M e t a l s on L i g n i n A d s o r p t i o n o f c o p p e r ( I I ) and z i n c ( I I ) i o n s from u n b u f f e r e d aqueous s u s p e n s i o n s o f l i g n i n was f o l l o w e d by p o l a r o g r a p h i c d e t e r m i n a t i o n o f the c o n c e n t r a t i o n o f those i o n s r e m a i n i n g i n s o l u t i o n , u s i n g v a r y i n g c o n centrations of C u and Z n i o n s e q u i l i b r a t e d w i t h l i g n i n p r e p a r a t i o n f o r 20-24 h . The amount o f the m e t a l i o n adsorbed by one gram o f l i g n i n ( F i g u r e s 3, 4) was s i m i l a r f o r both m e t a l s . The b i n d i n g i n c r e a s e s i n the sequence: k r a f t l i g n i n < enzymatic l i g n i n < brown r o t wood, f o r b o t h copper and z i n c i o n s , and the shapes of the a d s o r p t i o n i s o t h e r m s were s i m i l a r . The p r e s e n c e of two l i m i t i n g r e g i o n s , most c l e a r l y demonstrated f o r the a d s o r p t i o n o f z i n c i o n s on brown r o t wood, i n d i c a t e s a t l e a s t two d i f f e r e n t b i n d i n g c e n t e r s . The sequence of the b i n d i n g a b i l i t y o f l i g n i n f o r m e t a l i o n s d i f f e r s from t h a t found f o r b i n d i n g n i t r o s a m i n e and i n d i c a t e s t h a t b i n d i n g s i t e s f o r copper and z i n c a r e d i s t i n c t from those t h a t i n t e r a c t w i t h n i t r o s a m i n e . Enzymatic l i g n i n s may c o n t a i n r e s i d u a l c e l l u l o s e and some amount of adsorbed p r o t e i n s a n d / o r p e p t i d e s t h a t adsorb copper and z i n c . E l e m e n t a l n i t r o g e n and p r i m a r y amino groups have been d e t e c t e d i n our l i g n i n p r e p a r a t i o n s , but a s y s t e m a t i c study o f the e f f e c t o f t h e s e r e s i d u e s on Cu2+ and Z n b i n d i n g i s not y e t a v a i l a b l e . 2 +

2 +

2 +

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

17.

Properties

PADEN ET A L .

of Wood

Lignin

245

20 h pmoles/g

10 h

nrtrosodierhylamin Figure 1. Effect of high concentrations of solutions of strong electrolytes on the adsorption of N-nitrosodiethylamine on lignins. Dependence of the number of pmoles of the nitrosamine adsorbed on equilibrium concentration of free, unbound N-nitrosodiethylamine. Lignin was produced by digestion of Hurricane-pulverized, ball-milled aspen wood with an 80:20 mixture of enzyme-containing supernatants from Schizophyllum commune and Trichoderma reesei cultures (final sugar content 4%). Samples were exposed for 30 min to 1 M CaClt (Ca ), NaCl (Na ); LiCl (Li*), or water (0). Suspension was centrifuged, supernatant discarded, lignin suspended in solutions containing varying concentrations of N-nitrosodiethylamine, equilibrated for 24 h, and remaining concentration of the nitrosamine determined in the supernatant. 2+

+

Figure 2. Effect of treatment with EDTA on the ability of lignin to adsorb Nnitrosodiethylamine. Dependence of the number of pmoles of the nitrosamine adsorbed on equilibrium concentration of free N-nitrosodiethylamine. Lignin was produced as in Figure 1 with a final sugar content of 16%. The lignin was suspended in 0.1 M EDTA for 1 week. The suspension was then treated as in Figure 1.

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

246

UNCONVENTIONAL SOURCES OF DIETARY FIBER

brown rot

200

Mmoles/g L III redigest 100

2 mM copper ion Figure 3. Adsorption of cupric ions on lignins of various origin and similar materials. Dependence of the number of μ/noles of Cu ions adsorbed on equilibrium concentration of the free Cu ions. "Brown rof refers to birch wood decayed by Polyporus Betulinum and digested with Celluclast (without milling pretreatment). One sample of lignin (L III redigest) was produced from Hurricane-pulverized, ball-milled aspen wood and digested with both Celluclast and the Schizophyllum and Trichoderma enzyme mixture (final sugar content 12%). Another sample of Lignin (L III) was produced from Hurricane-pulverized, ball-milled aspen wood and then digested with the Schizophyllum and Trichoderma mixture (final sugar content 4%); Kraft lignin was purchased from Westvaco. Samples of lignin were suspended in solutions containing varying concentrations of copper sulfate, equilibrated for 24 h, and remaining concentration determined in the supernatant. 2+

2+

brown rot

2 mM zinc ion Figure 4. Adsorption of zinc ion on various lignins and similar materials. The same samples of lignin as in Figure 3 were equilibrated with a zinc sulfate solution.

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

17.

PADEN ET A L .

Properties of Wood

Lignin

247

The A d s o r p t i o n o f B i l e A c i d s on L i g n i n s The a d s o r p t i o n o f the b i l e a c i d s from aqueous s o l u t i o n s o f t h e i r s a l t s was f o l l o w e d by measuring the c o n c e n t r a t i o n o f the unadsorbed a c i d i n the s u p e r ­ n a t a n t , u s i n g the measurement o f the a b s o r p t i o n s p e c t r a o f p o l y e n y l i c c a r b o c a t i o n s formed from b i l e a c i d s i n 72% s u l f u r i c a c i d (8). For a given b i l e a c i d , e . g . , c h o l i c a c i d , much s m a l l e r d i f f e r e n c e s i n a d s o r p t i o n to a v a r i e t y o f l i g n i n s and wood r e l a t e d m a t e r i a l s was f o u n d , as compared t o a d s o r p t i o n o f n i t r o s a m i n e s . Bile a c i d s are thus l e s s s u i t a b l e f o r d i s c r i m i n a t i n g a d s o r p t i v e properties of l i g n i n preparations. Nevertheless, comparing the a d s o r p t i v e c a p a c i t i e s f o r a g i v e n mass o f a d s o r b e n t f o l l o w s a s i m i l a r sequence f o r the a d s o r p t i o n of b i l e a c i d s as wa both cases, c e l l u l o s adsorbers. A l l o f the l i g n i n s , b o t h k r a f t and e n z y ­ m a t i c , showed s i g n i f i c a n t l y g r e a t e r a b s o r p t i v i t y than wood ( F i g u r e 5 ) . The sequence o f a d s o r p t i o n p r o p e r t i e s o f c h o l i c , d e o x y c h o l i c and t a u r o c h o l i c a c i d s onto l i g n i n ( F i g u r e 6) f o l l o w s the same p a t t e r n as r e p o r t e d by Eastwood {9) and Kay (10), b u t i t i s d o u b t f u l i f these d i f f e r e n c e s are s i g n i f i c a n t . The s m a l l d i f f e r ­ ences i n a d s o r p t i v i t y o f t h e s e b i l e a c i d s , as w e l l as t h o s e o f l i t h o c h o l i c and g l y c o c h o l i c , i n d i c a t e t h a t the s i d e - c h a i n and the number o f the hydroxy groups p l a y a minor r o l e i n the a d s o r p t i v i t y , p r o b a b l y due to a f l a t o r i e n t a t i o n o f the r i g i d s t e r o l m o l e c u l e a t the l i g n i n surface. The a d s o r p t i o n o f b i l e s a l t s t o l i g n i n s i s p r a c ­ t i c a l l y complete i n about 5 h . When samples o f l i g n i n w i t h adsorbed b i l e a c i d were s e p a r a t e d from the s u p e r ­ n a t a n t , suspended i n water and shaken f o r 3 h , 50% o f the b i l e a c i d was d e s o r b e d . Experimental T r e m b l i n g aspen wood (Populus t r e m u l o i d e s M i c h x . ) was reduced to a powder i n a H u r r i c a n e P u l v e r i z e r . Samples were p r e p a r e d from e x t r a c t e d p u l v e r i z e d wood by b a l l - m i l l i n g f o r 10 days i n a r o t a t i n g c e r a m i c m i l l equipped w i t h an a i r - c o n d i t i o n i n g u n i t which p r e v e n t e d the temperature o f the c o n t e n t s o f the m i l l from r i s i n g above ambient. D i g e s t i o n o f the samples w i t h c e l l u l a s e was done w i t h a m i x t u r e o f S c h i z o p h y l l u m commune and Trichoderma r e e s e i c u l t u r e f i l t r a t e s at 30°C f o r 3 days. One sample ( L i g n i n I I I r e d i g e s t ) was d i g e s t e d a second time w i t h a commercial enzyme p r e p a r a t i o n , C e l l u c l a s t (Novo I n d u s t r y , Denmark), a t 5 0 ° C f o r 2 d a y s . The i s o ­ l a t e d l i g n i n s u s p e n s i o n was s t o r e d a t 4 ° C u n t i l analysis. ΑΐΪΙβΓίϋθΠ Cfiemlctf

Society Library 1155 16th St. N. W. In Unconventional Sources of Dietary Fiber; Furda, I.; D. C. 20038 ACS Symposium Series; Washington, American Chemical Society: Washington, DC, 1983.

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Figure 5. Adsorption of cholic acid on cellulose, wood and kraft, and enzyme lignins. Dependence of the number of pmoles of cholic acid adsorbed on equilibrium concentration of the free cholic acid. Lignin (L 5) was produced from Wiley-milled, ball-milled aspen wood and digested with the Schizophyllum and Trichoderma mixture (final sugar content 4%); Kraft lignin was purchased from Westvaco; microcrystalline cellulose (Avicel R) was obtained from the FMC Corporation (Newark). Samples of lignin, wood, or cellulose were suspended in unbuffered solutions of sodium cholate, equilibrated for 24 h, and remaining concentration determined in the supernatant.

J

ι

1

2 mM bile acid

Figure 6.

Adsorption of cholic, deoxycholic, and taurocholic acid on enzyme lignin (L 5). Lignin L 5 was obtained and treated as in Figure 5, solutions of sodium salts of cholic, deoxycholic, and taurocholic acids were equilibrated with lignin for 24 h, and remaining concentration in the supernatant determined.

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

17.

PADEN ET AL.

Properties of Wood

Lignin

249

One sample (L5) was produced from W i l e y - m i l l e d , b a l l - m i l l e d wood which had been d i g e s t e d w i t h the m i x t u r e o f enzymes found i n S. commune and Τ . r e e s e i c u l t u r e f i l t r a t e s (_5) . T h i s l i g n i n sample was d r i e d under vacuum a t ambient temperature and then ground t o a f i n e powder w i t h a mortar and p e s t l e . Brown r o t wood was p r e p a r e d from b i r c h wood which had been decayed by P o l y p o r u s B e t u l i n u m and d i g e s t e d with C e l l u c l a s t without m i l l i n g pretreatment. K r a f t l i g n i n ( l o t no. RLX 4290:20B) was o b t a i n e d from Westvaco, C h a r l e s t o n Research C e n t e r , N o r t h C h a r l e s t o n , S C . M a c r o c r y s t a l l i n e c e l l u l o s e ( A v i c e l R) was o b t a i n e d from FMC, Newark. In the study o f n i t r o s a m i n e a d s o r p t i o n , l i g n i n s u s p e n s i o n s were s t o r e d a t 4 ° C f o r l e s s than 15 d a y s Samples were expose f o r 30 min o r t o 0. were c e n t r i f u g e d and s u p e r n a t a n t s were d i s c a r d e d . L i g n i n samples were then suspended i n u n b u f f e r e d s o l u t i o n s c o n t a i n i n g v a r y i n g concentrations of N - n i t r o ­ s o d i e t h y l a m i n e and e q u i l i b r a t e d f o r 25 h . The r e m a i n ­ i n g c o n c e n t r a t i o n o f n i t r o s a m i n e was determined i n the s u p e r n a t a n t by p o l a r o g r a p h y (.3) . A d s o r p t i o n o f c o p p e r ( I I ) and z i n c (II) i o n s was determined by suspending l i g n i n samples i n u n b u f f e r e d s o l u t i o n s c o n t a i n i n g v a r y i n g c o n c e n t r a t i o n s o f copper s u l f a t e or zinc s u l f a t e . After equilibration for 20-24 h , the c o n c e n t r a t i o n r e m a i n i n g i n the s u p e r n a t a n t was determined by p o l a r o g r a p h y . Samples o f l i g n i n , wood, o r c e l l u l o s e were s u s ­ pended i n phosphate b u f f e r (pH 7 . 0 , μ = 0.5) c o n t a i n i n g v a r y i n g c o n c e n t r a t i o n s o f sodium c h o l a t e , sodium d e o x y c h o l a t e o r sodium t a u r o c h o l a t e . The c o n c e n t r a t i o n r e m a i n i n g i n the s u p e r n a t a n t a f t e r e q u i l i b r a t i o n f o r 20-24 h was determined by measuring the a b s o r p t i o n s p e c t r a o f p o l y e n y l i c c a r b o c a t i o n s formed from b i l e a c i d s i n 72% s u l f u r i c a c i d {8) . A d s o r p t i o n o f selenium(IV) i o n s from u n b u f f e r e d s o l u t i o n s o f k r a f t l i g n i n was examined i n p r e l i m i n a r y studies. P o l a r o g r a p h i c d e t e r m i n a t i o n (11) o f selenium(IV) r e m a i n i n g i n s o l u t i o n a f t e r 25 h e q u i l i ­ b r a t i o n i n d i c a t e d t h a t k r a f t l i g n i n adsorbs a n e g l i ­ g i b l e quantity of selenium. A polarographically r e d u c i b l e o r g a n i c compound which was e x t r a c t e d from the l i g n i n p r e v e n t e d a more d e t a i l e d a n a l y s i s o f the adsorption properties of selenium(IV).

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

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Literature Cited

1.

2. 3.

4. 5. 6. 7.

8. 9. 10.

11.

L a i , Y.Z; Sarkanen, Κ. V. "Lignins, occurrence, formation, structure and reactions"; Κ. V. Sarkanen and C. H. Ludwig, Eds; Wiley-Interscience: New York, NY, 1971; p 165. Pew, J . C . Tappi 1957, 40, 553. Rubio, M. A . ; Pethica, B.A.; Zuman, P.; Falkehag, S. I . ; "Dietary Fibers: Chemistry and Nutrition"; G. E. Inglett and S. I. Falkehag, E d s . ; Academic Press: New York, NY, 1979, p 251. Fukazawa, K.; Revol, J.-F.; Jurasek, L.; Goring, D.A.I. Wood Sci. Tech., in press. Desrochers, M.; Jurasek, L.; Paice, M.G. Appl. Environ. Microbiol. 1981, 41, 222. Allsop, Α.; Misra Marton, J . "Lignins structure and reactions"; Κ. V. Sarkanen and C. H. Ludwig, Eds.; Wiley-Interscience: New York, NY, 1971, p 639. Smith, R.; Zuman, P. unpublished data. Eastwood, Μ. Α . ; Hamilton, D. Biochem. Biophys. Acta 1968, 152, 165. Kay, R. M.; Strasberg, S. M.; Petrunka, C. N.; Wayman, M. "Dietary Fibers: Chemistry and Nutri­ tion"; G. E . Inglett and S. I. Falkehag, Eds.; Academic Press: New York, NY, 1979, p 57. Frank, A . S . ; Zuman, P. Anal. Chem., submitted for publication.

RECEIVED October 29,

1982

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

18 Chemical and Physical Properties of Tobacco Fiber VIDA D. SHEEN and S. J. SHEEN University of Kentucky, Department of Plant Pathology, Lexington, KY 40646

Fiber complexes of ten tobacco cultivars as by-products of the leaf protein extraction process con tain levels of parable to tha that in wheat bran. Compositional analyses revealed lesser concentrations of cellulose, lignin, and lipid but a greater amount of hemicellulose in tobacco than in alfalfa. In reference to wheat bran, tobacco fiber has comparable amounts of Na, P, S, Mn, and Cu but at least 20-fold more Ca and severalfold more Fe. Among tobacco cultivars, dark green tobaccos accumulate more starch and protein than burley and Turkish types. Quantitative variation of cellulosic components and minerals is also evident among tobaccos. Average bulk fiber volume of tobacco cultivars is 14.2 mL/g as compared to 9.5 and 4.5 mL/g for alfalfa fiber and wheat bran, respectively. The water retention capacity of tobacco fiber (9 g water/g) is nearly double that of alfalfa fiber and eight times greater than wheat bran. Cation exchange capacity ranks the highest for tobacco, followed by alfalfa and then wheat bran. Sodium taurocholate binding capacity is comparable among the three fiber sources although the fiber complex of flue-cured tobaccos absorbs more than others. The above properties of tobacco fiber complex suggest its potential as a source of dietary fiber. Epidemiological evidence i n causative r e l a t i o n s h i p s between f i b e r - d e p l e t e d d i e t s and c e r t a i n c h a r a c t e r i s t i c a l l y Western diseases has stimulated research i n t e r e s t i n the p h y s i o l o g i c a l and physico-chemical p r o p e r t i e s of various d i e t a r y f i b e r s i n the past decade ( 1). D i e t a r y f i b e r s are composed of c e l l u l o s e , l i g n i n , and n o n c e l l u l o s i c polysaccharides, a l l of which are the

0097-6156/83/0214-0251$06.00/0 © 1983 American Chemical Society

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components of plant c e l l w a l l . Pure c e l l u l o s e exerts l i t t l e p h y s i o l o g i c a l f u n c t i o n , whereas l i g n i n and n o n c e l l u l o s i c polysaccharides are capable of binding f a t t y a c i d s , b i l e a c i d s , and carcinogens (2, 3). Substances such as s t a r c h , p r o t e i n , l i p i d , p h y t i c a c i d , and minerals are u s u a l l y associated with d i e t a r y f i b e r i n forming a d i e t a r y f i b e r complex that a l t e r s physico-chemical p r o p e r t i e s of the f i b e r . Among the common foods of p l a n t o r i g i n , breakfast c e r e a l s and wholemeal breads containing wheat bran are high d i e t a r y - f i b e r foods f o r both h e a l t h conscious people and f o r the treatment and c o n t r o l of diabetes (4). Enrichment of foods with f i b e r from c e r e a l h u l l , c i t r u s pulp, guar gum and woody t i s s u e s i s p r e s e n t l y i n the experimental stage. Although f i b e r s i n the n a t u r a l foods of human d i e t are primarly derived from l e a f y vegetables, the p u r i f i e d d i e t a r y f i b e r s of l e a f t i s s u e o r i g i n are not commer­ c i a l l y available. The recent achievemen young tobacco plants has advanced the p o s s i b i l i t i e s f o r u t i l i z ­ ing tobacco plant m a t e r i a l s i n human foods (5). Young tobacco plants grown under high density produced l e a f biomass two to three times greater than s t a l k weight (6). Fibrous residue of young tobacco p l a n t s from the l e a f p r o t e i n e x t r a c t i o n process can be a p o t e n t i a l d i e t a r y f i b e r i f the r e s i d u e , a f t e r removal of p l a s t i c pigments, possesses d e s i r a b l e physicochemical p r o p e r t i e s . The present study was t h e r e f o r e under­ taken to evaluate the d e c o l o r i z e d tobacco f i b r o u s residues f o r chemical and p h y s i c a l p r o p e r t i e s and to compare them with wheat bran and a l f a l f a f i b r o u s residue. Because of a broad genetic v a r i a t i o n i n morphology and chemical composition of p l a n t s w i t h i n N i c o t i a n a tabacum, the present study compared a number of tobacco types. M a t e r i a l s and Methods A f i e l d experiment i n v o l v i n g ten tobacco c u l t i v a r s representing f l u e - c u r e d , dark f i r e - c u r e d , burley, Turkish and Maryland types was conducted i n Lexington, Kentucky, i n e a r l y s p r i n g 1980. About 110-120 p l a n t s per p l o t (1.5 χ 1 m) per c u l t i v a r were e s t a b l i s h e d by d i r e c t - s e e d i n g and c u l t u r e d accord­ ing to conventional p r a c t i c e s i n the burley growing r e g i o n . When p l a n t s reached 50 cm or t a l l e r i n the middle of June, a l l plants w i t h i n a 0.36 m area were cut about 12 cm from the ground as one sample with three samples taken per p l o t . A t o t a l of 30 samples were separately processed to obtain deproteinized and d e c o l o r i z e d f i b e r complex. D e p r o t e i n i z a t i o n of f r e s h tobacco p l a n t s was accomplished by homogenization i n a high capacity Waring blender with an equal weight of i c e - c o l d 2% sodium m e t a b i s u l f i t e s o l u t i o n . The homogenate was s t r a i n e d through three l a y e r s of cheese-cloth and one l a y e r of M i r a c l o t h and, subsequently, the green f i b r o u s 2

