Chemistry and Function of Pectins 9780841209749, 9780841211452, 0-8412-0974-X

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
Terminology......Page 8
Structure......Page 9
Conformation......Page 10
Physical Properties......Page 11
Chemical Properties......Page 15
Literature Cited......Page 16
2 Analytical Methods for Determining Pectin Composition......Page 19
Quantitative Analysis of Galacturonic Acid in Pectin......Page 20
Determination of Methyl, Acetyl, and Feruloyl Substitution in Pectin......Page 23
Neutral Sugar Composition of Pectin......Page 24
Acknowledgment......Page 25
Literature Cited......Page 26
3 A Critical Reexamination of Molecular Weight and Dimensions for Citrus Pectins......Page 28
Experimental......Page 29
HPSEC......Page 30
Results and Discussion......Page 32
Literature Cited......Page 42
4 Structural Studies of Apple Pectins with Pectolytic Enzymes......Page 44
Neutral Sugar Distribution......Page 45
Distribution of Methoxylated and Free Carboxyl Groups......Page 50
Conclusion......Page 53
Literature Cited......Page 54
5 Sugar Beet Pectins: Chemical Structure and Gelation through Oxidative Coupling......Page 55
Experimental methods......Page 56
Results and discussion......Page 57
Outlook......Page 64
Literature cited......Page 66
6 Interactions of Counterions with Pectins Studied by Potentiometry and Circular Dichroism......Page 67
Characterization of the samples......Page 68
Characteristics of the samples......Page 69
Counterion activity coefficients......Page 70
Influence of the polymer concentration on calcium activity coefficient......Page 71
Circular dichroism measurements......Page 72
Conclusion......Page 74
Literature cited......Page 77
7 Ionic Effects on the Conformation, Equilibrium, Properties, and Rheology of Pectate in Aqueous Solutions and Gels......Page 79
pH-induced Conformational Transition......Page 80
Energetics (thermodynamics) of interaction with divalent ions......Page 81
Aggregation of pectate......Page 85
Rheology of mixed H+/Ca2+ pectate gels......Page 88
Experimental......Page 90
Literature Cited......Page 92
8 Pectin Internal Gel Strength: Theory, Measurement, and Methodology......Page 94
Factors Affecting Internal Gel Strength......Page 97
III. Instrumentation......Page 100
Nondestructive Tests......Page 102
Destructive Tests......Page 104
Acknowledgments......Page 105
Literature Cited......Page 106
Jelly Grade/SAG Method......Page 109
Present Status of the SAG Method......Page 110
Instrumental Methods......Page 111
Comparison of Various Methods for Pectin Grading......Page 114
Conclusion......Page 121
Literature Cited......Page 122
10 Synergistic Gelation of Alginates and Pectins......Page 123
MATERIALS AND METHODS......Page 124
RESULTS AND DISCUSSION......Page 125
Literature Cited......Page 137
11 Control of Pectin Synthesis and Deposition during Plant Cell Wall Growth......Page 139
Channel 1. Production and Movement of Nucleotide Sugar Donors......Page 140
Channel 2. Synthesis and Compartmentalization of the Pectin Polymers with in the Endomembrane System......Page 142
Channel 3. Movement of Vesicles and Fusion with the Plasmamembrane for Assembly and Deposition within the Wall......Page 143
Literature Cited......Page 144
12 Comparative Analysis of Pectins from Pericarp and Locular Gel in Developing Tomato Fruit......Page 146
Materials and Methods......Page 147
Results......Page 149
Discussion......Page 154
Literature Cited......Page 159
13 Polygalacturonases in Higher Plants......Page 162
Literature Cited......Page 178
14 Paramagnetic Ion Spin-Spin Coupling as Direct Evidence for Cooperative Ion Binding to Higher Plant Cell Walls......Page 180
Materials and Methods......Page 181
Results and Discussion......Page 182
Literature Cited......Page 191
15 Softening of Cooked Snap Beans and Other Vegetables in Relation to Pectins and Salts......Page 194
Effects of salts present before cooking......Page 195
Effects of salts present before or after cooking......Page 196
The effects of adding salts after cooking......Page 198
Addition of salts before cooking, corrected for Ca++ displacement......Page 199
Discussion......Page 201
Literature Cited......Page 202
Experimental Procedures......Page 204
Results......Page 208
Conclusions......Page 213
Literature Cited......Page 220
17 Pectin Methylation Changes and Calcium Ion Effects on the Texture of Fresh, Fermented, and Acidified Cucumbers......Page 221
Literature Cited......Page 232
18 Enzymic Lysis of Pectic Substances in Cell Walls: Some Implications for Fruit Juice Technology......Page 234
Apple juice......Page 235
Apricot nectar......Page 245
Literature cited......Page 250
19 Effects of Pectin on Human Metabolism......Page 252
Lipid Effects......Page 254
Glycemic Effects......Page 261
Vitamin Bioavailability......Page 264
Effect and Fate of Pectin in the Gastrointestinal Tract......Page 265
Conclusion......Page 266
Literature Cited......Page 267
Materials and Methods......Page 270
Results and Discussion......Page 272
Literature Cited......Page 278
Author Index......Page 279
A......Page 280
E......Page 281
J......Page 282
P......Page 283
R......Page 284
T......Page 285
W......Page 286

Citation preview

Chemistry and Function of Pectins

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

ACS

SYMPOSIUM

S E R I E S 310

Chemistry and Function of Pectins Marshall L. Fishman, EDITOR Agricultural Research Service

Josep Campbell Institute for Research and Technology

Developed from a symposium sponsored by the Division of Agricultural and Food Chemistry at the 189th Meeting of the American Chemical Society, Miami Beach, Florida, April 28-May 3, 1985