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

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SHEEN AND SHEEN

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residue was washed with a s u f f i c i e n t volume of c o l d water to remove r e s i d u a l s o l u b l e p r o t e i n . The d e p r o t e i n i z e d green f i b r o u s residue was washed with s e v e r a l volumes of c o l d 80% isopropanol u n t i l cream c o l o r e d . In a d d i t i o n , three samples of f r e s h a l f a l f a (before the flower bud stage) which'were d e p r o t e i n i z e d and d e c o l o r i z e d by the same process and wheat bran from a commercial m i l l were included f o r comparison. A l l samples were sieved through a 9-mesh screen and oven-dried (70 C) overnight before chemical and p h y s i c a l c h a r a c t e r i z a t i o n . A l l f i b e r complexes were analyzed f o r p r o t e i n , s t a r c h , l i p i d , c e l l u l o s e , l i g n i n , and ash concentrations. Protein quantity was c a l c u l a t e d from the d i f f e r e n c e i n f r e e and t o t a l amino a c i d contents before and a f t e r 6Ν HC1 d i g e s t i o n . Ninhyd r i n r e a c t i o n of amino acids was c o l o r i m e t r i c a l l y determined at 570 nm with known q u a n t i t i e s of amino a c i d s as standards (7) Starch was quantitated method of Gaines and Meud and a f t e r overnight Soxhlet e x t r a c t i o n of f i b e r complex with hexane measured l i p i d content. C e l l u l o s e and l i g n i n q u a n t i t a ­ t i o n on an ash-free b a s i s was performed through g r a v i m e t r i c methods (9). Ash content was the r e s i d u a l weight of c e l l u l o s e and l i g n i n a f t e r c h a r r i n g i n a muffle furnace at 550 C f o r 4 hr. The d i f f e r e n c e of the above s i x components from 100% was considered as apparent h e m i c e l l u l o s e which includes h e m i c e l l u ­ l o s e , p e c t i c substances, gums, and mucilages, i f present. The same samples were analyzed f o r n e u t r a l and a c i d detergent f i b e r (NDF and ADF) content according to Van S o e s t s methods (10). Mineral elements except phosphorus and molybdenum i n the tobacco f i b e r were analyzed by use of Varian AA-6 Atomic Absorption Spectrophotometer. Samples were wet-ashed i n n i t r i c and p e r c h l o r i c acids (9:1, v/v) u n t i l completely d i g e s t e d . A f t e r evaporation to dryness, the ashes were s o l u b l i z e d i n IN HC1 f o r analyses. Q u a n t i t a t i o n of phosphorus was a c o l o r i m e t r i c method using a Technicon Autoanalyzer and the c o l o r r e a c t i o n of F i s k e and Subbarow (11). Molybdenum was quantitated by the method of E i v a z i et a l (12) which determines, with good r e p r o d u c i b i l i t y , a quantity as low as 0.02 ppm i n plant m a t e r i a l s . P h y s i c a l p r o p e r t i e s measured i n the present study included bulk volume, water r e t e n t i o n c a p a c i t y , and c a t i o n exchange c a p a c i t y . Bulk volume measurement was the method of Montgomery and Baugardt (13). whereas water r e t e n t i o n c a p a c i t y was the amount of water r e t a i n e d by the f i b e r against a c e n t r i f u g a l f o r c e of 14,000 g f o r one hr a f t e r a 24 hr hydration p e r i o d at 20 C (14). For determination of c a t i o n exchange c a p a c i t y , the f i b e r complex was soaked i n 2 Ν HC1 f o r 24 hr followed by wash­ ing with deionized water. Hydrogen ions were d i s s o c i a t e d from the f i b e r complex with 2N n e u t r a l NaCl s o l u t i o n which was then t i t r a t e d against 0.05N NaOH (14). Binding of sodium taurocholate by the f i b e r was the method of Kritchevsky and Story (15). A 50 or 100 mg sample of f i b e r 1

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254

UNCONVENTIONAL SOURCES OF DIETARY FIBER

was incubated i n 5 ml of HPLC-grade water c o n t a i n i n g 0.15 M NaCl and 8 mM (40 μ moles) o r 40 mM (200 μ moles) sodium taurocholate at 37 C f o r one hr with gentle shaking. For the c o n t r o l , 5 mL of 0.15 M NaCl s o l u t i o n devoid of sodium taurocholate was added to the t e s t i n g sample. Sodium taurocholate i n the same NaCl s o l u t i o n with a concentration s e r i e s of 40 mM, 20 mM, 10 mM, 5 mM and 2.5 mM was processed by the same procedure to e s t a b l i s h a c a l i b r a t i o n curve. A f t e r i n c u b a t i o n , the sample was f i l t e r e d through a 0.2 μπι microporous f i l t e r and the f i l t r a t e was ready f o r HPLC a n a l y s i s . V a r i a n Model 5000 L i q u i d Chromatograph equipped with a Vari-Chrom UV-Vis Detector was used. The HPLC c o n d i t i o n s were: Micro Pak MCH-10 column (30 cm X 4 mm I.D.); eluent, 70% MeOH and 30% 0.15 M NaCl s o l u t i o n , i s o c r a t i c ; flow r a t e , 1 mL/min; d e t e c t i o n at 210 nm. With a 10 μL i n j e c t i o n and 15 min c y c l e , sodium taurocholate can be detected w i t h i n 5 min C o n t r o l samples d i d no s i m i l a r to that of sodiu N i c o t i n e contamination i n tobacco f i b e r was assayed by the GLC equipped with an a l k a l i n e flame i o n i z a t i o n d e t e c t o r . The e x t r a c t i o n procedure and GLC c o n d i t i o n s have been d e t a i l e d i n a recent paper ( 6 ) . The s e n s i t i v i t y o f d e t e c t i o n i s below 1 ppm. A l l data were subjected to v a r i a n c e analyses. Whenever v a r i a t i o n among f i b e r sources was s t a t i s t i c a l l y s i g n i f i c a n t , the l e a s t s i g n i f i c a n t d i f f e r e n c e (LSD) a t the 5% and 1% l e v e l of p r o b a b i l i t y was c a l c u l a t e d f o r s t a t i s t i c a l i n f e r e n c e of the results. Results Chemical composition of tobacco and a l f a l f a d e p r o t e i n i z e d and d e c o l o r i z e d f i b e r complexes and wheat bran i s given i n Table I. Dark green tobaccos i n c l u d i n g f l u e - c u r e d (NC 95, NC 2326 and Coker 139), dark f i r e - c u r e d (Ky 171 and Ky 151) and Maryland (MD 609) types had comparable amounts of p r o t e i n with a mean of 15 g/100 g dry wt. The average value of the three burleys (Ky 16M, Burley 21 and Ky 14) was 9.46 g/100 g dry wt with Burley 21 showing s i g n i f i c a n t l y l e s s than the other two. Turkish tobacco Xanthi nc resembled burley i n p r o t e i n content. Alfalfa f i b e r had a p r o t e i n concentration s i m i l a r to that of dark green tobacco, whereas wheat bran contained a s i g n i f i c a n t l y higher l e v e l among the f i b r o u s samples under comparison. For s t a r c h content, a s i g n i f i c a n t c o n t r a s t e x i s t e d between dark green tobacco and b u r l e y o r Turkish type with the exception of MD 609 resembling burley i n low s t a r c h concentration. I n c i d e n t a l l y , Maryland and b u r l e y tobaccos are a i r - c u r e d types. That s t a r c h content was low i n a l f a l f a f i b e r and reached as high as 30% weight i n wheat bran was not unexpected owing to the nature of the corresponding p l a n t p a r t s . There was l i t t l e d i f f e r e n c e i n l i p i d c o n c e n t r a t i o n among the f i b r o u s residues of tobacco and a l f a l f a with a mean of 2.82 g/100 g dry wt. More than three

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983. 0.99 1.34

1.20 1.63

2.44 3.31

LSD 0.05 LSD 0.01

2.44 3.06 2.78 2.40 3.38 3.55 2.67 2.48 3.09 2.32 2.80 9.54

4.30 2.01 2.22 2.42 1.38 0.90 0.33 0.09 0.18 0.88 0.60 30.67

13.54 15.48 15.61 15.88 14.27 12.21 6.02 10.14 8.41 15.23 13.12 18.51

Lipids

Tobacco NC 95 NC 2326 Coker 139 Ky 171 Ky 151 Ky 16M Burley 21 Ky 14 Xanthi nc MC 609 Alfalfa Wheat bran

Starch

Protein

Lignin

41.51 41.50 47.25 41.85 42.15 47.89 45.15 42.07 46.98 51.06 50.71 11.56 2.28 3.10

53.46 53.36 55.91 50.42 55.36 56.06 53.84 51.84 61.04 55.74 61.84 47.05 5.26 7.15 3.62 4. 93 1.09 1.48

2.53 3.44

1.07 1.46

ADF

35. 52 33. 33 26. 08 36. 62 32. 75 35. 86 39. 71 42. 15 36. 27 25. 63 25. 05 24. 11

NDF

5. 68 4. 13 8. 25 4. 85 5. 10 5. 38 5. 77 4. 55 6. 24 7.15 9. 91 2.94

Apparent hemicellulose

31.38 33.88 32.21 28.09 33.83 32.68 32.71 30.60 35.19 34.00 44.23 10.17

g/100 g dry wt

Cellulose

7.14 8.11 12.55 10.75 9.31 9.41 12.79 9.94 10.79 14.79 4.20 5.80

Ash

Chemical Composition of Tobacco and A l f a l f a Fibrous Residues and Wheat Bran.

Fiber source

Table I.

256

UNCONVENTIONAL SOURCES OF DIETARY FIBER

times t h i s amount was found i n wheat bran. Ash content v a r i e d widely among tobacco f i b r o u s samples which had an average q u a n t i t y of 10.56 g/100 g dry wt. The ash content i n a l f a l f a f i b e r and wheat bran was about one-half or l e s s than that i n tobacco f i b e r . On the same weight b a s i s , wheat bran had only 1/3 to 1/4 the c e l l u l o s e q u a n t i t y present i n tobacco and a l f a l f a f i b e r s . A l f a l f a f i b e r was a l s o r i c h i n l i g n i n with a q u a n t i t y three times greater than that i n wheat bran. L i g n i n q u a n t i t y i n tobacco f i b e r was in-between those of a l f a l f a f i b e r and wheat bran; however, s i g n i f i c a n t d i f f e r e n c e s e x i s t e d w i t h i n and between tobacco types. Such a range of v a r i a t i o n was a l s o evident f o r the q u a n t i t y of apparent h e m i c e l l u l o s e i n tobacco samples. Apparent h e m i c e l l u l o s e content i n a l f a l f a f i b e r and wheat bran was i n d i f f e r e n t to the tobacco c u l t i v a r s being on the low concen t r a t i o n end of the v a r i a t i o n ADF was present among p a t t e r n of v a r i a t i o n . The tobacco c u l t i v a r means f o r NDF and ADF q u a n t i t y were 54.70 and 44.74 g/100 g dry wt, r e s p e c t i v e l y . These values were s i g n i f i c a n t l y higher than those of wheat bran. A l f a l f a f i b e r had the highest amounts of both detergent f i b e r s although the amounts were not s t a t i s t i c a l l y d i f f e r e n t from those of the tobacco c u l t i v a r s ranked high i n detergent f i b e r content. I t has been shown that f i b e r - r i c h d i e t s may impair the u t i l i z a t i o n of a number of e s s e n t i a l minerals i n c l u d i n g calcium, i r o n , z i n c , copper, and phosphorus (16). This can be c o r r e c t e d by d i e t a r y i n t a k e of minerals from a v a r i e t y of foods, on the one hand, and the endogenous source of e s s e n t i a l minerals i n the d i e t a r y f i b e r , on the other. Eleven mineral elements were t h e r e f o r e q u a n t i t a t e d i n tobacco and a l f a l f a f i b e r s and wheat bran (Table I I ) . In r e f e r e n c e to wheat bran, tobacco f i b e r contains comparable or s l i g h t l y higher l e v e l s of sodium, phosphorus, s u l f u r , manganese and copper but at l e a s t 20-fold more calcium and s e v e r a l - f o l d more i r o n . Iron c o n c e n t r a t i o n v a r i e d n e a r l y f i v e - f o l d among tobaccos. Wheat bran was r i c h i n potassium, magnesium, and molybdenum. A l f a l f a f i b e r showed a c l o s e resemblance to tobacco i n mineral content except f o r calcium, magnesium, and copper, which were s i g n i f i c a n t l y l e s s . Rasper (17) studied p h y s i c a l c h a r a c t e r i s t i c s of d i e t a r y c e r e a l f i b e r i n c l u d i n g those from wheat bran and reported that bran f i b e r had bulk volume around 3 to 5 mL/g depending upon p r e p a r a t i o n method. The wheat bran sample i n the present experiment gave the bulk volume of 4.53 mL/g which i s i n good agreement with the values c i t e d i n l i t e r a t u r e . The bulk volume of a l f a l f a and tobacco f i b e r s doubled and t r i p l e d the wheat bran value (Table I I I ) . Among tobaccos, Xanthi nc gave the l a r g e s t bulk volume and NC 2326 the l e a s t . The v a r i a t i o n seemed to be s i g n i f i c a n t f o r f l u e - c u r e d tobaccos but not f o r b u r l e y s . The average water r e t e n t i o n c a p a c i t y of tobacco f i b e r was 9 g water/g with a range of 7.40 to 10.86. Therefore, tobacco f i b e r held

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

LSD 0.05 LSD 0.01

NS NS

0.49 0.39 0.44 0.45 0.42 0.53 0.64 0.48 0.51 0.52 0.35 0.35

Na

0.29 0.39

0.28 0.24 0.90 0.49 0.38 0.69 0.71 0.51 0.55 0.96 0.11 1.46

Κ %

0.45 0.61

1.98 2.03 2.53 2.71 2.68 2.68 2.95 3.08 2.92 3.12 1.22 0.11

Ca

0.22 0.30 0.43 0.30 0.41 0.26 0.27 0.34 0.31 0.36 0.14 0.20 0.09 0.13

0.06 0.08

0.06 0.08

S

0.18 0.20 0.22 0.26 0.27 0.31 0.40 0.38 0.35 0.32 0.13 0.23

Ρ

0.12 0.16 0.20 0.18 0.23 0.19 0.22 0.32 0.27 0.25 0.16 0.54

Mg

0.43 0.85 0.91 0.65 0.54 0.23 0.72 0.49 0.57 0.75 0.50 1.25 0.49 0.67

2 3 46 65 34 46 407 553

Mo

22 21 43 21 29 12 21 38 32 32 7 20

m

Cu

58 54 40 30 56 37 51 116 59 78 36 184

PP

Zn

115 104 212 103 92 99 111 99 118 171 21 114

Μη

542 743 1510 701 659 293 880 738 861 1407 357 175

Fe

Quantities of Mineral Elements i n Tobacco and A l f a l f a Fibrous Residues and Wheat Bran.

Tobacco NC 95 NC 2326 Coker 139 Ky 171 Ky 151 Ky 16M Burley 21 Ky 14 Xanthi nc MD 609 Alfalfa Wheat bran

Fiber source

Table I I .

I

§"

g

M

X w w

00

UNCONVENTIONAL SOURCES OF DIETARY FIBER

258

Table I I I .

Fiber source

Tobacco NC 95 NC 2326 Coker 139 Ky 171 Ky 151 Ky 16 M Burley 21 Ky 14 Xanthi nc MD 609 Alfalfa Wheat bran LSD 0.05 LSD 0:01

Physical Properties of Tobacco and A l f a l f a Fibrous Residues and Wheat Bran

Bulk volume (mL/9)

Water retention capacity (g water/g fiber)

Cation exchange capacity (meq NaOH/g) Χ 10"

14.90 11.33 14.77 14.87 14.00 13.80 13.97 14.80 16.33 13o23 9o47 4.53

9.57 8.40 10.40 7.40 10.86 9.18 8.47 8.55 8.02 9.13 4.92 1.18

21.83 17.50 21.50 20.00 19.00 20.67 21.50 18.50 15.67 19.67 15.33 6.17

0.93 1.25

0.89 1.21

2.47 5.43

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

2

18.

SHEEN AND SHEEN

Tobacco

Fiber

259

twice the volume of water than i t s a l f a l f a counterpart and s i x ­ f o l d or more volume than wheat bran. The ranking of c a t i o n exchange c a p a c i t y among the f i b e r sources followed the same order of the above p h y s i c a l p r o p e r t i e s with tobacco f i b e r being the highest and wheat bran the l e a s t . The average value of c a t i o n exchange c a p a c i t y f o r tobacco c u l t i v a r s was 19.58 X 10"~ meq NaOH/g. The d i f f e r e n c e between tobacco f i b e r and wheat bran was about t h r e e - f o l d . The c a t i o n exchange c a p a c i t y of wheat bran was one-half the value of the a l f a l f a sample. Results of sodium taurocholate binding from two experiments employing d i f f e r e n t combinations of sodium taurocholate and f i b r o u s m a t e r i a l was given i n Table IV. There was a s i g n i f i c a n t d i f f e r e n c e among tobacco types but not w i t h i n a given type. Both experiments revealed that f l u e - c u r e d tobaccos had higher sodium taurocholate binding c a p a c i t y than the other tobacco types, and the binding of f i b e r . By averagin capacity was 2.47 μ moles/50 mg and 6.07 μ moles/100 mg f o r two experiments. These values were ranked the highest, followed by a l f a l f a ; the lowest was wheat bran. The d i f f e r e n c e between the l a t t e r two was not s t a t i s t i c a l l y s i g n i f i c a n t . The f i b e r of flue-cured tobacco was s i g n i f i c a n t l y higher i n binding c a p a c i t y than a l f a l f a f i b e r and wheat bran i n most cases. However, the high binding c a p a c i t y of f l u e - c u r e d tobacco cannot be c o r r e l a t e d with the quantity of any chemical c o n s t i t u e n t s given i n Table I. Young tobacco p l a n t s u s u a l l y contain about 0.5% of dry wt as n i c o t i n e although n i c o t i n e quantity v a r i e s widely among genotypes (18). N i c o t i n e contamination i n tobacco f i b r o u s residue ranged from 180 to 340 ppm with a mean of 260+53 ppm (data not shown). Samples of Ky 171 f i b e r which contains 256 ppm of n i c o t i n e were repeatedly washed with c o l d water (1:10, w/v) and the washed residues were analyzed f o r n i c o t i n e quantity by GLC. On the average, each washing removed two-thirds the n i c o t i n e i n the residue; and a f t e r s e v e r a l washings, n i c o t i n e l e v e l approached 1 ppm (Figure 1). This p o i n t s to the f e a s i b i l ­ i t y of lowering n i c o t i n e contamination i n the f i b r o u s residue to a n e g l i g i b l e l e v e l . The washing process d i d not a l t e r the concentration of p r o t e i n and n o n c e l l u l o s i c components. 2

Discussion P u r i f i e d p l a n t f i b e r s f o r d i e t a r y purposes are combinations of c e l l u l o s e , l i g n i n , h e m i c e l l u l o s e , gums and pectinaceous sub­ stances that exert chemical and p h y s i c a l a c t i o n s i n the g a s t r o ­ i n t e s t i n a l t r a c t . D i e t a r y f i b e r complexes such as wheat bran contain not only the f i b e r components but a l s o other organic compounds that c o n t r i b u t e to n u t r i t i o n . In comparison to wheat bran, the d e p r o t e i n i z e d and d e c o l o r i z e d f i b e r complexes of tobacco and a l f a l f a have favorable chemical composition as d i e t a r y f i b e r s . T h e i r low concentrations i n s t a r c h and l i p i d s

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

UNCONVENTIONAL

260

Table IV.