American Chemical Society, Washington, DC 1986

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

Library of Congress Cataloging-in-Publication Data Chemistry and function of pectins. (ACS symposium series, ISSN 0097-6156; 310) "Developed from a symposium sponsored by the Division of Agricultural and Food Chemistry at the 189th Meeting of the American Chemical Society, Miami Beach, Florida, April 28-May 3, 1985." Bibliography: p. Includes index. 1. Pectin—Congresses. I. Fishman, Marshall L., 1937 Joseph J., 1939. III. America Division of Agricultural and Foo Chemistry IV. Series. TP248.P4C48 1986 ISBN 0-8412-0974-X

664'.25

86-7983

Copyright © 1986 American Chemical Society All Rights Reserved. The appearance of the code at the bottom of the first page of each chapter in this volume indicates the copyright owner's consent that reprographic copies of the chapter 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., 27 Congress Street, Salem, M A 01970, 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 a 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. Registered names, trademarks, etc., used in this publication, even without specific indication thereof, are not to be considered unprotected by law. P R I N T E D IN T H E U N I T E D STATES OF A M E R I C A

American Chemical Society Library 1155 16th St., N.W.

In Chemistry and Function of Pectins; Fishman, M., et al.; D.C.Society: 20036 ACS Symposium Series; Washington, American Chemical Washington, DC, 1986.

ACS Symposium Series M . Joan Comstock, Series Editor Advisory Board Harvey W. Blanch

Donald E. Moreland

University of California—Berkeley

USDA, Agricultural Research Service

Alan Elzerman Clemson University

J. T. Baker Chemical Company

John W. Finley

James C. Randall

Nabisco Brands, Inc.

Exxon Chemical Company

Marye Anne Fox

W. D. Shults

The University of Texas—Austin

Oak Ridge National Laboratory

Martin L. Gorbaty

Geoffrey K. Smith

Exxon Research and Engineering Co.

Rohm & Haas Co.

Roland F. Hirsch

Charles S.Tuesday

U.S. Department of Energy

General Motors Research Laboratory

Rudolph J. Marcus

Douglas B. Walters

Consultant, Computers & Chemistry Research

National Institute of Environmental Health

Vincent D. McGinniss

C. Grant Willson

Battelle Columbus Laboratories

IBM Research Department

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

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-read the supervision of th 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, because symposia may embrace both types of presentation.

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

PREFACE THE PHYSICAL, BIOCHEMICAL, AND FUNCTIONAL PROPERTIES of

pectin

are of great interest to a diverse cross section of scientists and technologists because pectin can be classified as a polyelectrolyte, a complex polysaccharide, an important food fiber, a major plant cell wall component, and a ubiquitous nutritional factor and gelling agent in foods. An explosion in pectin research is evidenced by the more than 200 references cited in 1983 and 1984. Nevertheless, a appeared in over 10 years assemble chapters from leading scientists and technologists in the pectin research field to produce a state-of-the-art multidisciplinary book that will advance pectin research through a cross fertilization of sound research ideas. In any book of this nature, some duplication as well as contradictory interpretations of the same phenomena will occur. These problems should provide the impetus for new and continued research leading to a better understanding of the chemical and functional properties of pectins. Although we tried very hard to spread evenly the coverage of every aspect of pectin research in this book, unavoidably some areas are not covered adequately. We thank the authors for their excellent oral presentations at the symposium and their cooperation in completing the written manuscript in a timely manner. We also thank the reviewers for their incisive and constructive criticism. We sincerely hope the next book on pectin research does not have to wait 10 years to appear in print.

MARSHALLL.FISHMAN Agricultural Research Service North Atlantic Area Eastern Regional Research Center U.S. Department of Agriculture Wyndmoor, PA 19118

JOSEPH J. JEN Campbell Institute for Research and Technology Campbell Place Camden, NJ 08101 January 1986 ix In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

1 An Introduction to Pectins: Structure and Properties James N. BeMiller Department of Chemistry and Biochemistry, Southern Illinois University at Carbondale, Carbondale, IL 62901

Pectin, a polysaccharide, is composed primarily of essentially linear polymers of D-galactopyranosyl­ uronic acid unit linkages; the polyme various degrees with methanol. This regular structure is interrupted, however, with L-rhamnopyranosyl units and with side chains containing other neutral sugars. The polymer chains may also be partially acetylated. The most important physical property of pectin is its ability to form spreadable gels. Gel formation results when the polymer chains interact over a portion of their length to form a three-dimensional network. This aggregation of chains occurs through hydrogen bonding, divalent cation crossbridging, and/or hydrophobic interactions. Terminology I n 1944, d e f i n i t i o n s f o r p e c t i n s were e s t a b l i s h e d by t h e Committee f o r t h e R e v i s i o n o f t h e Nomenclature o f P e c t i c Substances O ) ; b u t s i n c e t h e n , t e r m i n o l o g y has changed somewhat, and m o d i f i e d d e f i n i t i o n s have been used. A t t h i s t i m e , t h e r e a r e no u n i v e r s a l l y agreed upon and a c c e p t e d d e f i n i t i o n s . The d e f i n i t i o n s presented here a r e t h o s e g e n e r a l l y i n c u r r e n t commercial use (2). P e c t i c acids are galacturonoglycans [poly(a-Dg a l a c t o p y r a n o s y l u r o n i c a c i d s ) ] w i t h o u t , o r w i t h oTTly a n e g l i g i b l e c o n t e n t o f , methyl e s t e r groups. P e c t i c a c i d s may have v a r y i n g degrees o f n e u t r a l i z a t i o n . S a l t s o f p e c t i c a c i d s a r e c a l l e d pectates. P e c t i n i c acids are galacturonoglycans with various, but g r e a t e r t h a n n e g l i g i b l e , c o n t e n t s o f m e t h y l e s t e r groups. P e c t i n i c a c i d s may have v a r y i n g degrees o f n e u t r a l i z a t i o n . S a l t s of p e c t i n i c a c i d s a r e c a l l e d p e c t i n a t e s . 0097-6156/ 86/0310-0002$06.00/0 © 1986 American Chemical Society

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

1.

BEMILLER

Pectins: Structure and Properties

P e c t i n s a r e m i x t u r e s o f p o l y s a c c h a r i d e s t h a t o r i g i n a t e from p l a n t s , c o n t a i n p e c t i n i c a c i d s as major components, a r e water s o l u b l e , and a r e a b l e t o form g e l s under s u i t a b l e c o n d i t i o n s (See s e c t i o n on P h y s i c a l P r o p e r t i e s ) . I n t h i s c h a p t e r , as i s f r e q u e n t l y done, t h e term p e c t i n w i l l be used i n a g e n e r i c sense t o d e s i g n a t e t h o s e w a t e r - s o l u b l e g a l a c t u r o n o g l y c a n s o f v a r y i n g methyl e s t e r c o n t e n t and degree o f n e u t r a l i z a t i o n t h a t a r e capable o f f o r m i n g g e l s under s u i t a b l e c o n d i t i o n s (See s e c t i o n on P h y s i c a l P r o p e r t i e s ) , i . e . , o t h e r p o l y s a c c h a r i d e s t h a t may be p r e s e n t i n commercial m i x t u r e s w i l l be ignored. P e c t i n s a r e s u b d i v i d e d a c c o r d i n g t o t h e i r degree o f e s t e r i f i c a t i o n (DE), a d e s i g n a t i o n o f t h e p e r c e n t o f c a r b o x y l groups e s t e r i f i e d w i t h methanol. P e c t i n s w i t h DE > 50? a r e high-methoxyl p e c t i n s ( H M - p e c t i n s ) ; t h o s e w i t h DE < 50? a r e low-methoxyl p e c t i n s ( L M - p e c t i n s ) The degree of a m i d a t i o c a r b o x y l groups i n t h e Properties). Structure For a more d e t a i l e d d i s c u s s i o n o f t h e c h e m i c a l s t r u c t u r e o f p e c t i n s , see r e f e r e n c e 3. P e c t i n , a s t r u c t u r a l , c e l l - w a l l polysaccharide of a l l higher p l a n t s , l i k e most o t h e r p o l y s a c c h a r i d e s , i s b o t h p o l y m o l e c u l a r and p o l y d i s p e r s e , i . e . , i t i s heterogeneous w i t h r e s p e c t t o b o t h c h e m i c a l s t r u c t u r e and m o l e c u l a r weight (jjO. From m o l e c u l e t o m o l e c u l e , i n any sample o f p e c t i n , b o t h t h e number and percentage of i n d i v i d u a l monomeric u n i t t y p e s w i l l v a r y , and t h e average c o m p o s i t i o n and d i s t r i b u t i o n o f m o l e c u l a r w e i g h t s can v a r y w i t h the s o u r c e , t h e c o n d i t i o n s used f o r i s o l a t i o n , and any subsequent t r e a t m e n t s . Because both parameters determine p h y s i c a l p r o p e r t i e s , v a r i o u s f u n c t i o n a l t y p e s o f p e c t i n can be produced by c o n t r o l l i n g t h e s o u r c e , i s o l a t i o n p r o c e d u r e , and subsequent treatment(s) P e c t i n i s p r i m a r i l y a polymer o f D - g a l a c t u r o n i c a c i d . The p r i n c i p a l and key f e a t u r e o f a l l p e c t i n m o l e c u l e s i s a l i n e a r chain of (1+4)-linked a-D-galactopyranosyluronic a c i d u n i t s , making i t an a-D-galacturonan [ a p o l y ( a - D - g a l a c t o p y r a n o s y l u r o n i c a c i d ) or an a - D - g a l a c t u r o n o g l y c a n ] . ~" I n a l l n a t u r a l p e c t i n s , some o f t h e c a r b o x y l groups a r e i n the methyl e s t e r form. Depending on t h e i s o l a t i o n c o n d i t i o n s , t h e r e m a i n i n g f r e e c a r b o x y l i c a c i d groups may be p a r t l y o r f u l l y n e u t r a l i z e d , i . e . , p a r t l y o r f u l l y p r e s e n t as sodium, potassium o r ammonium c a r b o x y l a t e groups. The r a t i o o f e s t e r i f i e d D-galacturonic acid units t o t o t a l D-galacturonic a c i d units i s "Sailed t h e degree o f e s t e r i f i c a t i o n " " ( D E ) and s t r o n g l y i n f l u e n c e s the s o l u b i l i t y , g e l f o r m i n g a b i l i t y , c o n d i t i o n s r e q u i r e d f o r g e l a t i o n , g e l l i n g temperature, and g e l p r o p e r t i e s o f t h e preparation. I n p e c t i n from some s o u r c e s , some o f t h e u n i t s occur as 0-2 o r 0-3 a c e t a t e s . Such e s t e r i f i c a t i o n hampers g e l a t i o n , so much so t h a t complete i n h i b i t i o n o f g e l a t i o n o c c u r s when one out o f e i g h t