SOURCES OF DIETARY FIBER

Binding In V i t r o of Sodium Taurocholate to Tobacco and A l f a l f a Fibrous Residues and Wheat Bran

Fiber source

Experiment 1 μ moles/50 mg

Tobacco NC 95 NC 2326 Coker 139 Ky 171 Ky 151 Ky 16M Burley 21 Ky 14 Xanthi nc MD 609 Alfalfa Wheat bran

3.65 3.30 2.78 1.89 1.91 2.12 1.86 2.54 2.48 2.21 2.16 2.09

7.89 7.13 7.88 6.44 4.78 6.56 4.37 4.99 5.14 5.56 4.93 4.84

0.88 1.20

1.83 2.49

LSD 0.05 LSD 0.01

Experiment 2 μ moles/100 mg

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

SHEEN AND SHEEN

Tobacco

Fiber

NUMBER OF WATER-WASHINGS

Figure 1. Effects of water-washing on the quantity of nicotine (O), protein (Ah and apparent hemicellulose (A) in the fibrous residue of Ky 171 tobacco.

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

262

UNCONVENTIONAL SOURCES OF DIETARY FIBER

and high amounts of c e l l u l o s i c components seem to make them more s u i t a b l e than wheat bran as l o w - c a l o r i e d i e t a r y f i b e r s . This i s f u r t h e r supported by the p h y s i c a l p r o p e r t i e s , e s p e c i a l l y f o r tobacco f i b e r , that a l a r g e bulk volume i n accompaniment with high water r e t e n t i o n and c a t i o n exchange c a p a c i t y would be d e s i r a b l e . In a d d i t i o n , the absorption of b i l e s a l t (sodium taurocholate) by fobacco f i b e r i s as e f f e c t i v e as, or more so than wheat bran which suggests that tobacco f i b e r may a l s o have the c a p a b i l i t y of r e g u l a t i n g serum c h l o r e s t e r o l l e v e l s . Nevert h e l e s s , the favorable chemical and p h y s i c a l p r o p e r t i e s of p l a n t f i b e r complex measured i n v i t r o s t i l l r e q u i r e confirmation from i n v i v o experimentation since the p h y s i o l o g i c a l performance and t e c h n o l o g i c a l f u n c t i o n a l i t y of d i e t a r y f i b e r i n f i b e r - e n r i c h e d foods are i n f l u e n c e d by many f a c t o r s . As suggested by Van Soest and Wine (10), the q u a n t i t a t i v e d i f f e r e n c e between NDF of h e m i c e l l u l o s e quantity l i g n i n content. Taking wheat bran i n the present study (Table I) as an example, the c a l c u l a t e d values f o r h e m i c e l l u l o s e and ADF are 35.49 and 13.12 g/100 g,which are i n good agreement with Southgate's f i n d i n g s (19). However, the c a l c u l a t e d value of h e m i c e l l u l o s e i s almost 50% greater than that of apparent hemicellulose. This i s not s u r p r i s i n g since wheat bran i s r i c h i n s t a r c h known to contaminate the NDF f r a c t i o n . On the b a s i s of NDF and ADF q u a n t i t i e s , the c a l c u l a t e d value of h e m i c e l l u l o s e i n the a l f a l f a sample i s 11.13 g/100 g, while the average amount f o r tobacco f i b e r i s 9.96 + 2.82 g/100 g. The r e s p e c t i v e values of apparent h e m i c e l l u l o s e are 225% and 345% greater. I t has been reported that the ADF-reagent sometimes leaves s u b s t a n t i a l amounts of h e m i c e l l u l o s e i n the ADF-residue (20). Furthermore, Van Soest's detergent methods do not measure water-soluble components, whereas apparent h e m i c e l l u l o s e includes these substances i f they are not removed by 80% isopropanol washing. A l l these f a c t o r s can c o n t r i b u t e to the discrepancy between c a l c u l a t e d and apparent h e m i c e l l u l o s e values. This l e d to the suggestion that the pretreatment of f i b r o u s m a t e r i a l with l i p i d e x t r a c t i o n and p r o t e o l y t i c and a m y l o l y t i c enzymes would minimize erroneous r e s u l t s (19). One can a l s o a n t i c i p a t e d i f f e r e n c e s between tobacco and a l f a l f a and among tobacco c u l t i v a r s i n chemical composition and s t r u c t u r e v a r i a t i o n of c e l l w a l l as w e l l as the nature of complex formation i n v o l v i n g c e l l w a l l c o n s t i t u e n t s and other substances. I t would be h i g h l y d e s i r a b l e to compare the sugar c o n s t i t u e n t s of tobacco f i b e r components with those of conventional d i e t a r y f i b e r s . The high concentration of i r o n and calcium i n tobacco f i b e r would be d e s i r a b l e from a n u t r i t i o n a l viewpoint. The b i o a v a i l a b i l i t y of mineral elements i n d i e t a r y f i b e r s has been discussed i n the l i t e r a t u r e (16). Aside from f i b e r per se, p h y t i c a c i d was found to be the major determinant of b i v a l e n t metal d e f i c i e n c i e s (21). Phytate content i s high i n wheat bran and other

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

18.

SHEEN AND SHEEN

Tobacco

Fiber

263

d i e t a r y f i b e r s of seed o r i g i n since i t i s a source of g l u c u r o nate precursor f o r synthesis of c e l l w a l l polysaccharides during germination (22). Among the hundreds of compounds i n tobacco l e a f , phytate has never been mentioned i n a l a r g e volume of tobacco l i t e r a t u r e s (18). In analogy to phytate i n wheat bran, tobacco p l a n t s accumulate 2 to 5% of dry weight as o x a l i c a c i d , predominately i n the form of calcium s a l t , i n mature leaves (18). Calcium oxalate i s i n s o l u b l e i n water. Young p l a n t s of seven b u r l e y c u l t i v a r s under high d e n s i t y growth had oxalate content i n the range of 0.6 to 1.4% (23). The d i e t a r y intake of oxalate poses a concern f o r calcium and magnesium a v a i l a b i l i t y although i t was documented that oxalate can be formed i n v i v o from d i e t s containing ascorbate, g l y c i n e , g l y c o l a t e , and g l y o x y l a t e (24). Oxalate content i n tobacco f i b e r was not determined i n the present study. However processes would remov remain i n the f i b r o u s residue.and w i l l not f u r t h e r deplete calcium i n d i e t s because o x a l i c a c i d i s already bound to calcium. Many e d i b l e p l a n t s are r i c h i n o x a l i c a c i d . For example, s p i n ach contains 8 to 10% dry weight as o x a l i c a c i d (25). F i v e cups of t e a per day i n the E n g l i s h d i e t c o n t r i b u t e s to an intake of 75 mg o x a l i c a c i d (26). Oxalate content i n tobacco f i b e r i s probably i n the same range as that of c e r t a i n common f o o d s t u f f s . Another concern of p o s s i b l e adverse e f f e c t s of tobacco f i b e r complex as d i e t a r y f i b e r i s n i c o t i n e contamination. Nico t i n e i s regarded as h i g h l y t o x i c . Gasseline et al (27) r e ported that the o r a l LD5Q of an a d u l t human has been estimated to be 30 to 60 mg (0.5 to 1.0 mg/kg body wt). They also s t a t e d that tobacco i s much l e s s t o x i c than expected from i t s n i c o t i n e content because i n t e s t i n a l absorption of n i c o t i n e as present i n tobacco i s so slow that metabolic i n a c t i v a t i o n sometimes keeps pace with absorption and the e l i m i n a t i o n of n i c o t i n e i s completed w i t h i n 16 hours. About 80 to 90% of the n i c o t i n e i n human u r i n e has already been d e t o x i f i e d . Since n i c o t i n e i s very waters o l u b l e , the washing of tobacco f i b r o u s residue could lower n i c o t i n e to a n e g l i g i b l e l e v e l as demonstrated i n the present study. I f the water-washed tobacco f i b e r contains 10 ppm of n i c o t i n e , a 10% i n c o r p o r a t i o n i n f i b e r - e n r i c h e d formulation r e s u l t s i n 1 ppm of n i c o t i n e i n the formulated foods. The presence of n i c o t i n e i n minute q u a n t i t i e s was reported i n tomato and other solanaceous vegetables (28). With the GLC method employing an a l k a l i n e flame i o n i z a t i o n detector (6), a broad array of f r e s h and preserved foods was analyzed f o r n i c o t i n e contamination. N i c o t i n e was detected at 2 to 15 ppm (on a dry wt b a s i s ) i n tomato, potato, eggplant, and green pepper but not i n a l f a l f a and wheat bran. Most i n t e r e s t i n g i s the f i n d i n g of n i c o t i n e i n t e a , e x p e c i a l l y i n s t a n t t e a , at l e v e l s of 15-24 ppm. N i c o t i n e q u a n t i t y i n tobacco can be d r a s t i c a l l y lowered by genetic means (29). V a r i e t a l d i f f e r e n c e i n oxalate content i n

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tobacco l e a f a l s o i n d i c a t e s p o s s i b l e genetic r e g u l a t i o n (23). I f tobacco i s grown as a food crop, the concentration of these undesirable l e a f c o n s t i t u e n t s can be lowered by means of breed­ ing. The p o t e n t i a l of tobacco u t i l i z a t i o n as a source of s o l u b l e p r o t e i n s and the d e p r o t e i n i z e d residue derived from the p r o t e i n e x t r a c t i o n process as animal feed and s a f e r smoking m a t e r i a l has been mentioned i n recent years (_5, 6^, 3Q). The present study suggests that a f t e r d e c o l o r i z a t i o n tobacco f i b r o u s residue may be a p o t e n t i a l source of d i e t a r y f i b e r . A d d i t i o n a l research i s c e r t a i n l y needed to evaluate the n u t r i t i o n a l and p h y s i o l o g i c a l s i g n i f i c a n c e of tobacco f i b e r i n experimental animals. Some of these s t u d i e s are p r e s e n t l y being undertaken. Acknowledgments The i n v e s t i g a t i o connection with a p r o j e c t of the Kentucky A g r i c u l t u r a l Experiment S t a t i o n , Lexington, Kentucky, and i s published with the approval of the D i r e c t o r . The authors are g r a t e f u l to the A n a l y t i c a l Laboratory of the Agronomy Department, U n i v e r s i t y of Kentucky f o r performing the q u a n t i t a t i o n of mineral elements. Literature Cited 1.

2.

3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

B u r k i t t , D. P. "Dietary f i b r e : current developments of importance to h e a l t h " ; Heaton, K. W., Ed.; Technomic P u b l i s h i n g Company, Inc.: Westport, CT. 1979, pp. 35-44. Heaton, K. W. "Dietary f i b r e : current developments of importance to h e a l t h " ; Technomic P u b l i s h i n g Company, Inc.: Westport, CT. 1979, p. 158. I n g l e t t , G. E.; Falkehag, S. I. "Dietary f i b e r s : chemistry and n u t r i t i o n " ; Academic Press: New York, 1979, p. 285. Anderson, J . W. Cereal Foods World 1977, 22, 12-15. Wildman, S. G. Crops and S o i l Magazine; January, 1979, pp. 7-9. Sheen, S. J . B e i t r . Tabakforsch. I n t r . 1982, i n press. Hamilton, J . L.; Lowe, R. H. Tob. S c i . 1978, 22, 89-93. Gaines, T. P.; Meudt, W. J . Tob. S c i . 1968, 12, 13O-133. Bacot, A. M. U.S.D.A. T e c h n i c a l B u l l e t i n No. 1225, 1960, p. 126. Van Soest, P. J . ; Wine, R. H. J . Assoc. O f f . Anal. Chem. 1967, 50, 50-55. F i s k e , C. H.; Subbarow, Y. J . B i o l . Chem. 1925, 66, 375-400. E i v a z i , F.; Sims, J . L.; C r u t c h f i e l d , J . Comm. S o i l S c i . Plant Anal. 1982, 13, 135-150. Montgomery, M. J . ; Baumgardt, B. R. J . Dairy S c i . 1965, 48, 1623-1628. McConnell, Α. Α.; Eastwood, Μ. Α.; M i t c h e l l , W. D. J . S c i . Food A g r i c . 1974, 20, 1457-1464.

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18. 15. 16.

17.

18.

19.

20.

21. 22. 23. 24. 25. 26. 27.

28.

29. 30.

SHEEN AND SHEEN

Tobacco

Fiber

265

K r i t c h e v s k y , D.; Story, J . A. J . Nutr. 1974, 104, 458-462. Reinhold, J . G. "Proc. 9th I n t . Cong. Nutr."; Chavez, Α.; Bourges, H.; Basts, S.; Eds.: Karger: B a s e l , 1975, pp. 98-105. Rasper, V. F. " D i e t a r y f i b e r s : chemistry and n u t r i t i o n " ; I n g l e t t , G. E.; Falkenhag, S. I . , Eds.; Academic Press: New York, 1979, pp. 93-115. Tso, T. C. "Physiology and biochemistry of tobacco p l a n t s " ; Dowdes, Hutchinson and Ross, Inc.: Stroudsburg, PA. 1972, p. 393. Southgate, D. A. T. " D i e t a r y fibre: current developments of importance to h e a l t h " ; Heaton, K. W., Ed.; Technomic P u b l i s h i n g Company, Inc.: Westport, CT. 1979, pp. 9-19. Theander, 0.; Aman, P. " D i e t a r y fibers: chemistry and n u t r i t i o n " ; I n g l e t t G E.; Falkehag S I . Eds.; Academic Press: New York, 1979 Maga, J . A. J . A g r i Matheson, Ν. K.; St. Clair, M. Phytochem. 1971, 10, 12991302. Brumagen, D. M.; H i a t t , A. J . Plant and S o i l 1966, 24, 239249. Rodricks, J . W.; Pohland, A. E. "Food s a f e t y " ; Roberts, Η. R., Ed.; John Wiley and Sons: New York, 1981, pp. 181-237. Regan, W. S.; Lambeth, V. N.; Brown, J . R.; B l e v i n s , D. G. Am. Soc. Hort. S c i . 1968, 93, 485-492. Zarembski, P. M.; Hodgkinson, A. B r i t . J . Nutr. 1962, 16, 627-634. G o s s e l i n , R. E.; Hodge, H. C.; Smith, R. P.; Gleason, M. N. " C l i n i c a l t o x i c o l o g y of commercial products, acute poisoning"; 4th ed.; The Williams and W i l k i n s Co.: Baltimore, 1976, pp. III-1-323. Mothes, H. K.; Schutte, H. R. " B i o s y n t h e s i s der A l k a l o i d e " , Deutscher V e r l a g der Wissenschaften VEB:Berlin, 1969, p. 730. Legg, P. D.; C h a p l i n , J . F.; C o l l i n s , G. B. J . H e r e d i t y , 1969, 60, 213-217. Sheen, S. J . ; Lowe, R. H.; Burton, H. R. B e i t r . Tabakforsch. I n t r . 1982, 11, 170-179.

RECEIVED January 11,

1982

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

19 Products of Wheat Straw Biodegradation by Cyathus stercoreus T H O M A S P. A B B O T T , C H R I S T O P H E R R O N A L D D. P L A T T N E R

J A M E S , and

U.S. Department of Agriculture, Agricultural Research Service, Northern Regional Research Center, Peoria, IL 61604

The residual biodegradation products of wheat straw lignin attacked by Cyathus stercoreus for thirty days were identified. Low-molecualr-weight nonvolatile materials constituted less than 5% of the total C released from labeled lignin. In this portion, syringic acid, vanillic acid, p-hydroxy benzoic acid, acetic acid, and 2-methoxy-succinic acid were found. The majority of the water-soluble biodegradation products (89%) are lignin-carbohydrate complexes with molecular weights greater than 1000. Analysis of the gases resulting from lignin breakdown and metabolism reveals primarilyCO with smaller amounts of ethanol, methanol, and acetone. The gas phase, flushed out with air and recovered, constitutes 50% of the total lignin breakdown products. 14

2,

This chapter not subject to U.S. copyright. Published 1983, American Chemical Society

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

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UNCONVENTIONAL SOURCES OF DIETARY FIBER

In the f i e l d of biomass u t i l i z a t i o n , separating l i g n i n from c e l l u l o s e and h e m i c e l l u l o s e by b i o l o g i c a l means has aroused a great deal of i n t e r e s t . Degradation of l i g n i n during the process of s e p a r a t i o n i s not n e c e s s a r i l y detrimental i f the l i g n i n can be converted to u s e f u l chemicals. To f u l l y understand the b i o l o g i c a l approach to modifying and s o l u b i l i z i n g l i g n i n , the biodégradation products of l i g n i n must be i d e n t i f i e d and quantitated. Cyathus stercoreus (Schw.) de Toni NRRL 6473 degrades l i g n i n p r e f e r e n t i a l l y during a 62-day fermentation of wheat straw, but the biodégradation products have not been c h a r a c t e r i z e d (1). Other workers have i d e n t i f i e d r e s i d u a l phenolics i n biodégradation broths of s e v e r a l woods and s y n t h e t i c or i s o l a t e d s o l u b l e l i g n i n s (2). More recent p u b l i c a t i o n s have i d e n t i f i e d f u n c t i o n a l group changes i n degraded l i g n i n s rather than s p e c i f i c low molecular weight compound o x i d i z e d species as a r e s u l Current knowledge i n l i g n i n biodégradation supports a mechanism of random o x i d a t i o n , generating aromatic compounds with v a r i o u s oxygen-containing s i d e groups, a l i p h a t i c c a r b o x y l i c a c i d s from subsequent r i n g cleavage and u l t i m a t e l y , carbon d i o x i d e (4-7). The purpose of the present work was to i d e n t i f y and q u a n t i t a t e the r e s i d u a l products a f t e r a 30-day fermentation of C^. stercoreus on wheat straw. The approach to t h i s problem i s l i m i t e d by a few, s i g n i f i c a n t c o n s i d e r a t i o n s . I d e a l l y , p r e l i m i n a r y e x t r a c t i o n of the straw e l i m i n a t e s i n t e r f e r i n g substances, such as f a t t y a c i d s and other e x t r a c t a b l e s present i n l a r g e q u a n t i t y , which overwhelm minute amounts of biodégradation products and thereby hinder t h e i r i d e n t i f i c a t i o n . However, the fungus does not grow w e l l when a l l s o l u b l e n u t r i e n t s are removed, as w i l l be shown i n l a t e r s e c t i o n s . A l s o , s t e r i l i z a t i o n by a u t o c l a v i n g may generate s o l u b l e l i g n i n s that cannot be a t t r i b u t e d to fungal a c t i o n , and t h e r e f o r e c o n t r o l s must be run. More s p e c i f i c a l l y , r e p r o d u c i b i l i t y of fermentation on a s o l i d substrate i n o c u l a t e d with a 1 cm plug of slowgrowing stercoreus i s d i f f i c u l t , e s p e c i a l l y because t h i s fungus i s s e n s i t i v e to excess water and w i l l not grow i n submerged c u l t u r e , although a high l e v e l of moisture i s necessary f o r fungal growth. A f t e r the fermentation i s complete, extracted water-soluble degradation products l o s e much of t h e i r water s o l u b i l i t y i f taken to dryness i n a i r or i f heated. Freeze-drying overcomes t h i s problem, but molecular weight d i s t r i b u t i o n s should be determined before f r e e z e - d r y i n g because polymer chains can be f r a c t u r e d by growing solvent c r y s t a l s (8). 2

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

19.