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

3

4

CHEMISTRY AND

FUNCTION OF

PECTINS

D - g a l a c t o p y r a n o s y l u r o n i c a c i d u n i t s are m o n o e s t e r i f i e d w i t h a c e t i c a c i d at 0-2 or 0 - 3 . The presence of a c e t y l groups, t h e r e f o r e , makes c e r t a i n p o t e n t i a l s o u r c e s of commercial p e c t i n , e.g., sugar beet ( 5 ) , s u n f l o w e r , and p o t a t o , l e s s d e s i r a b l e . N e u t r a l s u g a r s , p r i m a r i l y L-rhamnose, are a l s o p r e s e n t . However, t h e r e i s disagreement over t h e i r d i s t r i b u t i o n i n the l i n e a r c h a i n . One group of workers (6) r e p o r t e d t h a t , i n c i t r u s , a p p l e , and s u n f l o w e r p e c t i n s , L-rhamnopyranosyl u n i t s are more or l e s s e v e n l y i n s e r t e d i n t o t h e g a l a c t u r o n a n c h a i n i n the f o l l o w i n g manner: 0-a-D-GalpA-(1+2)-0-L-Rhap-(1+4)-0-a-D-GalpA. The c o n f i g u r a t i o n " " o f the L-rhamnopyranosyl l i n k a g e " i s unknown, but c a l c u l a t i o n s have shown t h a t i t s h o u l d be b e t a i n o r d e r t o p r o v i d e the n e c e s s a r y degree of k i n k i n g i n the s t r u c t u r e (6) (See s e c t i o n on P h y s i c a l P r o p e r t i e s ) . Another group (7) r e p o r t e d t h a t the L-rhamnopyranosyl u n i t s are q u i t e unevenly d i s t r i b u t e d w i t h i n t h e ~ g a l a c t u r o n a n backbone. These workers r e p o r t (8 regions [poly(a-D-galactopyranosyluroni " h a i r y " r e g i o n s . " A c c o r d i n g to them, the l a t t e r r e g i o n s c o n s i s t of rhamnogalacturonan sequences t h a t c o n t a i n h i g h l y branched a r a b i n o g a l a c t a n s i d e c h a i n s and g a l a c t u r o n a n sequences w i t h s h o r t s i d e c h a i n s composed of D - x y l o s e . The same may a l s o be t r u e of sugar beet p e c t i n ( 5 ) . The d a t a of the former group (9) a l s o i n d i c a t e s t h a t , however the L-rhamnopyranosyl u n i t s o c c u r i n the c h a i n , the l e n g t h of the p o l y T a - D - g a l a c t o p y r a n o s y l u r o n i c a c i d ) sequences between L-rhamriopyranose i n t e r r u p t i o n s (whether s i n g l e u n i t s or b l o c k s of u n i t s ) i s r a t h e r c o n s t a n t and t h a t the sequences are about 25 u n i t s l o n g i n each p e c t i n ( c i t r u s , a p p l e , s u n f l o w e r ) s t u d i e d . The t o t a l c o n t e n t of n e u t r a l sugars v a r i e s w i t h t h e s o u r c e , the e x t r a c t i o n c o n d i t i o n s , and subsequent t r e a t m e n t s . F e r u l i c a c i d i s e s t e r i f i e d t o the n e u t r a l sugar s i d e c h a i n s of p e c t i n from s p i n a c h (j_0) and sugar beet (5,11). Conformation G e l s are formed when polymer m o l e c u l e s i n t e r a c t over a p o r t i o n of t h e i r l e n g t h t o form a network t h a t e n t r a p s s o l v e n t and s o l u t e m o l e c u l e s . The j u n c t i o n zones t h a t r e s u l t from t h e s e c h a i n i n t e r a c t i o n s must be of l i m i t e d s i z e . I f they are too l a r g e , a p r e c i p i t a t e , r a t h e r than a g e l , r e s u l t s . The i n s e r t e d L-rhamnopyranosyl u n i t s may p r o v i d e the n e c e s s a r y i r r e g u l a r i t i e s T k i n k s ) i n the s t r u c t u r e and l i m i t the s i z e of the j u n c t i o n zones. The presence of " h a i r y " r e g i o n s may a l s o be a f a c t o r t h a t l i m i t s the e x t e n t of c h a i n a s s o c i a t i o n . As w i l l be d i s c u s s e d f u r t h e r i n the s e c t i o n on P h y s i c a l P r o p e r t i e s , j u n c t i o n zones are formed between r e g u l a r , unbranched p e c t i n c h a i n s when the n e g a t i v e charges on the c a r b o x y l a t e groups are removed ( a d d i t i o n of a c i d ) , h y d r a t i o n of the m o l e c u l e s i s reduced ( a d d i t i o n of a c o s o l u t e ) , and/or polymer c h a i n s are b r i d g e d by d i v a l e n t c a t i o n s ( c a l c i u m i ons), A n a l y s i s of p r o t o n n.m.r. s p e c t r a and m a t h e m a t i c a l model b u i l d i n g suggests that i n d i v i d u a l a-D-galactopyranosyluronic a c i d u n i t s have the ^CK ln(Y) = a + a- Κ 1 av ' 0 1 av v

(1)

n

2

Κ gn

(R

a n d

gw

z-average,

), r a d i i of gyration. These are defined by equations

R

4-6

= Σ C. / Σ (C./R ·) · ι . ι' g i

(*)

y

gn

Ί

R gw = Σ. C.ι R g i. / Σ. C R

(5)

2

= Σ C. R . / Σ C. R ι gi ι gi

gz

±

(6)

i

Where R ^ i s the radius of gyration of species i and

i s i t s con­

centration. For each DM, R

< R < R , which i s the expected order, gn gw gz' I n t e r e s t i n g l y , f o r the radius of gyration at peak p o s i t i o n ( R ) r

gp

ό

(maximum concentration of p e c t i n ) , R

< R < R f o r Rgp < 135 A gn gP gw > 247 A. Typical chromatograms are 6 r

whereas R

< R

gw gp shown i n F i g . 3.

< R

gz

for R

Q

gp

y v

As indicated by Table I , pooling data without regard to column type, s a l t concentration i n the mobile phase, or p e c t i n form gave data with a standard deviation ranging from 3 - 15% of the mean f o r the number-, weight- or Z-average R^.

In case of the number average

radius of gyration, the standard deviation ranged from 3.5 - 10%. Since, i t was our i n t e n t i o n to compare molecular weight and size values from SEC with those from end group t i t r a t i o n s , and osmometry, (comparisons of log R

gn

reduced the heterogenity i n variance (16))

values were analyzed for variance at the (p^ 0.05) confidence l e v e l . Such analysis was used to determine i f R^ values were affected s i g n i ­ f i c a n t l y by: (1) the concentration of s a l t i n the mobile phase; (2) whether the form of the carboxylate ion was hydrogen or sodium; (3) the pore size d i s t r i b u t i o n (psd) of the HPSEC columns.