ABBOTT ET AL.

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269

Products

M a t e r i a l s and Methods Sample P r e p a r a t i o n . Three wheat straw samples were t e s t e d . One was a l o c a l l y obtained, d r i e d and baled wheat straw; the other two were Red River 68 and Butte HRS, grown i n an environmental chamber. While monitoring the p l a n t growth and l i g n i n production i n the straw as reported by Stone et a l . (9), we grew the p l a n t s f o r 38 days (Butte HRS) or 48 days (Red River 68) and cut them f o r l i g n i n l a b e l i n g j u s t p r i o r to heading out of the g r a i n . In order to l a b e l the l i g n i n , the roots were cut o f f and the f r e s h l y cut s u r f a c e was immediately immersed i n a 10-ml s o l u t i o n of uniformly l a b e l e d C-phenylalanine i n a growth chamber u n t i l the s o l u t i o n was n e a r l y a l l taken up. The cut surface was then immersed 1-2 cm i n d i s t i l l e d water f o r 3 days, d r i e d at 45° C to constan 1-mm screen. The groun and d r i e d , then soaked i n water at 4° C f o r 16 h, washed, and f i n a l l y t r e a t e d with protease at room temperature f o r 3 h, water washed and f r e e z e - d r i e d . The protease used was Subtilopeptidase-A ( B a c t e r i a l , Type VII) from Sigma Chemical Company i n a pH 7 borate b u f f e r at a c o n c e n t r a t i o n of 1 mg/ml. T o t a l C content, as decompositions per minute per mg (DPM/mg), was determined by p y r o l y s i s of the straw i n an oxygen atmosphere to CO2 and trapping of the CO2 i n 10 ml of a s c i n t i l l a t i o n c o c k t a i l c o n s i s t i n g of 1,300 ml toluene, 1,200 ml methanol, 300 ml 2-aminoethanol, 0.0375 g l,4-[2-(4-methyl-5-phenyl-l,3-oxazolyl) benzene and 30.0 g 2,5-diphenyloxazole. Unlabeled wheat straw obtained l o c a l l y was hexane washed, d r i e d , soaked at 4° C i n water that was decanted, and then e i t h e r fermented d i r e c t l y or a f t e r f u r t h e r water washing. A l s o , at the end of the 4° soak the straw was broken up i n a Waring Blendor to prevent t r a p p i n g of water i n s i d e the hollow straw stem. Fermentation was accomplished i n a i r at 25° a f t e r i n o c u l a t i o n with a 1-cm plug of C^. stercoreus that had been grown f o r 1 week on a potato dextrose agar p l a t e . ll+

2

ll+

2

Separation of Biodégradation Products. The scheme devised f o r s e p a r a t i o n and recovery of biodégradation products i s i l l u s t r a t e d i n F i g u r e 1. The ether phase e x t r a c t , l a b e l e d C i n Figure 1, would c o n t a i n a c i d i c m a t e r i a l s such as v a n i l l i n , syringealdehyde and s i m i l a r compounds i f they were present. Some p o l y f u n c t i o n a l a c i d i c compounds remain i n the a c i d i f i e d water phase D. An a l t e r n a t e route using i o n exchange r e s i n i s b e t t e r s u i t e d f o r s e p a r a t i o n and i d e n t i f i c a t i o n of p o l y f u n c t i o n a l a c i d i c compounds, such as hydroxybenzoic a c i d . These compounds w i l l end up i n the water e x t r a c t l a b e l e d Ε i n Figure 1. Unless other s o l u b l e compounds mask t h e i r presence, these a c i d s can a l s o be i d e n t i f i e d i n the o r i g i n a l extracted fermentation broth.

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UNCONVENTIONAL SOURCES OF DIETARY FIBER

40g Unlabeled or 0.5g C Labeled U

Wheat Straw Fermented 30 Days

2. 0.45/im membrane

A

Retentates

Filtrate

Ethanol

Adjust pH to 10 Ether

Β Ether Phase

F

H2O Phase HCIto pH 4

Insoluble

Soluble (alternate r o u t e ) j

Ether

Ether Phase

o n

e x c n a n g e

| H +

,

" " ^ - ^ Ether

Ether Phase

D H 0 Phase 2

Ε H2O Phase

Dialysis

Dialysate

Figure 1.

Retentate

Separation of biodegraded wheat straw.

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

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ABBOTT ET A L .

Wheat Straw Biodégradation

Products

271

C h a r a c t e r i z a t i o n Methods. Labeled m a t e r i a l s i n s o l u b l e e x t r a c t s were q u a n t i t a t e d by counting 100 u l a l i q u o t s of the v a r i o u s s o l u t i o n s i n the same 10 ml s c i n t i l l a t i o n c o c k t a i l used to trap CO2. The q u a n t i t y o f C i n evolved gases was measured by d a i l y 15-min f l u s h e s o f the sealed fermentations with a i r i n t o the same s c i n t i l l a t i o n c o c k t a i l . The gas-phase components were i d e n t i f i e d by t h e i r r e t e n t i o n times on a 5 f t X 1/8 inch Porapak Q column. To trap enough gas f o r i d e n t i f i c a t i o n , the evolved gases from 2 days of fermentation were f l u s h e d with helium i n t o a 1 f t X 1/8 i n c h Porapak Q f i l l e d s t a i n l e s s - s t e e l column f i l l e d with Porapak Q and immersed i n l i q u i d N2. The column e x i t gas passed through a bubbler c o n t a i n i n g s c i n t i l l a t i o n f l u i d . After the 15-min f l u s h the column was capped on the entrance s i d e and a new bubbler was attached. The column was then allowed to warm to room temperature. Trappe warmed, and when gas e v o l u t i o i n t o a Tracor Model 560 gas chromatograph (GC); the entrance side of the trap column was attached to the i n j e c t i o n port and the e x i t s i d e to a 4 f t by 1/8 i n c h Poropak Q column. The b a s e l i n e had been e s t a b l i s h e d p r e v i o u s l y but r e s i d u a l CO2 came o f f as helium c a r r i e r gas was turned on and the oven was programmed from 40° to 180° a t 5°/min. Samples from the water phase were t e s t e d f o r v o l a t i l e s other than CO2. They were tested on the same Poropak Q column as gas-phase samples, but the column was programmed from 100 to 170° a t 10°/min and held at 170° f o r 20 min. To ensure that the v o l a t i l e s other than CO2 were from l i g n i n degradation, the same l i q u i d N2 trapping procedure was followed. The column was attached a t room temperature to a GC with a thermoconductivity (TC) detector l e a d i n g to a flowing s c i n t i l l a t i o n f l u i d trap f o r gases as they e l u t e d . R e s u l t s of the a n a l y s i s of v o l a t i l e s by GC were confirmed by high-pressure l i q u i d column chromatography (HPLC) and i n f r a r e d s p e c t r a of the major components. Less v o l a t i l e low-molecular-weight biodégradation products, were c h a r a c t e r i z e d by gas chromatography-mass spectroscopy (GCMS). Higher molecular weight compounds were c h a r a c t e r i z e d by gel permeation chromatography (GPC), membrane f i l t r a t i o n , d i a l y s i s and n u c l e a r magnetic resonance (NMR). The methylated (CH2N2) low-molecular-weight compounds were analyzed on a Kratos MS-30 equipped with a Perkin Elmer Sigma 3 gas chromatograph and a 3 f t X 1/8 inch OV-1 column. Retention times of the sample peaks i d e n t i f i e d by GC-MS were compared to those of known standards on a 6 f t X 1/8 inch OV-1 column on a Tracor model 560 GC f o r c o n f i r m a t i o n . L i g n i n content was measured by the UV method (10). C o n t r o l samples of wheat straw o x i d i z e d by the standard nitrobenzene method (11) and separated by the scheme i l l u s t r a t e d i n F i g u r e 1 were a l s o t e s t e d by the GC-MS method to confirm the v a l i d i t y of the s e p a r a t i o n and recovery techniques. 1I+

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272

UNCONVENTIONAL SOURCES OF DIETARY FIBER

To c h a r a c t e r i z e high-molecular-weight biodégradation products, an Amicon Model 202 D i a f i l t r a t i o n Apparatus was used s e q u e n t i a l l y with XM300, PM10 and UM2 membranes at 10, 17 and 55 p s i N2 pressure, r e s p e c t i v e l y . The samples tested were water-soluble biodégradation products that had never been f r e e z e d r i e d . Concentrations of the v a r i o u s f r a c t i o n s were determined on a l i q u o t s so that GPC curves could be determined on samples that had always been i n s o l u t i o n . High-molecular-weight biodégradation products f o r NMR were i s o l a t e d by d i a l y s i s and membrane f i l t r a t i o n . D i a l y s i s bags of regenerated c e l l u l o s e were c h a r a c t e r i z e d with standard dextrans and r a f f i n o s e . GPC curves of a d e x t r a n - r a f f i n o s e mixture before and a f t e r s e p a r a t i o n by d i a l y s i s i n d i c a t e s a nominal molecular weight of 3,000 or greater was r e t a i n e d f o r NMR a n a l y s i s . High-molecular-weight compounds i s o l a t e d by t h i s method were f r e e z e - d r i e d , and t h e i WH 90 i n D2O. Gel permeatio determined with a Waters 244 HPLC on 2 columns of E - l i n e a r μ-Bondagel, 7.8 mm X 30 cm each. Water was the solvent at 1 ml/min and ambient temperature. Detection was by r e f r a c t i v e index and UV absorbance at 254 mn. Results and D i s c u s s i o n Recovery of the gas phase of fermented wheat straw and separation of the r e s i d u e by the method i l l u s t r a t e d i n Figure 1 gave a m a t e r i a l balance of the v a r i o u s f r a c t i o n s by weight C content, as shown i n Tables I and I I . Although only 4.6% of the o r i g i n a l 40 g of straw i s e x t r a c t e d as water-soluble m a t e r i a l a f t e r the fermentation and 9.3% of the r a d i o a c t i v e straw by weight, a higher percentage of the o r i g i n a l C (13.3%) i s i n the recovered water-soluble m a t e r i a l (Table I ) . Thus there i s a higher c o n c e n t r a t i o n of l i g n i n degradation products i n the fermentation b r o t h than the c o n c e n t r a t i o n of l i g n i n i n the wheat straw. A u t o c l a v i n g unfermented straw y i e l d s 0.8% of the o r i g i n a l r a d i o a c t i v i t y i n the water phase. C a r e f u l drying to avoid l o s s of moderately v o l a t i l e substances such as v a n i l l i n involved f r e e z e - d r y i n g the water-soluble m a t e r i a l from the 40-g straw fermentations. A d d i t i o n a l l y , -^C-labeled water s o l u b l e s were counted by t e s t i n g a l i q u o t s (100 u l ) i n s c i n t i l l a t i o n f l u i d , both before and a f t e r f r e e z e - d r y i n g f o r 54 h past apparent dryness. Less than 4.3% of the o r i g i n a l C was l o s t on f r e e z e drying and r e c o n s t i t u t i o n , and no C was detected i n the -70° f r e e z e - d r y i n g t r a p . Some l o s s of both weight and C w i l l occur from d i s s o l v e d CO2, methanol and ethanol. From the r e s u l t s i n Tables I and I I i t can a l s o be concluded that the gas phase contains approximately 13% of the o r i g i n a l C. The t o t a l l i g n i n i s degraded 27% a f t e r 30 days, h a l f i n the gas phase and h a l f i n the water phase of the fermentation, before e i t h e r phase i s separated f o r i d e n t i f i c a t i o n . I d e n t i f i c a t i o n of the components of the gas phase by r e t e n t i o n time i s i l l u s t r a t e d i n Figure 2. 1I+

ll4

ll+

ll+

1 4

1I+

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

19.

ABBOTT ET A L .

Wheat Straw Biodégradation

273

Products

A t y p i c a l flame i o n i z a t i o n d e t e c t i o n (FID) curve i s shown i n Figure 2 (curve 2). Gases and water-phase v o l a t i l e s i d e n t i f i e d by r e t e n t i o n time are ethanol, methanol, acetone and a c e t i c a c i d . A comparison of counts c o l l e c t e d over 10° i n t e r v a l s i n the programmed chromatograph to the r e t e n t i o n times of peaks detected by both FID and TC i n d i c a t e s that a l l of the gases i n a d d i t i o n to CO2 have at l e a s t part of t h e i r o r i g i n i n the Rel a b e l e d l i g n i n (compare F i g u r e 2, curve 3 to F i g u r e 2, curves 1 and 2). R e s i d u a l ethanol i n the water phase was determined by HPLC to be 0.02% or 0.033 g i n a 30-day, 40-g straw fermentation with f r e e ( f i l t e r e d ) atmospheric gas exchange. CO2 and water were evident i n the i n f r a r e d spectra of fermentation gases, with no evidence of s i g n i f i c a n t amounts of hydrocarbon gases. As i n d i c a t e d by weight and C a n a l y s i s (Table I I ) , few i f any low-molecular-weight aldehydic phenols were i s o l a t e d i n ether phase C. Therefore tested by adding 0.05 g straw and c a r r y i n g i t through the e x t r a c t i o n and f r e e z e - d r y i n g procedure. The added v a n i l l i n was e a s i l y determined i n ether e x t r a c t C by GC. Only a t r a c e was found i n e t h a n o l - s o l u b l e e x t r a c t F, but even t h i s t r a c e , 0.1 mg, was detectable, i n c o n t r a s t to the e x t r a c t s of biodegraded wheat straw that contained no a l d e h y d i c phenols. The absence of v a n i l l i n , syringealdehyde, and s e v e r a l other phenols i n the water-soluble biodégradation products was confirmed by t h i n l a y e r chomatography (TLC) i n 95/5 benzene/methanol, u s i n g TJV and radiocarbon d e t e c t i o n . Of a l l the compounds examined, only carboxylated phenols do not migrate away from the o r i g i n i n t h i s s o l v e n t . Ether e x t r a c t i o n procedures have been used a l s o to i s o l a t e aldehydic phenols from a l k a l i n e nitrobenzene o x i d i z e d l i g n i n to c h a r a c t e r i z e the l i g n i n i n v a r i o u s p l a n t s . Fe(NH^)2(S0i+)2 washed ether should be used to prevent peroxide o x i d a t i o n of the aldehyde groups (12) . Using Fe(NHi ) 2(S0if) 2 washed ether throughout, we demonstrated again that aldehydic phenols were not present i n the ether e x t r a c t s of biodegraded wheat straw i n which over 2 g of l i g n i n i s degraded, but these phenols are e a s i l y found and q u a n t i t a t e d when 2 g of wheat straw i s o x i d i z e d by the standard nitrobenzene method and the phenols are recovered as i l l u s t r a t e d i n F i g u r e 1. Table I I I l i s t s the compounds found i n nitrobenzene o x i d i z e d wheat straw, as w e l l as s i m i l a r r e s u l t s f o r e x t r a c t s of the biodégradation products. The absence of low-molecular-weight biodégradation products i n the ethanol e x t r a c t i s confirmed by the o b s e r v a t i o n that the gas chromatographic curve f o r the ethanol s o l u b l e s ( s i l y l a t e d ) of fermented wheat straw i s i d e n t i c a l to the curve f o r s i m i l a r d e r i v a t i z e d e x t r a c t s of unfermented wheat straw c a r r i e d through the same e x t r a c t i o n procedure. To t e s t f o r p o l y s u b s t i t u t e d phenols i n the water s o l u b l e biodégradation b r o t h , the methylated (CH2N2) d e r i v a t i v e of f r e e z e - d r i e d e x t r a c t A (Figure 1) was e a s i l y produced and y i e l d e d interprétable mass s p e c t r a . S e v e r a l phenolic standards were 1

ll+

+

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

274

Table I.

UNCONVENTIONAL

SOURCES

OF

DIETARY

FIBER

Quantitation of Biodégradation Products

Material Balance by Weight

Starting material

Total Sample

Lignin

Basis

Basis

Total Sample Basis A l l Soluble Nutrients Removed

6.4% H 15.6% lignin

Hexane extract 4° Water extract, decanted

0.4%,

0.16

5.5%,

2.2 g

0.4%,

-0-

g 4.8%,

g

0.3 g

4° Water extract and water wash Fermented filtered H 0 solubles

0.16

5.6%,

2.25

g

2.6%,

1.02

g

2.3%,

0.939 g

2

4.6%,

1.92

g

7.1%,

0.44

g

0.45 urn f i l t e r e d H 0 solubles 2

EtOH solubles after fermentation

1.3%,

0.53

g

6.4%,

0.3+0.1 g

6

Fermented extracted dry straw

69.5%, 27.8 g

Lost or i n gas phase

12.5%, 5.0 g

65.2%, 4.07

g

16.5%, 1.03 g

78.8%, 31.52

6.3%,

Trapped gases

Average of 5 determinations on 40 g wheat straw ^Lignin determinations by UV method (10) Average of 2 determinations. ^Decomposition per minute. e

Not corrected for fungal mass.

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

g

2.5 g

19.

ABBOTT E T A L .

14

C

Balance

Sample 1

Sample 2

Butte HR

Butte HR

38.03

25.04

Wheat Straw Biodégradation

d

Products

6

DPM X10" , Percent

Sample 3 RR 68

Sample 4 RR 68

43.8

5.0, 14.0%

5.47, 14.4%

3.33, 13.3%

5.27, 12.1%

0.25, 0.7%

0.13, 0.3%

25.0, 65.8%

6.2% 5.04, 13.3%

4.72, 13.2%

16.9, 67.3%

24.8, 56.5

12.3%

17.6%

1.27, 5.1%

5.90, 13.5%

12.8, 35.8%

-6.4% 20.2, 56.7%

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

276

UNCONVENTIONAL SOURCES OF DIETARY FIBER

TABLE I I .

Separation

of Water-Soluble Biodégradation Products

by Ether E x t r a c t i o n (Figure 1)

Percent of Water-Soluble Products i n Ether E x t r a c t of Basic H 0

Ether E x t r a c t of A c i d i c H 0

2

By weight 14 By

C label

2

0.41

0.96

0.03

0.4

a

A s i l l u s t r a t e d by the

b

A s i l l u s t r a t e d by the e x t r a c t l a b e l e d C i n Figure 1.

Curve 5

Ethanol (Methanol

2

I JUL_j Curve 4

100

120

140 160 Temperature, °C

180

600 ^400

t

-

Q "

18,800

m Curve 3

200 0»— Curve 2 Methanol Curve 1 40

Ethanol

A 60

80

A c e

A 100

120

,

t o n e

l

Ethyl ether

λ 140

160

Temperature, °C

Figure 2. Gas chromatographic curves for volatiles. Key: Curve 1, standard mix in water of less than 1% each of methanol, ethanol, diethyl ether, and acetone flushed with He, trapped and tested on Poropak Q as were the fermentation gases (FID); Curve 2, gas phase of eight-day fermentation trapped and tested as described in text (FID); Curve 3, collected and counted GC effluents (see text); Curve 4, standard mix of 0.1% each methanol, ethanol, acetone, and acetic acid; and Curve 5, water phase of fermentation.

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

19.

ABBOTT ET A L .

Table I I I .

Wheat Straw Biodégradation

277

Products

GC-MS I d e n t i f i e d TMS D e r i v a t i v e s o f Biodegraded and

O x i d i z e d Wheat Straw Q

Extract of E x t r a c t s o f Biodegradabl EtOH E x t r a c t

b

Extract

0

Wheat Straw

P a l m i t i c acid'TMS

No monomeric

Phenol-TMS

L i n o l e i c acid^TMS

phenols

L a c t i c acid'TMS

Pentose·TMS Tetramer and A h i g h molecular pentamer weight (422) hydrocarbon, p o l y d i m e t h y l not TMS d e r i v a t i z e d siloxane which dominates the artifacts. GC curve i n s i z e . The same Probably a t r i t e r p e n e . 422 molecular weight compound as i n column 1.

G l y c o l i c acid^TMS p-Hydroxybenzaldehyde *TMS Vanillin-TMS Syringealdehyde *TMS 2 isomers o f

Nitrobenzene

a

K r a t o s MS-30 equipped w i t h a P e r k i n Elmer Sigma 3 gas chromatograph. An OV-1 surface-coated open t u b u l a r c a p i l l a r y column was h e l d a t 70° f o r 3 min, then programmed a t 4°/min t o 250° a f t e r sample i n j e c t i o n . ^ I s o l a t e d as i l l u s t r a t e d i n F i g u r e 1. I s o l a t e d as i l l u s t r a t e d by e x t r a c t C i n F i g u r e 1.

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

278

UNCONVENTIONAL SOURCES OF DIETARY FIBER

methylated f o r comparison to the unknown. Since methylated e x t r a c t A had not been separated i n t o v a r i o u s components before methylation, the gas chromâtograph curve shows many peaks (Figure 3, curve 1). The smaller peaks i n the area of methylated p o l y f u n c t i o n a l phenols are t h e r e f o r e d i f f i c u l t to i d e n t i f y from t h e i r mass spectrum alone. A technique c a l l e d mass chromatography was used wherein d i s t r i b u t i o n of s e v e r a l major masses that appear i n a standard spectrum i s p l o t t e d underneath the gas chromatograph curve as i n F i g u r e 3. In t h i s way, coincidence of s e v e r a l masses that are present i n a standard mass spectrum under a s i n g l e peak i n the gas chromatograph curve i d e n t i f i e s the l o c a t i o n of that standard. T h i s , i n conjunction with r e t e n t i o n time, confirms the presence of a compound i n the unknown, whereas the absence of such coincidence at a given r e t e n t i o n time confirms the absence of a compound to very low l e v e l s of- d e t e c t a b i l i t technique, methylated v a n i l l i methoxy s u c c i n n i c a c i d and s y r i n g i c a c i d were found to be present. The p a l m i t i c a c i d , l i n o l e i c a c i d and t r i t e r p e n e s found e a r l i e r were the dominant m a t e r i a l s i n the water s o l u b l e s examined by t h i s technique, as can be seen i n Figure 3. The magnitude of these three m a t e r i a l s prevents p u t t i n g a l a r g e r sample on the GC-MS instrument to enhance the i n t e r p r e t a t i o n of small peaks. Larger samples p o s s i b l y could burn out f i l a m e n t s i n the mass d e t e c t i o n system. For t h i s reason, p r e - e x t r a c t i o n of the t r i t e r p e n e s and f a t t y a c i d s as much as p o s s i b l e was attempted with hexane. The f a t t y a c i d s and t r i t e r p e n e s c o n s t i t u t e the i n t e r f e r i n g substances r e f e r r e d to i n the i n t r o d u c t i o n . Since s e p a r a t i o n of these lower molecular weight compounds i s not e a s i l y achieved by ether e x t r a c t i o n , they are not q u a n t i t a t e d by the s e p a r a t i o n scheme of F i g u r e 1. Separation of higher molecular weight m a t e r i a l s f o r c h a r a c t e r i z a t i o n was a l s o necessary. Both of these goals were achieved by membrane f i l t r a t i o n , d i a l y s i s and GPC c h a r a c t e r i z a t i o n . Membrane f i l t r a t i o n r e s u l t s i n Table IV gave us considerable i n s i g h t i n t o the molecular weight d i s t r i b u t i o n of r e s i d u a l biodégradation products. We concluded from these r e s u l t s that few of the r e s i d u a l biodégradation products are present as lowmolecular-weight s p e c i e s . Compounds of l e s s than 1,000 nominal molecular weight c o n s t i t u t e 11% of the water-soluble biodégradation products, with monomeric aromatics only a p o r t i o n of t h i s . GPC curves of the v a r i o u s membrane-separated f r a c t i o n s confirm that the high-molecular-weight f r a c t i o n s c o n t a i n the m a j o r i t y of u l t r a v i o l e t absorbing species (Figures 4 and 5). F i g u r e 6 shows the C NMR spectrum of high-molecularweight biodégradation products from membrane f i l t r a t i o n and d i a l y s i s and the NMR spectrum of a l i g n i n - c a r b o h y d r a t e complex published by Himmelsbach and Barton (13). The spectrum of the biodégradation products shows predominantly carbohydrate polymers. The combination of weight and ^ C - l a b e l d i s t r i b u t i o n among s e q u e n t i a l membrane f i l t e r s , the UV absorbance of these species, 1 3

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

19.

ABBOTT ET A L .

Wheat Straw Biodégradation

279

Products

Mass 165

Curve 3

Mass 181 Curve 4

\jL„J_JijMJLJLJ^ Mass 196

Curve 5

70

150

100 Temperature, °C

Figure 3. GC-mass spectra curves. Key: Curve 1, total ionization vs. temperature curve for methylated water-soluble biodégradation products; Curve 2, a portion of Curve 1 for searching; Curve 3, plot of mass 165 ions received while Curve 2 was being collected; Curve 4, plot of mass 181 ions received while Curve 2 was being collected; and Curve 5, plot of mass 196 ions received while Curve 2 was being collected.

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

UNCONVENTIONAL SOURCES OF DIETARY FIBER

280

Table IV.

Membrane F i l t r a t i o n of Biodégradation Products

Percent of H 0 Solubles

Percent of H 0 Solubles

2

2

Retained b y

a

Membrane

14

Weight f o r the 40 g

f o r the

C Labeled L i g n i n

Fermentation

Wheat Straw Fermentation

Χ M 300

13.2

22.8

PM 10

10.9

23.9

UM 2

48.8

42.0

93.9

95.5

Through UM 2 Recovery

a

Nominal molecular weights r e t a i n e d by the XM300 membrane i s >300,000, by the PM10 membrane i s 10,000 to 300,000, and by the UM2 membrane i s 1,000 to 10,000.

A

J

.

v

V

Pu

Β

w

J 1

1

220

110

1 1 1 .

1 6 8 . 6 4.5

Calibrated Dextran Molecular Weight χ 1 0 - 3

I

ι

ι

ι

ι

0

5

10

15

20

.

1 25

Elution Volume, ml

Figure 4. Gel permeation chromatograms of the total water-soluble biodégradation product. Detection for Chromatogram A is by refractive index change, and Chromatogram Β is by UV absorbance at 254 nm.

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

19.

ABBOTT ET A L .

Wheat Straw Biodégradation

Products

Figure 5. Gel permeation chromatograms of the membrane filtration fractions using the same conditions as for the unseparated sample described in Figure 4.

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

281

282

UNCONVENTIONAL SOURCES OF DIETARY FIBER

I

I 160

180

I 140

I 120

I 100

I 80

I 60

I 40

I 20

L 0

PPM

13

Figure 6. C-NMR spectra of a lignin-carbohydrate complex A (13), and the high molecular weight water-soluble biodégradation products isolated by dialysis (B).

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

19.

ABBOTT ET A L .

Wheat Straw Biodégradation

Products

283

and the NMR spectrum i n F i g u r e 6 lead to the c o n c l u s i o n that the overwhelming m a j o r i t y of r e s i d u a l biodégradation products are l i g n i n - c a r b o h y d r a t e complexes. Conclusions Although some low-molecular-weight o x i d i z e d aromatic species can be found i n biodégradation products of Cyathus stercoreus on wheat straw, the predominant r e s i d u a l biodégradation species are l i g n i n - c a r b o h y d r a t e complexes. The other major product of degraded l i g n i n i s carbon d i o x i d e . Lesser amounts of ethanol, methanol, acetone, a c e t i c a c i d , 2-methoxy s u c c i n n i c a c i d , v a n i l l i c a c i d , s y r i n g i c a c i d and p-hydroxy benzoic a c i d were a l s o found. These f i n d i n g s are c o n s i s t e n t with current t h i n k i n g on mode of a t t a c k of f u n g i on l i g n o c e l l u l o s i c s as described e a r l i e r . I f random o x i d a t i o n cleave f r a c t i o n s from l i g n o c e l l u l o s i c s cannot be metabolized; however, as these a r e broken down to lowmolecular-weight m a t e r i a l s by continued oxygenation, these f r a c t i o n s a r e r a p i d l y metabolized to CO2. Thus, the r e s i d u a l fermentation b r o t h would c o n t a i n predominantly i n d i g e s t i b l e high-molecular-weight species and a few low-molecular-weight species and a s i g n i f i c a n t amount of CO2 would have been generated. We f i n d such a composition. We g r a t e f u l l y acknowledge Charles L. Swanson f o r h i s g e l permeation chromatography work and Gordon Adams f o r a s s i s t a n c e i n the fermentations. Literature Cited 1. 2. 3.

4. 5. 6. 7. 8. 9.

Wicklow, D. T.; Detroy, R. W.; Jessee, B. A. Appl. Environ. M i c r o b i o l . , 1980, 40, 169. Christmen, R. F.; Oglesby, R. T., i n L i g n i n s , Ch. 8, John Wiley and Sons, Inc., N.Y., N.Y., 1971. K i r k , T. K.; H i g u c h i , T.; Chang, Η., Ed. L i g n i n Biodegradation: M i c r o b i o l . , Chem., and P o t e n t i a l A p p l i c a t i o n s . V o l . I and V o l . I I , CRC Press, Boca Raton, F l o r i d a , 1980. K i r k , T. K.; S h u l t z , E.; Connors, W. J . ; Lorenz, L. F.; Zeikus, J. G. Arch. M i c r o b i o l . , 1978, 117, 277. Crawford, R. L.; Crawford, D. L.; D i z i k e s , G. J . Arch M i c r o b i o l . , 1981, 129, 204. Haider, K.; Trojanowski, J . Arch. M i c r o b i o l . , 1975, 105, 33. Bar-Lev, S. S.; K i r k , T. K. Biochem. Biophys. Res. Commun., 1981, 99, 373. E l i a s , H-G. Macromolecules. V o l . I I , Plenum Press, N.Y., N.Y., 1977, 548. Stone, J . Ε.; B l u n d e l l , M. J . ; Tanner, K. G. Can. J . Chem., 1951, 29, 734.

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

284 10. 11. 12. 13.

UNCONVENTIONAL SOURCES OF DIETARY FIBER Bagby, M. O.; Cunningham, R. L.; Maloney, R. L. Tappi, 1973, 56, 162. Roadhouse, F. E.; MacDougal, D. Biochem. J., 1956, 63, 33. Hedges, J . I.; Parker, P. L. Geochim. Cosmochim. Acta, 1976, 40, 1019. Himmelsbach, D. S.; Barton I I , F. E. A g r i c . Food Chem., 1980, 28, 1203.

RECEIVED October 29,

1982

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

20 Biphenyl Hydroxylase, Arylhydrocarbon Hydroxylase, and Epoxide Hydrolase Activities in Intestinal and Liver Microsomes of Rats Fed Selected Types of Dietary Fibers J. C. O P D Y C K E

1

and J. C. S T R E E T

Utah State University, Department of Animal, Dairy, and Veterinary Science, Toxicology Program, Logan, UT 84322

"Dietary fiber" has been shown to be protective against induction of colon cancer by some chemical carcinogens. On tective action ma lism. The affects of diets containing different types of purified fiber on intestinal and liver microsomal metabolism of biphenyl, benzo(a)pyrene and epoxides were assessed. Adult, male Wistar rats were individually fed chemically defined, nutrient density balanced rations containing either no dietary fiber, or15%pectin, Metamucil, lignin or cellulose for 30 days;a similar group was fed commercial laboratory chow diet as reference. The microsomal hydroxylations of biphenyl and benzo(a)pyrene (BaP) were compared following incubation with appropriate preparations of liver and intestinal mucosa. The hydroxybiphenyl products were determined by fluorimetric HPLC, hydroxylated BaP was determined by fluorimetry, and epoxide hydrolase activity was determined using 3-(p-nitrophenoxy)-1 ,2-propene oxide as a substrate. Activities were higher with liver microsomes than the intestinal microsomal preparations for each assay. Fiber-fed rats showed significantly higher hepatic but not intestinal epoxide hydrolase levels than did control rats. Hepatic arylhydrocarbon hydroxylase levels were higher in synthetic diet-fed rats than lab chowfed animals. Variations in intestinal biphenyl hydroxylase activities were not significant with respect to fiber type regardless of the specific metabolite measured. Hepatic biphenyl hydroxylase activities were significantly higher with synthetic diet-fed rats. 1

Current address: Mobay Chemical Corporation, Kansas City, MO

64120

0097-6156/83/0214-0285$06.00/0 © 1983 American Chemical Society

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

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SOURCES OF DIETARY FIBER

Some c u r r e n t hypotheses about the e t i o l o g i e s of colon cancer and c h o l e s t e r e m i c disease impute a s p e c i a l f u n c t i o n t o d i e t a r y f i b e r and to some o f i t s components; that f u n c t i o n i s g e n e r a l l y a s s o c i a t e d w i t h disease prevention. Evidence f o r the p r o t e c t i v e f u n c t i o n o f d i e t a r y f i b e r has been obtained both from epidemiol o g i c a l s t u d i e s ( 1 - 8 ) and from experiments w i t h animals. Animal experiments, i n p a r t i c u l a r , have been u s e f u l i n t e s t i n g v a r i o u s hypotheses about t h e r e l a t i v e e f f e c t i v e n e s s o f d i f f e r e n t kinds o f d i e t a r y f i b e r and about p o s s i b l e mechanisms o f f i b e r e f f e c t s . Thus, s t u d i e s o f b u l k i n g a c t i o n ( 9 - 1 0 ) , o f s e l e c t i v e secondary b i l e a c i d b i n d i n g ( 1 1 - 1 2 ) , o f increased i n t e s t i n a l t r a n s i t times ( J J 3 ) , o f a l t e r e d b a c t e r i a l a c t i v i t i e s and secondary b i l e a c i d production ( 1 4 - 1 5 ) , and s e l e c t i v e b i n d i n g o f c a r c i n o gens ( 1 6 - 1 9 ) have been c a r r i e d out i n l a b o r a t o r y animals. S e v e r a l r e l a t e d s t u d i e s have been conducted i n humans as w e l l Among the l a t t e r are i n v e s t i g a t i o n f i b e r content of d i e t ( 4 , 2 0 - 2 1 ) .

Because o f t h e p o s s i b l e i n t e r p l a y o f many f a c t o r s and p r o c e s s e s t h a t m i g h t o c c u r among t h e d i f f e r e n t c h e m i c a l s cons t i t u t i n g d i e t a r y f i b e r i t s e l f and carcinogens or promoters, we have been c o n d u c t i n g a s y s t e m a t i c s t u d y o f s u c h i n t e r a c t i o n s u s i n g a model e x p e r i m e n t a l d e s i g n w i t h t h e r a t . Our model cons i s t s of feeding purified diets containing different purified f i b e r s , i n h y d r a t e d form, f o r a 3 0 day p e r i o d t o a l l o w f o r t h o r o u g h b a c t e r i a l a d a p t a t i o n ( 2 2 ) . A t t h a t t i m e each r a t was t r e a t e d per os w i t h a s i n g l e dose o f a x e n o b i o t i c chemical s e l e c ted to e x e m p l i f y a s p e c i f i c aspect o f x e n o b i o t i c metabolism. T o x i c o k i n e t i c analyses were then u t i l i z e d to q u a n t i f y the c r i t i c a l parameters, such as a b s o r p t i o n and e l i m i n a t i o n r a t e constants f o r t h e p a r e n t compounds, t h e i r b a c t e r i a l m e t a b o l i t e s , and m e t a b o l i t e s undergoing enterohepatic c i r c u l a t i o n . By r e p e t i t i v e study w i t h d i f f e r e n t x e n o b i o t i c s w i t h i n the f i x e d d i e t a r y design, a comprehensive understanding o f the f i b e r r a n i m a l : c a r c i n o g e n i n t e r a c t i o n s i s being developed ( 2 2 - 2 4 ) . In order to f u l l y i n t e r p r e t the t o x i c o k i n e t i c experiments, a d d i t i o n a l b a s a l d a t a a r e r e q u i r e d f r o m t h i s d i e t model; i t i s p a r t i c u l a r y necessary to e s t a b l i s h parameters f o r the h e p a t i c and i n t e s t i n a l x e n o b i o t i c m e t a b o l i s m i n r a t s on the standard f i b e r d i e t s . T h i s paper p r e s e n t s our f i n d i n g s o f the e f f e c t s o f d i f f e r e n t d i e t a r y f i b e r types on h e p a t i c and i n t e s t i n a l metabol i s m o f reference x e n o b i o t i c s . Previous Work R e l a t i n g to S e l e c t i o n o f Substrates C h a r a c t e r i s t i c s o f I n t e s t i n a l vs H e p a t i c X e n o b i o t i c M e t a b o l i s m . I n t e s t i n a l mucosa has been shown both to m e t a b o l i c a l l y a c t i v a t e and to d e a c t i v a t e x e n o b i o t i c s . The mixed f u n c t i o n oxidase (MFO)

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

20.