No global

trends were i d e n t i f i e d because of interactions between combinations

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

Molecular Weight and Dimensions for Citrus Pectins

3. FISHMANETAL.

-1.0

29

-0.

Figure 3. Typical chromotograms f o r pectins with 0, 37 and 70% methylation e s t e r f i c a t i o n .

ο

Λ

TABLE I RADIUS OF GYRATION (R , A) FOR PECTINS

1

\ R NUMBER-AVERAGE

WEIGHT-AVERAGE

Z-AVERAGE

PEAK P0S.

0

51.7 + 2.2

65.2 + 4.1

84.5 + 11

53.7 ± 4.2

35

99.6 + 3.8

144 + 3.0

212 + 7.8

37

70.5 + 2.2

105 + 2.9

165 + 12

81.5 ± 8.2

57

131 + 9.4

207 + 17

309 + 40

258 ± 28

58-60

124 + 4.3

194 + 4.5

288 + 11

252 ± 33

70

126 + 6.0

201 + 10

301 + 12

247 ± 35

123 + 12

206 + 11

314 + 12

292 ± 35

101 + 3.3

149 + 4.0

225 + 15

135 ± 31

72-73 73G

â

123 ± 11

1

DATA AVERAGED OVER MOBILE PHASE CONCENTRATION (0.05M & 0.1M NaCl), OVER FORM (ACID & NEUTRALIZED) AND OVER COLUMN (E-1000 AND E-LINEAR) 24 DETERMINATIONS.

2

STANDARD DEVIATION OF POOLED DATA AS IN (1).

3

DATA FOR E-1000 COLUMN ONLY (12 DETERMINATIONS).

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

2

30

CHEMISTRY AND FUNCTION OF PECTINS

of mobile phase concentration, carboxylate counter i o n and column psd.

In another approach, a t constant degree of methylation, R

g n

values were separated at the (P^ 0.05) confidence l e v e l through differences i n t h e i r logs by the Bonferroni LSD method (17). within any horizontal row i n

Thus

T a b l e I I , means not f o l l o w e d by the

same l e t t e r were s i g n i f i c a n t l y d i f f e r e n t a t the (p Î CMOO>OOCMCOί H O C 0 H L O 0 0 r ^ r H r H r H r - » r H

cnNiHooHcOpCiai VO

·

(ooomoooNooHoo ο

Ο pu

U

Ό

ν ο ο ο ν ο U Ό Ό «î «J • · . oo m m ΓΗ οο > ί ο ο σ ι θ Η θ Μ Η ^ C T i U O r H r H r H r H r H

u ΡΩ

vu

t-H*

u 0>

PQ

^ ι η ι \o σι H H m

CO Γ—

Ο

ο m Γ** ι ο ι co co co m oo r- CM

§

Β » H

rH > j 0) w ! 00 Q ,

'S

1

βI W Ο Q M ι I IH H i ^ ÎH CO I Λ i

sa H H O ' « I Ο I EH W CM

H 2 0 ) . HM p e c t i n s w i l l form g e l s i n the p r e s e n c e of sugar and a c i d and a r e f u r t h e r d i v i d e d i n t o broad c a t e g o r i e s o f r a p i d s e t p e c t i n s , 75 to 72% DM, which g e l i n 20 t o 70 s e c ; medium s e t p e c t i n s , 71 t o 68% DM, which g e l i n 100 t o 150 s e c ; and slow s e t p e c t i n s , 66 t o 62% DM, which g e l i n 180 t o 250 s e c (Table I) ( 8 ) . y

T a b l e I . C a t e g o r i e s of H i g h M e t h o x y l P e c t i n ( C i t r u s ) w i t h Approximate Degree of M e t h y l a t i o n , S e t t i n g Times, and Temperatures

Degree o f methylation Ultra rapid Rapid s e t Medium s e t Slow s e t Reference

>75 75 71 66 -

Setting time (sec)

— 72 68 62

20 100 180 -

Setting temperature (°C)

— 70 150 250

97 92 83 -

95 87 72

(8)

T h i s r e v i e w w i l l d i s c u s s the needs o f the j e l l y m a n u f a c t u r e r to measure g e l s t r e n g t h , the t h e o r y o f g e l f o r m a t i o n , f a c t o r s a f f e c t i n g g e l s t r e n g t h , and i n s t r u m e n t a t i o n used t o measure g e l s t r e n g t h . J e l l y M a n u f a c t u r e r ' s Needs. D u r i n g the f i r s t h a l f o f t h i s c e n t u r y , t h e r e was no u n i f o r m method to measure the a b i l i t y o f p e c t i n t o form a g e l . C o n s e q u e n t l y , j e l l y m a n u f a c t u r e r s had t o c o n s t a n t l y a d j u s t the amount o f p e c t i n used per b a t c h o f j e l l y w i t h l i t t l e a s s u r a n c e t h a t the appearance and t e x t u r e o f the f i n i s h e d p r o d u c t would be o f a h i g h q u a l i t y . A f t e r the a d o p t i o n of the I n s t i t u t e o f Food T e c h n o l o g i s t ' s (IFT-SAG) method i n 1959, a l l p e c t i n s were s t a n d a r d i z e d on t h e i r a b i l i t y t o form a g e l . T h i s method has been used by p e c t i n and j e l l y m a n u f a c t u r e r s f o r more t h a n 25 y e a r s . The d e t a i l s are d i s c u s s e d l a t e r i n t h i s chapter. A l t h o u g h the IFT-SAG method has been the p e c t i n g r a d i n g s t a n d a r d , i t has some