OPDYCKE AND STREET

Intestinal and Liver Microsome Activities 287

of i n t e s t i n a l mucosa responds to inducers and i n h i b i t o r s d i f f e r e n t l y than h e p a t i c MFO. I n t e s t i n a l MFO a l s o d i f f e r s from h e p a t i c MFO i n i t s c o n s t i t u a t i v e l e v e l s , m e t a b o l i c p a t t e r n s , and subs t r a t e s p e c i f i c i t y (25-41 )» B i p h e n y l H y d r o x y l a t i o n . F a c t o r s a f f e c t i n g 2-,3-, and 4-biphenyl hydroxylase enzymes are: age, sex, species, n u t r i t i o n a l s t a t u s , genetic background, x e n o b i o t i c preexposure, and t i s s u e type. The 2- and 4-hydroxy biphenyl hydroxylases have been demonstrated to be d i f f e r e n t on the b a s i s of s e l e c t i v e i n d u c t i o n , conducted both i n v i v o and in v i t r o , and on d i f f e r e n t species s p e c i f i c i t i e s ( 4 2 jTT). Toftgard et al.(51 ) reported that changes i n d i f f e r e n t forms of cytochrome P-450 give r i s e to a l t e r e d proportions of biphenyl metabolites. I t i s now c l e a r that biphenyl 2-hydroxylase a c t i v i t y i s conferred only by cytochrome P-448-type i n d u c t i o n Rat h e p a t i c biphenyl 4-hydroxylas l e v e l s than 2-hydroxylase P-450 and P-448 (52). W i e b k i n e t al.(53-54) summarized advant a g e s f o r use o f b i p h e n y l as s u b s t r a t e i n t h a t i t s m e t a b o l i c p r o f i l e provides an i n d i c a t o r to the t o x i c i t y of substrate and p r o d u c t s , i t h i g h l i g h t s f u r t h e r m e t a b o l i s m o f p r i m a r y metabol i t e s , and i n d i c a t e s the r e l a t i o n s h i p s among d i f f e r e n t MFO inducers; f o r these reasons we chose to use biphenyl as an i n d i c a t o r s u b s t r a t e to a s s e s s o v e r a l l MFO m e t a b o l i s m , as s u g g e s t e d by B i l l i n g s and McMahon (55)* I n t e s t i n a l microsomal metabolism can be monitored v i a biphen y l h y d r o x y l a t i o n as w e l l . Biphenyl hydroxylase assays are more s e n s i t i v e than some other hydroxylase assays which has permitted measurements of the low c o n s t i t u a t i v e enzyme l e v e l s i n i n t e s t i n a l mucosa and some other extrahepatic t i s s u e s (56-59)» Arylhydrocarbon Hydroxylase. B e n z o ( a ) p y r e n e (BaP) i s c o n s i d e r e d an i d e a l model compound f o r m o n i t o r i n g arylhydrocarbon hydroxy l a s e (AAH) MFO l e v e l s i n r a t s . I t s m e t a b o l i s m has been w i d e l y i n v e s t i g a t e d i n both r a t l i v e r and e x t r a h e p a t i c t i s s u e s (4041.61 -70). Arylhydrocarbon h y d r o x y l a s e has been compared to b i phenyl hydroxylase i n the i n t e s t i n e s of r a t s (25.28). I n t e s t i n a l AAH has been shown to be induced up to 3 0 - f o l d w i t h cytochrome P448-type inducers, while being i n s e n s i t i v e to P-450-type induction. Therefore, we have used i n t e s t i n a l AAH i n conjunction with biphenyl hydroxylation, to evaluate the e f f e c t s of d i e t a r y f i b e r s on i n t e s t i n a l metabolism. E p o x i d e H y d r o l a s e . E p o x i d e h y d r o l a s e (EH) a c t i v i t y has been studied i n numerous t i s s u e s with many d i f f e r e n t substrates (71 72). Hepatic microsomal EH has been shown to be induced by polyc y c l i c aromatic hydrocarbons, but i s i n s e n s i t i v e to cytochrome P450 type inducers (73)» Epoxide hydrolase has been r e l a t e d to the a c t i v a t i o n of p o l y c y c l i c aromatic hydrocarbons i n t o r e a c t i v e carcinogens (70). Epoxide hydrolase a c t i v i t y has been determined

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

UNCONVENTIONAL SOURCES OF DIETARY FIBER

288

i n t h e stomach as w e l l as t h e i n t e s t i n e and o t h e r e x t r a h e p a t i c t i s s u e s (74-75). I n d i c a t i o n s o f the e f f e c t s o f s p e c i f i c d i e t f i b e r s on epoxide hydrolase a c t i v i t y could be d i r e c t l y a p p l i e d to s u b s t a n t i a t e or modify f i b e r : c a r c i n o g e n e s i s hypotheses. Methods and M a t e r i a l s D i e t a ry F i b e r s . The c e l l u l o s e used i n t h e s e e x p e r i m e n t s was SolkaFlok, KS-1016, a c e l l u l o s e derived from s u l f i t e processing o f wood ( b i r c h , beech, and maple) h a v i n g a p a r t i c l e s i z e o f 290 m i c r o n s and c o n t a i n i n g 0.15-0.2$ l i g n i n and 8-9$ h e m i c e l l u l o s e (Brown C o . , B e r l i n , N.H.). The l i g n i n used was I n d u l i n AT, w h i c h i s a K r a f t l i g n i n described as being 99$ l i g n i n and 1.0$ dry ash, f r e e of simple sugars, i n s o l u b l e i n water and s o l u b l e i n a l k a l i (Westvaco, N o r t h C h a r l e s t o n , S.C.) The l i g n i n was S o x h l e t ext r a c t e d f o r 30 hours usin rated from the l i g n i n i was t h e n suspended i n d e i o n i z e d w a t e r and f i l t e r e d t h r o u g h a Buchner funnel w i t h Whatman 1 f i l t e r paper. T h i s washing process was r e p e a t e d u n t i l the f i l t r a t e was l i g h t orange i n c o l o r . The h e m i c e l l u l o s e used was M e t a m u c i l ( S e a r l e L a b o r a t o r i e s , C h i c a go,111.), a p h a r m a c e u t i c a l p r e p a r a t i o n o f I s p a g h u l a husk f r o m P l a n t a g o o v a t a ( F o r s k ) w h i c h has been c h a r a c t e r i z e d (76). The p e c t i n used was an NF c i t r u s p e c t i n , f i n e l y ground to a powder with p a r t i c l e s i z e of 200 microns (Hercules,Inc., Wilmington,DL). Diet Preparations. The American I n s i t u t e of N u t r i t i o n (AIN) synt h e t i c d i e t f o r m u l a t i o n was used a l o n g w i t h g e l a t i n t o form h y d r a t e d g e l l e d d i e t s . The i n g r e d i e n t composition i n grams/100 grams d i e t was: d e x t r o s e 64.6, c a s e i n 16.2, d l - m e t h i o n i n e 0.2, AIN s a l t m i x t u r e 3·5, AIN v i t a m i n m i x t u r e 1.5, g e l a t i n 6.0, and l a r d 8.0 f o r the c o n t r o l d i e t ; f i b e r d i e t s contained 15$ l e s s of each i n g r e d i e n t plus 15g f i b e r . Dry i n g r e d i e n t s were premixed and the hydrated g e l l e d d i e t s were prepared weekly by adding water (I90ml/I00g) and l a r d t o the a p p r o p r i a t e amount o f p r e m i x , blending f o r 1 minute, l e t t i n g the d i e t s g e l f o r 2 hours at 5°C, and reblending the s e m i g e l l e d d i e t s to evenly d i s t r i b u t e i n s o l u b l e i n g r e d i e n t s . The p r e p a r e d d i e t s were s t o r e d a t 5°C i n t u b s w i t h p l a s t i c f i l m l a i d d i r e c t l y on the s u r f a c e o f the d i e t s to minimize evaporative l o s s . Animal Treatment. S i x groups of 11 , t h i r t y - d a y o l d , male W i s t a r r a t s (100-120g), were r a n d o m l y d i v i d e d i n t o i n d i v i d u a l cages. A f t e r f i v e days a c c l i m a t i o n the groups were fed: Purina Lab Chow ad l i b i t u m , c o n t r o l ( f i b e r - f r e e g e l l e d d i e t ) and the g e l l e d d i e t s which c o n t a i n e d 15$ p e c t i n , M e t a m u c i l , l i g n i n , and c e l l u l o s e , r e s p e c t i v e l y . Every second day each r a t was weighed, unused food was weighed and f r e s h d i e t placed i n feeders secured at the f r o n t o f each cage. A t t h e end o f 28 days, two r a t s from each group were s a c r i f i c e d each day f o r 5 consecutive days.

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

20.

OPDYCKE AND STREET

Intestinal and Liver Microsome

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289

Enzyme P r e p a r a t i o n . Each r a t was weighed, k i l l e d by c e r v i c a l d i s l o c a t i o n , the l i v e r removed and 2g homogenized i n 10 ml 0.05M T r i s . , 0.15M KCL, pH 7.8 b u f f e r . I n t e s t i n a l m i c r o s o m e s were p r e p a r e d a c c o r d i n g to S t o h s et al.(28). The e n t i r e s m a l l i n t e s t i n e o f each r a t was removed, washed w i t h b u f f e r , measured f o r length, s p l i t l o n g i t u d i n a l l y w i t h s c i s s o r s , l a i d mucosal s i d e up on a g l a s s p l a t e and s c r a p e d w i t h a g l a s s s l i d e . The m u c o s a l c e l l s were weighed and homogenized i n 10ml b u f f e r w i t h 20$ g l y c e r o l , 25mg t r y p s i n i n h i b i t o r and 2 u n i t s h e p a r i n . H e p a t i c and i n t e s t i n a l homogenates were c e n t r i f u g e d at 600g f o r 2 minutes and then at 13000g f o r 20 minutes, with the microsomes sedimented at 105,000g f o r 60 minutes. A s s a y s . P r o t e i n was d e t e r m i n e d a c c o r d i n g to Lowry ejb a l ( 7 7 ) ) . Biphenyl hydroxylase a c t i v i t determined w i t h 2 microsomal p r o t e i n , 400 micromole micromoles magnesium c h l o r i d e dehydrogenase i n 1 ml t o t a l volume. A f t e r a 2 minute preincubat i o n , 1 mM b i p h e n y l i n 10 m i c r o l i t e r s o f a c e t o n e was added to s t a r t the 15 minute incubation. The r e a c t i o n s were stopped w i t h 0.5 ml 6N HCL, e x t r a c t e d w i t h i s o o c t a n e and c h r o m a t o g r a p h e d by HPLC on M i c r o p a c NHp-C-10 ( V a r i a n ) w i t h i s o o c t a n e i a c e t o n i t r i l e : i s o a m y l a l c o h o l (100:4:4)(59). D e t e c t i o n o f the 2-, 3-, and 4 - h y d r o x y b i p h e n y l p r o d u c t s was by f l u o r e s c e n c e d e t e c t i o n w i t h e x c i t a t i o n at 275nm and e m i s s i o n at 38nm. Q u a n t i t a t i o n of 2-, 3-, and 4-hydroxybiphenyls was accomplished with peak height comparisons to 1 .25, 2.50, and 3*75 m i c r o g r a m s o f each s t a n d a r d ; s t a n dards were incubated and extracted with each set of determinat i o n s . Epoxide hydrolase a c t i v i t y was determined by the method of G i u l i a n o ejt a l ( 7 5 ) u s i n g 3-(p-nitrophenoxy)-1 ,2-propene oxide as substrate w i t h 0.5mg microsomal p r o t e i n . The d i o l was separated u s i n g ODS r e v e r s e phase HPLC w i t h 40$ a c e t o n i t r i l e i n water. D e t e c t i o n was accomplished u s i n g UV absorbance at 315nm. A r y l h y drocarbon hydroxylase a c t i v i t y was determined by the assay procedure of Dehnen e_t al.(78) on an AmincoBowman s p e c t r o f l u o r o m e t e r a f t e r i n c u b a t i o n s w i t h 0.5mg microsomal p r o t e i n and NADPH genera t i n g system. One u n i t a c t i v i t y i s defined as picomoles/mg/minute as based on the f l u o r e s c e n c e of 20 ng 3-hydroxybenzo(a)pyrene. Results and D i s c u s s i o n Results of body, l i v e r and l i v e r / b o d y weight r a t i o s a f t e r 30 days on s p e c i f i e d d i e t s a r e l i s t e d i n T a b l e I. Each o f the treatment groups showed an i n i t i a l weight l o s s upon i n t r o d u c i n g the p u r i f i e d d i e t s . However, a l l but the l i g n i n - f e d a n i m a l s q u i c k l y recovered to steady weight gains throughout the r e s t of the experiment. Lab Chow-fed r a t s were s i g n i f i c a n t l y h e a v i e r than the p u r i f i e d d i e t - f e d r a t s . Body weights i n pectin-and Metamucilfed r a t s were s i g n i f i c a n t l y (0.05 l e v e l ) h i g h e r than c o n t r o l - ,

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

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290

Table I.

Body and L i v e r Weights and L i v e r t o

Body Weight Ratios o f Fiber-Fed R a t s .

Diet Type

Body Weigjit

L i v e r Weight

g Control

(No F i b e r )

a

L i v e r Wt./Body Wt.

g

g

18

Lab Chow

300

15% P e c t i n

b

28

11.14

220°

18

15% Metamucil

224°

15% L i g n i n

15% C e l l u l o s e

b

1.63

.037

.004

7.73

1.19

.034

.003

12

7.83

1.19

.034

.006

153

15

5.61

0.64

.036

.003

172

10

6.10

0.50

.035

.002

Values are means + S.D. f o r groups of 11 r a t s . b

S i g n i f i c a n t l y d i f f e r e n t from a l l other groups (P=.05) .

c S i g n i f i c a n t l y d i f f e r e n t from the other groups f e d p u r i f i e d d i e t s (P=.05),

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

20.

OPDYCKE AND

STREET

Intestinal and Liver Microsome

Activities

291

l i g n i n - o r c e l l u l o s e - f e d a n i m a l s . L i g n i n - f e d a n i m a l s c o u l d be considered to be under weight. L i g n i n - and c o n t r o l - f e d a n i m a l s ate s i g n i f i c a n t l y l e s s p u r i f i e d d i e t than other treatment groups. L i v e r weights i n Lab Chow-fed r a t s were a l s o s i g n i f i c a n t l y higher than i n r a t s fed p u r i f i e d d i e t s . The l i v e r weights of p e c t i n - and Metamucil-fed r a t s were s l i g h t l y higher (not s i g n i f i c a n t l y ) than c o n t r o l - , l i g n i n - or c e l l u l o s e - f e d r a t s . L i v e r : t o : b o d y r a t i o s were not s i g n i f i c a n t l y d i f f e r e n t i n any of the treatment groups. The i n f l u e n c e o f s p e c i f i c d i e t a r y f i b e r s on s m a l l i n t e s t i n e length, on the amount of mucosal m a t e r i a l scraped from the i n t e s t i n e , and on the r a t i o s o f grams mucosa/cm o r cm/g mucosa i s i n d i c a t e d by the d a t a i n T a b l e I I , w h i l e T a b l e I I I l i s t s the i n t e s t i n a l microsomal p r o t e i n (expressed as t o t a l or mg/cm s m a l l i n t e s t i n e ) . Lab Chow and d i e t s c o n t a i n i n g p e c t i n o r M e t a m u c i l produced i n t e s t i n e lengths that were s i g n i f i c a n t l y longer than i n the r a t s f e d the c o n t r o l r e s u l t s support s i m i l a b e r g and J a k o b s e n (79). The s t a b l e g e l s formed by M e t a m u c i l and p e c t i n f i b e r s caused s i g n i f i c a n t i n t e s t i n a l t r a c t enlargement, even though f e d i n h y d r a t e d form. The r a t s f e d Lab Chow and the p e c t i n d i e t had the highest mucosal weights, which were s i g n i f i c a n t l y higher than i n c o n t r o l - , l i g n i n - , o r c e l l u l o s e - f e d animals. L i g n i n - f e d animals y i e l d e d the l e a s t amount of mucosal m a t e r i a l , which c o i n c i d e s w i t h t h e i r having the s h o r t e s t s m a l l i n t e s t i n e s and lowest body weights (Table IV). T o t a l i n t e s t i n a l microsomal p r o t e i n d i d not d i f f e r s i g n i f i c a n t l y (0.05 l e v e l ) among treatment groups, although p e c t i n d i e t and Lab Chow-fed r a t s had r e l a t i v e l y higher t o t a l microsomal p r o t e i n values. Both l i v e r and i n t e s t i n a l mucosa m i c r o s o m a l p r o t e i n v a l u e s a r e l i s t e d i n T a b l e IV. T h e r e were no s i g n i f i c a n t d i f f e r e n c e s between t r e a t m e n t groups f o r e i t h e r tissue. L i s t e d i n T a b l e V a r e the v a l u e s o f 2 - , 3 - , 4 - b i p h e n y l hyd r o x y l a s e a c t i v i t i e s i n l i v e r microsomes of f i b e r - f e d r a t s . V a l u e s a r e e x p r e s s e d as nanomoles (2-, 3-,or 4 - h y d r o x y b i p h e n y l ) / m g / l 5 m i n u t e i n c u b a t i o n ; the l e a s t d e t e c t a b l e amount was 0.05 moles/mg. The h e p a t i c m i c r o s o m a l l e v e l s o f 4 - b i p h e n y l hyd r o x y l a s e were on the o r d e r o f 4 0 - f o l d h i g h e r t h a n the 2-or 3biphenyl hydroxylase a c t i v i t i e s . This difference i s s l i g h t l y g r e a t e r t h a n the h i g h e r c o n s t i t u a t i v e 4 - b i p h e n y l h y d r o x y l a s e l e v e l s r e p o r t e d by o t h e r s (46,50,80). The y i e l d o f 2 - h y d r o x y b i p h e n y l was s i g n i f i c a n t l y h i g h e r w i t h h e p a t i c m i c r o s o m e s from l i g n i n - f e d a n i m a l s t h a n w i t h m i c r o s o m e s from any o f the o t h e r treatment groups. The s l i g h t l y higher b i p h e n y l hydroxylase values f o r the p u r i f i e d d i e t - f e d r a t s i s notable. L i g n i n - f e d r a t s a l s o showed the highest 3-hydroxybiphenyl y i e l d s although t h i s e f f e c t was not s i g n i f i c a n t . The l e v e l s of h e p a t i c microsomal 4-hydroxyb i p h e n y l h y d r o x y l a s e were s i g n i f i c a n t l y l o w e r i n Lab Chow-fed animals than l i g n i n - f e d animals. There were no s i g n i f i c a n t d i f f e r e n c e s among the p u r i f i e d d i e t t r e a t m e n t groups, however, l i g n i n - f e d r a t s again had s l i g h t l y higher l e v e l s of a c t i v i t y .

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

4.95 6.39 6.74 6.48

34.70 39.86 48.01 41.06

.004 .004 .003 .004

.029 .025 .021 .026

6 6

108° e

I n t e s t i n e s i g n i f i c a n t l y longer

°

(P=.05).

(P=.05).

Mucosal weight s i g n i f i c a n t l y greater

^

f o r groups of 11 rats.

4

96

.35

2.30

15% Cellulose

Means + S.D.

6

93

.35

1.98

15% Lignin

108

.51

2.78

.39

2.88

e

31.84

III

.42

.004

36.79 + 6.92

.032

0.028 + 0.005

94 + 4

cm

cm/g Mucosa

8

g

cm

15% Metamucil

b

Mucosal Wt./cm

Small I n t e s t i n e

3.15

b

3.52

2.65 + 0.47

g

Mucosa

a

Small I n t e s t i n e Length, Mucosal Weight and t h e i r Ratio i n Fiber-Fed R a t s .

15% Pectin

Lab Chow

Control (No Fiber)

Diet Type

Table I I .

20.

OPDYCKE AND STREET

Intestinal and Liver Microsome

293

Activities

Table I I I . Microsomal P r o t e i n i n Small I n t e s t i n e Mucosa of Fiber-Fed Rats.

Type

T o t a l mg

Control

(No F i b e r )

13.77 + 5.80

mg/cm Length

0.14 + 0.06

Lab Chow

14.81

7.88

.13

.07

15% P e c t i n

15.23

5.45

.14

.05

15% Metamucil

10.50

5.20

.19

.05

15% L i g n i n

10.09

6.74

.11

.08

15% C e l l u l o s e

11.27

6.42

.12

.07

a

Mean microsomal p r o t e i n + S.D. f o r groups o f 11 r a t s ; d i f f e r e n c e s were not s i g n i f i c a n t (P=.05).

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

294

UNCONVENTIONAL SOURCES OF DIETARY FIBER

Table IV.

Microsomal P r o t e i n i n L i v e r and I n t e s t i n a l

Preparations

Diet Type

from Fiber-Fed

mg P r o t e i n / g L i v e r

Rats.

a

mg P r o t e i n / g

Mucosa

C o n t r o l (No F i b e r )

11.

Lab Chow

14.5

4.6

4.26

2.46

15% P e c t i n

12.4

5.4

4.97

1.87

9.4

3.7

3.71

1.94

15% L i g n i n

12.1

7.4

5.11

3.83

15% C e l l u l o s e

15.1

4.3

4.91

2.30

15% Metamucil

Values are mean + S.D. f o r groups of 11 r a t s . treatments were not s i g n i f i c a n t (P=.05).

D i f f e r e n c e s among

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

20.