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

90

CHEMISTRY AND FUNCTION OF PECTINS

d e f i c i e n c i e s i n measuring i n t e r n a l g e l s t r e n g t h . So, m a n u f a c t u r e r s a r e l o o k i n g f o r a s u p p l e m e n t a l method f o r p e c t i n e v a l u a t i o n which would c o r r e l a t e w e l l w i t h s p r e a d a b i l i t y , be i n c l o s e agreement w i t h commercial p r a c t i c e , and g i v e r e p r o d u c i b l e r e s u l t s . The equipment s h o u l d be d u r a b l e , o p e r a t o r i n d e p e n d e n t , and i n e x p e n s i v e . Ideally, the p e c t i n s h o u l d be added t o t h e j e l l y t e s t b a t c h , a s i n commercial o p e r a t i o n s , so i t w i l l be i n c o n t a c t w i t h t h e a c i d and sugar d u r i n g the l a s t phase o f c o o k i n g . I f t h e t e s t were r e l a t i v e l y r a p i d , i t c o u l d p r o v i d e r e a l time feedback f o r c o n t r o l o f t h e next b a t c h o f jelly. F i n a l l y , t h e t e s t method s h o u l d r e l a t e t o t h e consumer's p e r c e p t i o n o f t e x t u r e , be i n a c c o r d a n c e w i t h t h e t h e o r y o f g e l f o r m a t i o n , and be based on fundamental t e s t p r i n c i p l e s . Theory o f G e l F o r m a t i o n . I t i s important t o understand the f o r c e s t h a t s t a b i l i z e a p e c t i n g e l s t r u c t u r e and t h e mechanisms o f p e c t i n g e l a t i o n b e f o r e examining t h e i n s t r u m e n t s used t o measure i n t e r n a l gel strength. P e c t i n wa c o l l o i d a l polygalacturoni o f m e t h y l e s t e r groups and h a v i n g c a p a b i l i t i e s o f f o r m i n g g e l s w i t h sugar and a c i d . P e c t i n i c a c i d s a r e composed p r i m a r i l y o f α 1,4 linked D-galacturonic acid u n i t s . In p l a n t s , p e c t i n i s thought o f as a m i x t u r e o f s t r u c t u r a l , c a r b o h y d r a t e m o l e c u l e s w i t h a g e n e r a l i z e d r a t h e r than s p e c i f i c c o m p o s i t i o n . P e c t i n s have a l o c a l i z e d d i s t r i b u t i o n o f c o v a l e n t l y attached n e u t r a l sugars forming h a i r y and smooth r e g i o n s ( 2 1 ) . Rees (22) p r o p o s e d f o u r l e v e l s o f s t r u c t u r e f o r p o l y s a c c h a r i d e s like pectin. P r i m a r y s t r u c t u r e r e f e r s t o t h e n a t u r e and mode o f l i n k a g e o f t h e component g a l a c t u r o n i c a c i d s . In p e c t i n , p o l y - D - g a l a c t u r o n a t e forms a r e g u l a r , b u c k l e d , t w o - f o l d c o n f o r m a t i o n (23) e x c e p t where a k i n k i s formed by t h e i n s e r t i o n o f L-rhamnose o r another sugar. Secondary s t r u c t u r e r e f e r s t o the p y r a n o s e r i n g shapes. N e l s o n ( 2 0 ) reviewed t h e e v i d e n c e t h a t t h e 4 - C - l c o n f o r m a t i o n i s f a v o r e d due t o m i n i m i z a t i o n o f t h e s t e r i c r e p u l s i o n between a x i a l s u b s t i t u e n t s . The r e s t r i c t i v e r o t a t i o n a l a n g l e s (anomeric c a r b o n one t o pendent oxygen) and (carbon four t o pendent oxygen) d e t e r m i n e t h e o v e r a l l c o n f o r m a t i o n o f t h e polysaccharide chain. T e r t i a r y s t r u c t u r e o f p e c t i n i s the s p e c i f i c , r i g i d r o d - l i k e geometry which i s f a v o r e d by n o n c o v a l e n t i n t e r a c t i o n s , r i g i d s e c o n d a r y s t r u c t u r e , e f f i c i e n t p a c k i n g , and i s i n h i b i t e d by l o s s o f c o n f o r m a t i o n a l energy, h y d r a t i o n , i n t e r m o l e c u l a r e l e c t r o s t a t i c r e p u l s i o n , and s t r u c t u r a l irregularities. T e r t i a r y s t r u c t u r e s may remain i n t a c t even a f t e r h y d r a t i o n i n s o l u t i o n o r i n a g e l network. Quaternary s t r u c t u r e i s the i n t e r a c t i o n o f s p e c i f i c , r i g i d u n i t s o f t e r t i a r y s t r u c t u r e t o form a h i g h e r l e v e l o f o r g a n i z a t i o n ( 2 2 ) . S t u d i e s on t h e f o r c e s t h a t s t a b i l i z e t h e p e c t i n g e l network have c o n t r i b u t e d t o o u r u n d e r s t a n d i n g o f t h e mechanism o f g e l a t i o n . Owens and Maclay ( 2 4 ) r e p o r t e d t h a t hydrogen bonding between h y d r o x y l s o f p e c t i n , water, and sugar was promoted by a d e c r e a s e i n pH, due t o a d e c r e a s e i n i n t e r m o l e c u l a r , e l e c t r o s t a t i c r e p u l s i o n . In a d d i t i o n , weak v a n d e r Waals f o r c e s between m e t h y l e s t e r groups c o n t r i b u t e t o t h e s t a b i l i z a t i o n o f t h e g e l network ( 2 4 ) . McCready and Owens (25) and M o r r i s e t a l . (26) proposed t h a t e s t e r groups make a p o s i t i v e c o n t r i b u t i o n t o i n t e r c h a i n a s s o c i a t i o n . M o r r i s e t a l . (26) o b s e r v e d a d e c r e a s e i n g e l s t r e n g t h o f 72% e s t e r p e c t i n

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

8.