Intestinal and Liver Microsome

OPDYCKE AND STREET

Table V.

295

Activities

Biphenyl Hydroxylase A c t i v i t i e s i n L i v e r Microsomes o f Fiber-Fed R a t s .

3

Nanomoles/mg/15 min Diet Type

2-OH

3-0H

Control (No F i b e r ) 0.43 + 0.10

Lab Chow

,30

15% P e c t i n

b

4-OH

0.40 + 0.13

.06

.36

.44

.15

15% Metamucil

.41

15% L i g n i n

15% C e l l u l o s e

b

15.98 + 5.75

b

.07

9.77

1.94

.32

.09

12.46

3.04

.08

.29

.07

13.82

4.00

.81°

.35

.62

.18

20.03

5.65

.30

.06

.28

.07

11.43

2.57

a

Values are mean hydroxylase a c t i v i t y + SE (N=10) i n nanomoles/mg p r o t e i n / 1 5 min.; min. d e t e c t a b l e amount was 0.05 nmole.

b

D i f f e r e n c e from l i g n i n - t r e a t e d animals s i g n i f i c a n t

C

D i f f e r e n c e from c o n t r o l and a l l other groups s i g n i f i c a n t

(P=.05).

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

(P=.05).

296

UNCONVENTIONAL SOURCES OF DIETARY FIBER

I n t e s t i n a l microsomal biphenyl h y d r o x y l a t i o n f o r f i b e r - f e d r a t s i s l i s t e d i n T a b l e VI. Due to the v a r i a b i l i t y o f the d a t a t h e r e were no s i g n i f i c a n t (0.05 l e v e l ) d i f f e r e n c e s among the groups o f f i b e r - f e d r a t s . L i g n i n f e e d i n g l e d to the h i g h e s t i n t e s t i n a l 2-biphenyl hydroxylase a c t i v i t i e s , while a l l the r a t s f e d p u r i f i e d d i e t s had h i g h e r l e v e l s t h a n the Lab Chow group. Among the groups fed p u r i f i e d d i e t s , the p e c t i n - f e d r a t s had the lowest 2-biphenylhydroxylase l e v e l s . P e c t i n feeding a l s o gave the highest i n t e s t i n a l 3-biphenylhydroxylase although that d i f f e r e n c e o n l y approached s i g n i f i c a n c e a t the 0.05 l e v e l . Lab Chow-fed r a t s had the l o w e s t 3 - b i p h e n y l h y d r o x y l a s e l e v e l i n i n t e s t i n a l microsomes. I n t e s t i n a l microsomes from f i b e r - f e d r a t s d i d not d i f f e r s i g n i f i c a n t l y (0.05 l e v e l ) i n 4-biphenyl hydroxylase a c t i v i t y . C e l l u l o s e - f e d animals had the lowest l e v e l , with the cont r o l s and l i g n i n - f e d r a t showin th highest i n t e s t i n a l microso mal 4-biphenylhydroxylas had r e l a t i v e l y low l e v e l 4-bipheny hydroxylas The r e s u l t s of a p r e l i m i n a r y i n t e s t i n a l microsomal biphenyl hydroxylase t r i a l are l i s t e d i n Table VII. These r a t s were on the same h y d r a t e d p u r i f i e d d i e t s c o n t a i n i n g 15$ f i b e r f o r 32 days before the microsomal enzyme determinations. One d i f f e r e n c e between these animals and those i n the primary experiment reported h e r e i n i s t h a t t h e y were s t a r t e d on the d i e t s a t 45 days o f age and weighed about 200g at the s t a r t . The biphenyl h y d r o x y l a t i o n i n c u b a t i o n was done with 5 mg i n t e s t i n a l microsomal p r o t e i n as compared to 2 mg p r o t e i n used i n the subsequent incubations. The b i p h e n y l h y d r o x y l a t i o n v a l u e s were not s i g n i f i c a n t l y d i f f e r e n t (0.05) i n any of the treatment groups although Lab Chow-fed r a t s had s l i g h t l y higher 4-hydroxy biphenylhydroxylase values. Benzo(a)pyrene hydroxylase a c t i v i t i e s i n h e p a t i c and i n t e s t i n a l microsomes of f i b e r - f e d r a t s are presented i n Table V I I I . There were no s i g n i f i c a n t d i f f e r e n c e s (0.05 l e v e l ) among any of the p u r i f i e d f i b e r treatment groups i n e i t h e r l i v e r or i n t e s t i n a l microsomes. Lab Chow-fed r a t s had the l o w e s t l i v e r m i c r o s o m a l benzo(a)pyrene h y d r o x y l a s e (AHH) l e v e l . The i n d i c a t i o n o f enhanced i n t e s t i n a l P-448-dependent h y d r o x y l a t i o n of biphenyl i n the p e c t i n - f e d r a t s (seen as elevated 3-hydroxybiphenyl formation i n microsomes, Table Vl) was not supported by these p r e l i m i n a r y data. Microsomal epoxide hydrolase a c t i v i t i e s are l i s t e d i n Table IX. There was s i g n i f i c a n t l y higher (0.05 l e v e l ) h e p a t i c microsomal e p o x i d e h y d r o l a s e a c t i v i t y i n p e c t i n - , M e t a m u c i l - , and c e l l u l o s e - t r e a t e d r a t s , as compared to c o n t r o l - , l i g n i n - and Lab Chow-fed animals. There was no s i g n i f i c a n t d i f f e r e n c e i n i n t e s t i n a l m i c r o s o m a l e p o x i d e h y d r o l a s e a c t i v i t i e s among any o f the treatment groups. Studies d i r e c t l y probing the i n t e r a c t i o n of d i e t a r y f i b e r s on f o r e i g n compound m e t a b o l i s m a r e l i m i t e d . Brown,_ejt al.(81 ) reported that r a t s fed b a s a l chow d i e t s had no l i v e r metabolism d i f f e r e n c e s compared to r a t s on d i e t s supplemented w i t h p e c t i n or

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

20.

OPDYCKE AND STREET

Table VI.

Intestinal and Liver Microsome

Activities

297

Biphenyl Hydroxylase A c t i v i t y i n I n t e s t i n a l Microsomes of F i b e r - F e d R a t s .

a

Nanomoles/mg/15 min Diet Type

C o n t r o l (No F i b e r )

2-OH

0.38 + 0.16

3-OH

4-OH

0.79 + 0.35

1.13 + 0.74

Lab Chow

.30

.09

.46

.14

.79

.24

15% P e c t i n

.19

.05

2.08

.74

.35

.09

15% Metamucil

.50

.01

.88

.13

.92

.45

15% L i g n i n

.68

.32

1.14

.26

1.32

1.06

15% C e l l u l o s e

.47

.07

.46

.13

.22

.08

a

Values are mean hydroxylase a c t i v i t y + SE f o r groups of 7 r a t s ; d i f f e r e n c e s among treatments were not s i g n i f i c a n t (P=.05).

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983. 3.22

S i g n i f i c a n t d i f f e r e n c e from c o n t r o l , P>.05.

5.73

C

0.62

Nanomoles/mg p r o t e i n i n mucosal microsomes/15 min.

2.86

0.25

6.39

4.41

1.24

0.50

0.64

1.40

.97

.97

.83

.41

0.95

n=2.

a

.21

.42

.15

.12

.73

.44

.85

.72

0.98

.12

.19

.11

.09

+ 0.09

Biphenyl Hydroxylase^ 3-OH

+ 0.22

2-OH

of 3 r a t s , except hydroxylase values where

5

2.56

0.27

^

103

15% C e l l u l o s e

7

2.55

0.70

7.28 + 0.90

Microsomal x) Protein (mg/g)

Values are means + S.D.

101

15% L i g n i n

8

2.51

2.30 + 0.45

Mucosal x) Weight (g)

Rats.

.73

.45

.33

1.19

0.77

and Biphenyl Hydroxylase A c t i v i t i e s

i n I t i t e s t i n a l Microsomes of Fiber-Fed

Small Intestine Parameters

3

119

15% Metamucil

6

100 + 3

108

(No Fiber)

I n t e s t i n e x) Length (cm)

Lab Chow

Control

Fiber Type

Table VII.

+

4-OH

.09

.07

.07

.56

0.11

20.

OPDYCKE AND STREET

Table V I I I .

Intestinal and Liver Microsome

Activities

299

Benzo(a)pyrene Hydroxylase A c t i v i t i e s i n

Hepatic and I n t e s t i n a l Mucosa Microsomes of Fiber-Fed

Rats.

a

Picomoles /mg/min Diet Type

Control

(No F i b e r )

Lab Chow

Liver

Small I n t e s t i n e

21

92

u

7.1

19.9

6.8

15% P e c t i n

193

81.1

17.9

4.7

15% Metamucil

263

98.1

18.7

5.3

15% L i g n i n

255

86.4

19.3

3.4

15% C e l l u l o s e

245

98.1

19.6

4.8

Values are means + SE f o r groups of 7. Q u a n t i t a t i o n was based on the fluorescence of 20ng 3-hydroxy-benzo(a)pyrene and s c a l e d to mg microsomal p r o t e i n . S i g n i f i c a n t d i f f e r e n c e from c o n t r o l s , P>.05.

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

300

UNCONVENTIONAL SOURCES OF DIETARY FIBER

Table IX.

Epoxide Hydrolase A c t i v i t i e s i n

L i v e r and I n t e s t i n a l Mucosa Microsomes of Fiber-Fed

Rats.

Nanomoles/mg/5 min Diet Type

Liver

Small I n t e s t i n e

C o n t r o l (No F i b e r )

38.0 4- 8.13

14.9 + 2.94

Lab Chow

45.0

7.24

13.1

3.28

15% P e c t i n

64.3

11.81

12.6

3.05

15%

62.0

13.32

16.3

3.24

15% L i g n i n

44.9

4.94

14.3

4.33

15% C e l l u l o s e

64.4

18.6

7.01

Metamucil

υ

U

22.1

Values are means + SE f o r groups o f seven s c a l e d to mg microsomal p r o t e i n f o r 3-(p-nitrophenoxy)-l,2-propene oxide as s u b s t r a t e . S i g n i f i c a n t l y d i f f e r e n t from c o n t r o l s , P>.05.

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

20.

OPDYCKE AND STREET

Intestinal and Liver Microsome

Activities

301

wheat bran as judged by a n t i p y r i n e and acetaminophen metabolism, Chadwick et al.(82), however, d e m o n s t r a t e d s i g n i f i c a n t enhancement o f l i n d a n e m e t a b o l i s m i n p e c t i n - f e d r a t s - P r o i a e t al.(83) r e c e n t l y reported that p e c t i n feeding lowered hepatic cytochrome P-450 l e v e l s w h i l e a t t h e same t i m e i n c r e a s i n g i n t e s t i n a l benzo(a)pyrene hydroxylase l e v e l s . I t remains to be evaluated to what e x t e n t t h e d i f f e r e n c e s o b s e r v e d i n d r u g o r o t h e r f o r e i g n compound _in v i v o metabolism i n r a t s f e d p e c t i n or other p u r i f i e d f i b e r s can be explained by d i f f e r e n c e s i n c o n s t i t u a t i v e l e v e l s of the relèvent enzymes i n l i v e r and i n t e s t i n a l mucosa. Our d a t a suggest that MFO h y d r o x y l a t i n g a c t i v i t i e s i n both t i s s u e s are not p a r t i c u l a r l y s e n s i t i v e to e f f e c t s o f feeding p u r i f i e d f i b e r s at high l e v e l s (the d i e t model used i n our work represents extremes i n f i b e r i n t a k e ) . The s i g n i f i c a n t e f f e c t on h e p a t i c e p o x i d e hydrolase seen with p e c t i n Metamuciland c e l l u l o s e - f e d r a t s (Table IX) may point towar to chemical carcinogenesis o r a t o r y t o compare G S H - t r a n s f e r a s e a c t i v i t i e s and t h e t i s s u e g l u t a t h i o n e s t a t u s i n s p e c i f i c f i b e r - f e d r a t s . These s t u d i e s may enhance o u r a p p r e c i a t i o n o f t h e c o n t r i b u t i o n s o f t h e nono x i d a t i v e enzymes i n f o r e i g n compound metabolism to the r e l a t i o n ships between n u t r i t i o n a l s t a t e and chemical carcinogenesis. Acknowledgment s T h i s r e s e a r c h was s u p p o r t e d , i n p a r t , by a g r a n t f o r the study of "Diet and Colon Cancer i n Man: The E f f e c t o f F i b e r (CA25580)" and a g r a n t f o r t o x i c o l o g y t r a i n i n g (ES-7097) from the N a t i o n a l I n s t i t u t e s o f H e a l t h . F a c i l i t i e s and o t h e r s u p p o r t by the Utah A g r i c u l t u r a l Experiment S t a t i o n are a l s o acknowledged.

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Intestinal and Liver Microsome

Activities

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60. Conney, A.H. Pharmacol. Rev. 1967, 19, 317. 61. Al-Turk, W.A.; Stohs, S.J.; Roche, E.B. Horm.Metabol. Res. 1980, 12, 404. 62. Gontovnick, L.S.; Bellward, G.D. Drug Metabol. Dispos. 1981, 9(3), 265. 63. Bock, K.W.; Clausbruch, U.C.; Winne, D. Med. Biol.1979, 57, 262. 64. Nebert,D.W.; Gelboin, H.V. Arch. Biochem. Biophys.1969, 134, 76. 65. Zampaglione,N.G.; Mannering, G. J. J. Pharm. Exp.Therap. 1973, 185, 676. 66. Vadi, H.; Moldeus, P.; Capdevia, J.; Orrenius, S. Cancer Res. 1975, 35, 2083. 67. Wattenberg, L.W. Cancer 1971, 6, 99. 68. Cantoni, L.W.; Rizzardini M. Belvedere G. Cantoni,R. Funelli.R.; Salmona 69. Stohs, S.J.; Wu, L.J. Res. Commun. Cem. . Pharmacol. 1981, 34(3), 461. 70. Guenthner, T.M.; Oesch, F. Trends in Pharmacol. Sci.1981, 5, 129. 71. Oesch, F. "Progress in Drug Metabolism"; Bridges, J.W.; Chassaud,L.F., Eds.; John Wiley, London, 1979, Vol. III, p. 253. 72. Oesch, F. ExcerptaMedica (Ciba Found.Sympos.) 1980,76, 169. 73. Oesch, F. Biol. Chem. 1976, 251, 79. 74. Suzuki,K.; Lee, I.P.Toxicol. Appl. Pharmacol. 1981, 58, 151. 75. Giuliano, K.A.; Lau, E.P.; Fall, R.R. J. Chromat.1980, 202, 447. 76. Kennedy, J.F.; Sandhu, J.S.; Southgate, D.A.T. Carbohy. Res. 1979, 75, 265. 77. Lowry,O.H.;Rosenbrough, N. J.; Farr, A.L.; Randall, R.T. Biol. Chem. 1951, 139, 265. 78. Dehnen, W.; Tomingas, R.; Roos, J. Anal. Biochem. 1973, 53, 373. 79. Rotenberg, S.; Jakobsen, P. E. Z.Tierphysiolgie 1979, 42, 299. 80. Haugen, D.A. Drug Metab. 1981, 9(3), 212. 81. Brown, R.C; Kelleher, J.; Losowsky, M.S. Brit. J. Nutr. 1979, 42, 357. 82. Chadwick, R.W.; Copeland, M.F.; Chadwick, C.J.Fd. Cosmet. Toxicol. 1978, 16, 217. 83. Proia, A.D.; McNamara; K.D.G. Edwards, and K.E.Anderson. ASPET Meeting Abstract 1981,240,158. RECEIVED January 20, 1983

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

INDEX Β

A Acetylation 106 Acidic xylan, methylation analysis .... 42* Agar 11 dietary 11 Alfalfa cation exchange capacities 140 effect on fecal bile acids 5 Alfalfa diet 1 colon carcinogenesis 8 colon tumor incidence 8* Alfalfa fiber ash content 256 cellulose quantity 256 lignin quantity 256 protein concentration 254 Alphacel, composition 225* Alphacel diet fecal material 226 hydrolyzable glucose in feces 226 Amino acids, pectin-lipoprotein interactions 188 Aminopolysaccharides 105-121 general properties 106-108 Anion binding by chitosan 115 Antibiotics, unabsorbable 116 AOM—See Azoxymethane Apparent hemicellulose 253 Apple cell wall materials, preparation 47 Apples cell wall materials 42 sugar composition 43/ structural chemistry of nonstarchy polysaccharides 33-47 Arabinan, methylation analysis and structure 38* Arabinogalactan, degradation by intestinal bacteria 126* Arabinose—See Sugars Arylhydrocarbon hydroxylase activities 287 activity in rat microsomes 285-301 Aspen wood, minerals 243* Atherosclerosis, prevention in children 72 Atomic absorption spectrophotometry, element analysis 160 Available carbohydrate, glycémie response 24 Azoxymethane induced colon carcinogenesis, dietary fiber effects 8

Bacteroides in polysaccharide degradation

Bacteroides thetaiotaomicron Bean-supplemented diet(s) glucose metabolism serum cholesterol serum cholesterol concentration

123-33 126*

127 51 57 54* 53-54

side effects 55 Bean supplements, tolerance 56 Bile acid adsorption on lignin 247 Bile acid binding 4, 187 Bile acids fecal excretion 5 in vitro affinity to dietary fibers .... 186 Bile salts 94 See also Sodium taurocholate enterohepatic circulation 4 Bioflavanoids 201 Biphenyl 4-hydroxylase 287 Biphenyl hydroxylase activities ...291,295* Biphenyl hydoxylase activity 287 determination 289 in rat microsomes 285-301 Biphenyl hydroxylation, activities 287 Blood cholesterol vs. lipid excretion .... 148* Blood glucose concentrations, reducing 17 Blood serum lipid patterns, psyllium effects 67 Blood serum triglyceride levels, psyllium effects 68 Blood glucose response studies 23/ Bowel cancer, beef effects 2 Brans 4 Broccoli 3 Brown rot wood, preparation 247, 249 Brussels sprouts 3

C Cabbage 3 fermentability 139 Calcium absorption 169/ chitin effects 161 chitosan effects 161, 179 Carbohydrate digestion, leguminous seed fiber effects 17-29

307

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308

UNCONVENTIONAL SOURCES OF DIETARY FIBER

Carbohydrate monomers, in citrus wastes 216* Carbohydrate(s) available, glycémie response 24 dietary, mixed with guar 24 as dietary fibers 3 Carcinogenesis, initiating agents 2 lesion development 2 promotional phase 14 Carcinogens 94 Carob 72 Carrageenan 4, 11 Carrageenan diet 1 colon tumor incidence 13/ Carrot alcohol insoluble residue, preparation 46 Carrot cell wall materials polysaccharide extraction 4 preparation 44 sugar analyses 34, 36/, 37* Carrot pectin isolation S 37 methylation analyses 39/ Carrot pulp, polysaccharide extractions 35/ Carrots hemicellulose fractions 38 polysaccharide fractions, methylated sugars 41/ polysaccharide subfractions 39 composition 40/ structural chemistry of nonstarchy polysaccharides 33-47 sugar composition of polysaccharide fractions 37/ Cation adsorption 140 Cation exchange capacity measurements 137 noncereal dietary fibers 140 Cationic bridges, formation 140 Cecum, lacteal dilation of lamina propria 177, 178/ Cell turnover 168 Cells in metaphase transition 168 Cellulose 41, 185 effect on dimethylhydrazineinduced tumors 11 effect on hemoglobin levels 180/ effect on hepatic epoxide hydrolase 301 effects on cholesterol digestion 146/ function 252 growth effects 168 in human digestion 221-38 hypocholesterolemic effects 51 Cellulose fraction of citrus wastes, monosaccharides 217/ Chemically purified fibers 4 Chitin 106, 156 acetylation 106

Chitin—Continued chemical properties 181 chemical structure 107/ commercial, derivation 156 effect on element absorption 155-82 effect on hemoglobin levels 180/ experimental diet composition 159/ growth effects 168 molecular weight 106 physiological activity 109-12 potential applications in foods 108 solubility 108 structure 157/ toxicity 109 Chitin experiments, histopathology .... 160 Chitosan 106 anion binding 115 bile acid excretion 109

chemical structure 107/ comparison with hypocholesterolemic resins 119/ effect on cholesterol absorption .... 112 effect on element absorption 155-82 effect on hemoglobin levels 180/ effect on liver triglycerides 109 effect on serum cholesterol 109 experimental diet composition 159/ growth effects 161, 168 hypocholesterolemic activity 109 possible mechanism 112-18 hypolipidemic activity, possible mechanism 112-18 in vitro and in vivo properties 120/ interaction with mixed micelles 117/ for lowering blood cholesterol 177 molecular weight 106 physiological activity 109-12 potential application in foods 108 solubility 108 structure 157/ toxicity 109 Chitosan experiments, histopathology 160 Cholesterol as major carrier 187 lowering effects 148 Cholesterol absorption 114/ Cholesterol ester excretion, pectin effects 144 Cholesterol esters, fatty acids profile 147/ Cholesterol-free diet, fecal steroid excretion 152/ Cholesterol levels, effect of fiber additives 111/ Cholesterol-rich lipoproteins, legumes effect 49-50 Cholestyramine 156 in vitro and in vivo properties 120/

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309

INDEX Cholestyramine diets Cholic acid Chondroitin lyase Chondroitin sulfate degradation by intestinal bacteria fermentation Citrus fiber diets colon tumor incidence dimethylaminobiphenyl-induced tumors effect on dimethylaminobiphenylinduced tumors Citrus fruits, components Citrus juice processing Citrus pectic polysaccharides Citrus pulp Citrus wastes analyzing and carbohydrate monomers citrus fraction hydrolysis composition 207/, extraction of pectic substances fractionation distribution glucose hydrolyzable polysaccharides neutral sugars pectic substance fractions protein total nitrogen uronic acid uronic acid analysis water-soluble pectins xylose Colon bacteria carbohydrate sources polysaccharide utilization Colon cancer and dietary fiber dietary fiber consumption mechanism of protective effect of dietary fiber protective factors in diet risk correlation studies Colon cancer etiology, concepts Colon carcinogenesis alfalfa diet animal model studies azoxymethane induced bile acid-binding conventional dietary fibers effect of caloric density and fecal bulk pectin diet presence of Salmonella

typhimurium unconventional dietary wheat bran diet Colon tumor incidence alfalfa diet carrageenan diet

1 247 127 ..