C R A N D A L L AND

WICKER

Pectin Internal Gel Strength

91

g e l s i n the presence o f 8M u r e a and proposed t h a t the g e l network i s s t a b i l i z e d by n o n c o v a l e n t f o r c e s analogous t o f o r c e s s t a b i l i z i n g tertiary structure i n proteins. Doesburg (27) d i s c u s s e d the phenomenon of g e l a t i o n and d e f i n e d g e l s as two-phase systems w i t h a d i s c o n t i n u o u s phase o f s o l i d m a t e r i a l which r e s t r i c t s a f i n e l y d i s p e r s e d o r c o n t i n u o u s aqueous phase. N e l s o n (20) proposed a g e l model i n which the p e c t i n m o l e c u l e s were random r i b b o n s i n the s o l state,- p r o g r e s s i n g t o l e s s random r i b b o n s o f lower water a c t i v i t y i n the second s t a t e , and were a g e l i n the t h i r d s t a t e where most of the p e c t i n c h a i n s were i n v o l v e d i n some t y p e o f c h a i n s t a c k i n g . In a HM p e c t i n g e l , a c i d , s u g a r , w a t e r , and p e c t i n i n t e r a c t t o form a g e l . At lower pH v a l u e s near pH 3, e l e c t r o s t a t i c r e p u l s i o n i s m i n i m i z e d due t o the g r e a t e r number o f n o n i o n i z e d c a r b o x y l groups which enhance the p r o b a b i l i t y of n o n c o v a l e n t a t t r a c t i o n among the m e t h o x y l , a l c o h o l , and c a r b o x y l groups ( 2 4 ) . The f u n c t i o n o f sugar has been l e s s c l e a r I t has been suggested t h a t the promotes hydrogen bondin Rees (30) suggested t h a t sugar c o n t r o l l e d the water a c t i v i t y . F u r t h e r p r o g r e s s towards u n d e r s t a n d i n g the i n t e r r e l a t i o n s h i p of s u g a r , a c i d , w a t e r , and HM p e c t i n has been made by O a k e n f u l l and S c o t t ( 3 1 ) . They proposed t h a t h y d r o p h o b i c i n t e r a c t i o n s a r e e s s e n t i a l t o g e l f o r m a t i o n i n HM p e c t i n . However, t h e c o n t r i b u t i o n of hydrogen bonding t o the s t a n d a r d f r e e energy o f f o r m a t i o n i s n e a r l y t w i c e t h a t o f h y d r o p h o b i c i n t e r a c t i o n s and hydrogen bonding a l o n e was not s u f f i c i e n t t o overcome the e n t r o p y b a r r i e r t o g e l a t i o n from the l o s s o f d i s o r d e r ( 3 1 ) . Even though s u c r o s e i n c r e a s e d h y d r o p h o b i c i n t e r a c t i o n s by 67% ( 3 2 ) , o t h e r p o l y o l s , such as s o r b i t o l were more e f f i c i e n t a t s t a b i l i z i n g h y d r o p h o b i c i n t e r a c t i o n s than s u c r o s e ( 3 2 ) . They f u r t h e r r e p o r t e d t h a t the f o r m a t i o n o f j u n c t i o n zones from 18 t o 250 g a l a c t u r o n i c a c i d u n i t s i n l e n g t h , between two p e c t i n m o l e c u l e s , s t a b i l i z e d the p e c t i n g e l network ( 3 2 , 33). The l e n g t h o f the j u n c t i o n zone depends on the h y d r o p h o b i c i n t e r a c t i o n s , w h i c h depend on the c o n c e n t r a t i o n of sugar o r p o l y o l and on the DM v a l u e o f p e c t i n ( 3 1 ) . C o n s i d e r a t i o n o f the g e l f o r m a t i o n f o r Ui p e c t i n s has been p u r p o s e l y o m i t t e d because of space l i m i t a t i o n s . R e f e r e n c e s h e l p f u l i n u n d e r s t a n d i n g the f o r m a t i o n o f LM g e l s a r e 2> 23, 27, 34, 35. Factors A f f e c t i n g I n t e r n a l Gel Strength S e v e r a l o f the f a c t o r s t h a t a f f e c t g e l s t r e n g t h w i l l be d i s c u s s e d such as pH, m o l e c u l a r w e i g h t , type o f p e c t i n , and c o n d i t i o n s used t o t e s t the g e l . The m a j o r i t y o f p e c t i n i s used i n the manufacture o f j e l l i e s and jams, so t h i s r e v i e w w i l l f o c u s on HM p e c t i n s . Jelly grade i s d e f i n e d as the number of grams o f sugar w i t h which one gram of p e c t i n w i l l form a 65% s o l u b l e s o l i d s g e l of s p e c i f i e d s t r e n g t h under s u i t a b l e a c i d c o n d i t i o n s ( 3 ) . O l l i v e r (36) p o i n t e d out two o m i s s i o n s i n t h i s d e f i n i t i o n . F i r s t , no r e f e r e n c e i s made t o a s t a n d a r d g e l s t r e n g t h f o r the " s e t " of the j e l l y . Second, from the d e f i n i t i o n , the amount o f sugar i s the v a r i a b l e ; however, i n p r a c t i c e , the sugar c o n c e n t r a t i o n i s h e l d c o n s t a n t a t 65% s o l u b l e s o l i d s ; t h e r e f o r e , i t i s the amount o f p e c t i n t h a t i s v a r i e d .