126/ 127 10/ 12/

11 206/ 206 185-89 201 208-12 21 212-15 209 213/ 215 211 214/ 210/ 211 209 214/ 209 208 215 124 125-27 94 6-13 3-6 3 2-3 2 8 6-13 8 7 1-16 7 5 8

6 fibers

1-16 8 8/ 13/

Colon tumor incidence—Continued citrus fiber diets 10/ pectin diet 8/ wheat bran diets 8/, 10/ Colon tumors, induction by dimethylhydrazine 7 Colonic Bacteroides 123-33 Commercial chitin, derivation 156 Comminution process, mechanical .... 242 Conventional dietary fibers in colon carcinogenesis 1-16 Copper absorption 171/ chitosan effects 168 Corn bran composition 225/ in human digestion 221-38 xylose:arabinose ratio 232 Corn bran fibers 232

Crypt epithelial cells, nuclear division

168

Cyathus stercoreus lignin degradation wheat straw biodégradation products

268 267-83

D Death, premature 71 Deoxycholic acid 5 Diabetes 17 therapeutic use of fiber 21-24 treatment with fiber diet 21 Diabetics high fiber dietary effects 87 low glycémie index diets 28 Dialysis mode, carbohydrate absorption studies 18 Dietary agar 11 Dietary carbohydrate, mixed with guar 24 Dietary fiber consumption, effect on colon cancer 6-13 Dietary fibers 3 analysis 95 benefits 156 from citrus wastes, characterization 205-18 classification 3-4 definition 105, 155, 185 degradation 124 effect on colon cancer 94 in vitro affinity to bile acids 186 mechanism altering plasma cholesterol 94 noncereal 135-41 protection against colon cancer 1 protective mechanism in colon carcinogenesis 3

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UNCONVENTIONAL SOURCES OF DIETARY FIBER

Dimethylaminobiphenyl-induced tumors citrus fiber diets citrus fiber effects wheat bran diets 1,2-Dimethylhydrazine, binding Dimethylhydrazine, colon tumor induction Dimethylhydrazine-induced tumors .... DMAB—See Dimethylaminobiphenyl DMH—See 1,2-Dimethylhydrazine and Dimethylhydrazine

12* 11 12* 4 7 11

Foods effect on digestive absorption glycémie index vs. sugar concentration Fructosyl-glycine as carbon source catabolism by Bacteroides effect on bacterial growth effect on glucose uptake structure Fruit fibers

24 26/ 27/ 123 129 129 129, 131/ 132 130/ 4

G

Ε Element absorption in rats, chitin and chitosan effects 155-82

Enterobacter

177

Epoxide hydrolase activities activity in rat microsomes

Escherichia coli

28 285-301

177

F Familial hypercholesterolemia, locust bean gum effects 71-89 Fecal bile acids alfalfa effects 5 wheat bran effects 5 Fecal bulk and colon carcinogenesis 5 psyllium effects 63 Fecal excretion of bile acids 5 Fecal fiber composition, psyllium effects 66 Fecal hydrolysis 237* Fecal mutagenic activity 5, 6 Fecal secondary bile acids 5 Fecal transit times, measurement 63 FHC—See Familial hypercholes­ terolemia Fiber analysis, Furda method 88 Fiber deficiency disorders 17 Fiber diets, use in diabetes treatment 21 Fiber-free diet fecal output 235 sugar production 227/ Fiber-rich foods, health benefits 58 Fiber supplemented foods, need 21 Fiber supplements, importance of mixing with food 21 Fibers effect in small intestine 18 water-soluble, cholesterollowering effects 50 Fleawort—See Psyllium Food emulsifiers 124, 128

Galactomannan-degrading system Galactose—See Sugars Gastric emptying rate, pectin effects .. Gastrointestinal tract psyllium

128 145

Glucosamine 108 Glucose—See also Sugars in citrus wastes 215 Glucose metabolism, bean supplemented diets 57 Glucose uptake, fructosyl-glycine effects 132 Glycémie index 24-25 definition 25 food fat correlations 25 food protein content 25 of foods 26/ vs. sugar concentration 27/ Glycosaminoglycans 187 Glycosylated hemoglobin determination 76 locust bean sum effects 82 Grapefruit—See Citrus wastes Guar carbohydrate absorption effects 18 cholesterol-lowering effects 50 crispbread preparation 51 effect on blood glucose 20/ effect on hyperlipidemic patients .... 72 hypocholesterolemic effects 51 mixing with dietary carbohydrates .. 24 urinary glucose effects 22/ use in diabetes treatment 21 Guar crispbread 24 Guar gum 128 bulking effects 94 effect on plasma cholesterol 96 effect on plasma lipoproteins 93-103 locust bean gum, degradation by intestinal bacteria 126* Guar gum diets effect on high-density lipoproteins 98/ effects on plasma cholesterol 98/ growth curves of rats 97/

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INDEX

311

Guar gum diets—Continued high-density lipoproteins Gums Gut physiology, leguminous fiber effects

101/ 4, 185

Isomaltol-galactoside breakdown catabolism by Bacteroides structure

123, 132 132 129 130/

18-21

H Hamlin washed orange pulp, fiber analysis 199* HDL—See High-density lipoproteins Heart disease 17 Heavy metal binding, on lignin 244 Hemicellulose 62, 185 apparent 253 Hemicellulose sources 235 Hemoglobin, determination 160 Heparin, fermentation 127 High carbohydrate/high-fiber diet High-density lipoproteins and dietary fiber 101 regulation mechanisms 102 wheat bran effects 94 High dietary fiber, as protective factor 3 High fat diets 2 High-fiber diets blood glucose effects 19/ carbohydrate absorption effects 17-29 gastrointestinal effects 19/ insulin requirements in diabetics .... 87 High-fiber foods diabetic treatment 25, 28 dietary effects 49 Human digestive process, diet effects 221-38 Human serum cholesterol, plant fiber effects 51* Hypercholesterolemic activity of chitosan, possible mechanism .112-18 Hypercholesterolemic effects locust bean gum 71-89 of plant fibers 50-51 Hyperlipidemic patients, pectin and guar 72 Hypocholesterolemic agents 145 Hypocholesterolemic resin, comparison with chitosan 119* Hypolipidemic activity of chitosan, possible mechanism 112-18

I Intestinal microsomal biphenyl hydroxylation, fiber diet 296 Intestinal microsomal metabolism, monitoring 287 Intestinal microsomal protein 293* Intestinal vs. hepatic xenobiotic metabolism 286-87 Iron absorption 168, 170/ mechanism of chitosan effects 181

L LBG—See Locust bean gum LDL—See Low-density lipoproteins LDLC—See Low density lipoprotein cholesterol Legumes and their soluble fiber 49-58 effect on cholesterol-rich lipoproteins 49-58 hypocholesterolemic effects 51 Leguminous seed fiber 17-29 effect on gut physiology 18-21

Lignin-carbohydrate complexes 283 Lignins 3, 185 See also Wheat straw absorptivity 247 bile acid adsorption 247 complex with iron 242 degradation by Cyathus stercoreus 268 as dietary fiber 3 function 252 growth effects 291 heavy metal binding 244 ion exchange effects 244 metal ion adsorption 246/ metal ion sources 242 minerals 243* nitrosoamine adsorption 245/ preparation 241 Lipid excretion vs. blood cholesterol .. 148* Lipidemias 67 Lipoproteins, low density 53 See also Low density lipoproteins Lipoprotein lipase activity, pectin effects 100 Lithocholic acid 5 Liver cholesterol, chitosan effects 109 Liver cholesterol levels fat effect 149* pectin effect 149* Liver lipid concentrations, chitosan effects 110* Liver lipid removal, pectin effect 151* Liver weights, diet effects 291 Locust bean gum effect on glycosylated hemoglobin .. 82 effect on lipoprotein cholesterol 80 effect on triglycerides 80 hypercholesterolemic effects 71-89 mechanism of action 88 origin 72 physiology 88

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UNCONVENTIONAL SOURCES OF DIETARY FIBER

312 Locust bean gum—Continued potential role in treating hypercholesterolemia 87 side effects 82 total cholesterol effects 77 Low density lipoprotein cholesterol, locust bean gum effects 80 Low density lipoproteins 53, 187 and cardiovascular disease 43 effect of citrus pectic polysaccharides 185-89 interaction with pectic polysaccharides 188-89 Low glycémie index diets, advantages 28

M Magnesium absorption 170/ chitin effects 16 Maillard products catabolism by colonic Bacteroides 123-33 utilization 128-33 Mannose—See Sugars Mechanical comminution process 242 Metal ion adsorption on lignin 246/ Metamucil, effect on hepatic epoxide hydrolase 301 Methyl cellulose, effect on blood glucose 20/ N-Methylated neomycin 116 Methylated sugars, from carrot polysaccharide fractions Alt Methylated wheat straw 279/ Methylation, linkage analysis of pectins 37 Methylation analyses 44 Microsomal epoxide hydrolase activities 296,300* Mineral utilization, psyllium effects .... 67 Mitotic figures, duodenum section 168 MNU—See Methylnitrosourea Monosaccharide production, influences 225 Mucopolysaccharides, catabolism by colonic Bacteroides 123-33

Ν Neomycin 116 Nitrobenzene oxidized wheat straw .... 273 compounds 277/ Nitrosoamine adsorption on lignin .... 245/ Noncereal dietary fibers 135-41 biological properties 139* cation exchange capacities 140 composition 138, 138* fermentability 138 in vitro properties 137 physicochemical properties 139* water holding capacity 138

Nonconventional dietary fibers in colon carcinogenesis Nonstarchy polysaccharides fractionation structural chemistry structural families

1-16 34 33-47 33

Ο Oat bran hypocholesterolemic effects plasma cholesterol levels tolerance Oat gum, cholesterol-lowering effects Oleic acid absorption Orange pulp, washed—See Washed orange pulp Oxalates

51 100 56 50 114*

263

Particulate fiber 21 Pectic polysaccharides 185 interactions with lipoproteins 188-89 methoxy content 189 rhamnose content 189 Pectin-lipoprotein interaction 187-88 Pectin(s) 4,41,185 blood cholesterol vs. lipid protein .. 148* bulking effects 94 cholesterol lowering influence 50, 186 composition 225* degradation by intestinal bacteria .. 126* degree of methylation 148* effect of dietary cholesterol 100 effect of gel formation 147 effect on blood glucose 20/ effect on gastric emptying rate 145 effect on hepatic epoxide hydrolase 301 effect on hyperlipidemic patients .... 72 effect on lipoprotein lipase activity 100 effect on liver lipid removal 151* effect on plasma lipoproteins 93-103 effect on plasma triglycerides 100 effect on rat metabolic processes .143-52 effectiveness of lowering cholesterol 148 effects on cholesterol digestion 146* in human digestion 221-38 linkage analysis 37 with low esterification 36 washed orange pulp as source 196 Pectin-bile salt interaction 186-87 Pectin diet(s) 1 colon carcinogenesis 8 colon tumor incidence 8* effect on high-density lipoproteins .. 98/ effects on plasma cholesterol 98/ growth curves of rats 97/ high-density lipoproteins 101/

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313

INDEX Phenolics 201 Phosphorus absorption 169/ chitin effects 161 chitosan effects 161 Phytate content, tobacco fiber 262 Plant fibers definition 49 hypocholesterolemic effects 50-51 Plant gums, catabolism by colonie Bacteroides 123-33 Plantago psyllium—See Psyllium Plasma cholesterol chitosan effects 112 guar gum effects 96 Plasma lipid concentrations, chitosan effects 110/ Plasma lipids, analysis 95 Plasma lipoproteins guar gum effects 93-10 pectin effects 93-10 Plasma triglycerides, pectin effects .... 100 Polar entrapment 116, 117/ Polygalacturonic acid degradation by intestinal bacteria .. 126/ fermentation 128 Poly glucosamines 106 Polysaccharide metabolism 127-28 Polysaccharide utilization by colon bacteria 125-27 Polysaccharides degradation by intestinal bacteria .. 126/ fermente rs 125, 126/ hydrolyzable, from citrus wastes ... 211 nonstarchy fractionation 34 structural chemistry 33-47 structural families 33 pectic 185 interactions with lipoproteins .188—89 methoxy content 189 rhamnose content 189 sulfated 11 water insoluble, cholesterollowering effects 50 Promotional phase, of carcinogenesis 14 Protein in citrus wastes 211 Protective effects 4 Psyllium blood serum lipid patterns 67 composition 63 copper adsorption 140 effect on proximal composition of feces 63, 66 fecal bulk effects 63 fecal fiber composition effects 66 fecal nitrogen losses 66 fece composition effects 64/ gastrointestinal effects 64/ gastrointestinal tract function effect 63

Psyllium—Continued laxative properties mineral utilization effects purified, effect on gastrointestinal function

62 67 61-68

R Rhamnose—See also Sugars in citrus wastes in pectic polysaccharides Rat metabolism, pectin effects

218 189 143-52

S Salmonella typhimurium absence

177

Serum cholesterol bean-supplemented diets 53-54 bean diet 54/ chitosan effects 109 plant fiber effects 50/, 51/ Serum cholesterol levels fat effect 149/ pectin effect 149/ Serum glucose concentrations, bean diet 55 Serum lipid concentrations, reducing .. 17 Serum triglyceride concentrations 54-55 Small intestine, fiber effects 18 Sodium taurocholate See also Bile acids and Bile salts binding to tobacco fiber 253, 259 Soluble pectin, determination 192 Soybean, cation exchange capacities .. 140 Spinach, oxalic acid content 263 Sterol ester hydrolase 147 Stool softeners 124 Sugar analyses, methods 44 Sugars in apple cell wall materials 43/ in carrot cell wall materials 34, 36/ methylated, from carrot polysac­ charide fractions 41/ in polysaccharide fractions of carrots 37/ Sulfated polysaccharides 4, 11

Τ Taurocholate, binding Tetracyclines Tissue lipoprotein lipase activity guar gum effects pectin effects

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

115 116 93-103 93-103

UNCONVENTIONAL SOURCES OF DIETARY FIBER

314 Tobacco fiber 251-64 ash content 256 bulk volume 256 calcium 262 cellulose quantity 256 essential minerals 256 iron 262 lignin quantity 256 mineral elements 253 nicotine contamination 254, 263 phytate content 262 protein calculations 253 sodium taurocholate binding 253 Tobacco plants, deproteinization 252 Total cholesterol, locust bean gum effects 77 Total pectin, definition 192 Total plasma cholesterol, from diets with and without cholestero Tragacanth carbohydrate absorption effects .... 18 effect on blood glucose 20/ Triglyceride levels effect of fiber additives Ill* psyllium effects 68 Triglyceride-lowering 56 Triglycerides, locust bean gum effects 80 Tumor formation, cause of inhibition 9 Tumorigenesis, promotional phase .... 9 Tumorigenic compounds, dilution in the colon 3 Tumors, induction 1

U Unabsorbable antibiotics Unconventional dietary fibers in colon carcinogenesis Uronic acid analysis, citrus wastes .... Uronic acid, in citrus wastes

116 1-16 209 214*

V Valencia washed orange pulp, compositional analysis Vegetable fibers Vitamin A Vitamin C

198* 4 196 196

W Washed orange pulp component composition sugar-free basis fiber particulate density Hamlin, fiber analysis nutrient analyses oil holding capacities origin pasteurization

191-203 195* 197* 201 199* 192 202* 191 193

Washed orange pulp—Continued pectin loss 201 structure 194/ sugar analyses 192 sugar value 196 utilization 201 Valencia, compositional analysis .... 198* water holding capacity 192, 202* Water holding capacity, wood cellulose 139 Water holding capacity studies 137 Water-insoluble polysaccharides, cholesterol-lowering effects 50 Water-soluble fibers, cholesterollowering effects 50 Water-soluble pectins, of citrus wastes 208

g cellulose quantity 256 effect on fecal bile acids 5 hypocholesterolemic effects 51 lignin quantity 256 Wheat bran diets colon tumor incidence 8, 8*, 10, 10* dimethylaminobiphenyl-induced tumors 12* effect on high-density lipoproteins .. 98/ effects on plasma cholesterol 98/ growth curves of rats 97/ high-density lipoproteins 101/ Wheat straw biodégradation products analysis of volatiles 271 high-molecular weight 280/ molecular weight distribution 278,280* separation 276* separation and recovery 270/ fermentation 269 labelling 269 low-molecular weight aldehydic phenols 273 methylated 279/ nitrobenzene oxidized 273 compounds 277/ separation of biodégradation products 269 Wheat straw biodégradation by Cyathus stercoreus 267-83 Wood cellulose copper adsorption 140 water holding capacity 139 Wood lignin 241-49 absorptivity 247 nitrosoamines adsorption 242-44 Wood lignin samples, preparation 247, 249

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

INDEX

Xylan acidic, methylation analysis composition in human digestion Xylan fibers

42* 225*, 232 221-38 232

Xylose absorption in citrus wastes

Zinc absorption

In Unconventional Sources of Dietary Fiber; Furda, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.