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

CHEMISTRY A N D FUNCTION OF

92

PECTINS

Pectin. The h e t e r o g e n e i t y o f p e c t i n i s d i f f i c u l t t o c o n t r o l d u r i n g m a n u f a c t u r e , not w e l l c h a r a c t e r i z e d , and i n f l u e n c e s g e l s t r e n g t h i n commercial j e l l y b a t c h e s . Pectin i s a naturally occurring p o l y s a c c h a r i d e which i s m a i n l y e x t r a c t e d from c i t r u s p e e l and a p p l e pomace. R e s e a r c h e r s have r e p o r t e d on d i f f e r e n c e s among the y i e l d and j e l l y grade o f p e c t i n s from v a r i o u s c i t r u s c u l t i v a r s (37, 38), between f r e s h and d r y c i t r u s p e e l ( 3 9 ) , and i n p e c t i n e x t r a c t i o n c o n d i t i o n s (40, 4 1 ) . F u r t h e r m o r e , t h e r e a r e d i f f e r e n c e s among a p p l e s t a r t i n g m a t e r i a l s (27, 42) and d i f f e r e n c e s between the r e s u l t a n t p e c t i n s made from c i t r u s and a p p l e ( 4 3 ) . In a d d i t i o n t o n a t u r a l v a r i a t i o n i n c o m m e r c i a l l y p r e p a r e d p e c t i n s , the DM o f p e c t i n i n f l u e n c e s i n t e r n a l g e l s t r e n g t h . The s e n s i t i v i t y o f p e c t i n t o d i v a l e n t c a t i o n s and the mechanism o f g e l f o r m a t i o n i s i n f l u e n c e d by the DM v a l u e . Furthermore, j e l l y grade can be i n c r e a s e d by d e c r e a s i n g the DM v a l u e by s a p o n i f i c a t i o n . Doesburg and G r e v e r s (44) showed t h a t the j e l l y grade was 25% h i g h e r i n a 53% DM p e c t i n than s t a r t i n g m a t e r i a l . Wile low DM p e c t i n w i t h a h i g h m o l e c u l a r weight (MW) which produces a g e l w i t h an e x t r e m e l y h i g h r u p t u r e s t r e n g t h . A d d i t i o n a l v a r i a b i l i t y e x i s t s because DM v a l u e s a r e heterogeneous among p e c t i n s . Walter and Sherman (46) found t h a t a t a s p e c i f i c DM v a l u e , the p h y s i c a l p r o p e r t i e s o f a HM p e c t i n a r e i n f l u e n c e d by the d i s p e r s i o n and l o c a t i o n o f f r e e c a r b o x y l s . Another HM p e c t i n w i t h a s i m i l a r DM, but d i f f e r e n t d i s t r i b u t i o n o f unmethoxylated c a r b o x y l s , p o s s e s s e d different physical characteristics. The DM i n t e r a c t s w i t h g e l pH t o d e t e r m i n e the maximum g e l s t r e n g t h measurement. In the t y p i c a l c u r v e shown i n F i g u r e 1, the optimum f i r m n e s s o c c u r s a t h i g h e r pH v a l u e s f o r h i g h e r DM p e c t i n s ·

M o l e c u l a r Weight. Swenson e t a l . (48) determined t h a t i n s t r u m e n t s used t o measure the b r e a k i n g s t r e n g t h o f p e c t i n g e l s a s s i g n a h i g h e r grade to h i g h l y p o l y m e r i z e d , h i g h e r MW p e c t i n s than do i n s t r u m e n t s measuring e l a s t i c i t y . C h r i s t e n s e n (49) s u p p o r t e d t h i s by showing t h a t the r e l a t i o n s h i p between b r e a k i n g s t r e n g t h and sag was not c o n s t a n t but depended on the MW o f the p e c t i n . P e c t i n s whose m o l e c u l a r w e i g h t s were near 135,000 were graded e q u a l l y by t h e s e two methods. When the MW was g r e a t e r than 135,000, the d e s t r u c t i v e measurements graded the p e c t i n s h i g h e r . M i t c h e l l and B l a n s h a r d (50) wrote p e c t i n g e l s have e i t h e r s h o r t and v e r y s t i f f o r l o n g e r and more f l e x i b l e c h a i n s . The e l a s t i c i t y modulus i s i n f l u e n c e d p r i m a r i l y by the s h o r t s t i f f c h a i n s and i s independent o f p e c t i n MW above a minimum. B r e a k i n g s t r e n g t h i s i n f l u e n c e d p r i m a r i l y by the l o n g e r , more f l e x i b l e c h a i n s which a r e l a r g e l y MW dependent and remain c r o s s l i n k e d a f t e r the s h o r t s t i f f c h a i n s have r u p t u r e d . M i t c h e l l (51, 52) a l s o found measurements o f the apparent e l a s t i c modulus a t s h o r t times ( l i k e the R i d g e l i m e t e r r e a d i n g ) t o be independent o f MW, above a c e r t a i n l i m i t i n g v a l u e . Thus, a p e c t i n w i t h a h i g h R i d g e l i m e t e r grade would be expected to have a g r e a t e r number of s h o r t s t i f f c h a i n s and a h i g h e r c r o s s l i n k d e n s i t y . This p r o p e r t y may be c o n t r o l l e d to a c e r t a i n e x t e n t by p e c t i n m a n u f a c t u r e r s and s h o u l d be s e l e c t e d f o r by p e c t i n u s e r s f o r products needing a h i g h e l a s t i c i t y c h a r a c t e r i s t i c .

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

8. CRANDALL AND WICKER

93

Pectin Internal Gel Strength

DEGREE OF METHYLATION

I

I

I

I

I

I

10

3.2

3.4

3.6

3.8

40

GEL pH F i g u r e 1. R e l a t i o n s h i p among g e l s t r e n g t h , degree o f m e t h y l a t i o n and g e l pH ( E h r l i c h , 7 ) .

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

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CHEMISTRY AND FUNCTION OF PECTINS

J e l l y pH. Another f a c t o r which i n f l u e n c e s t h e i n t e r n a l g e l s t r e n g t h i s the j e l l y pH. I n the l a b o r a t o r y , O l l i v e r (53) found pH d i f f e r e n c e s between 2.82 and 3.12 c o u l d cause a v a r i a t i o n i n j e l l y grade o f about 30%. The i n t e r a c t i o n s between j e l l y pH and g e l s t r e n g t h i s shown i n F i g u r e 1. A c i d s a r e used t o c o n t r o l the pH w i t h i n the g e l l i n g range. The optimium pH i s about 3.1 and 3.4 f o r slow s e t HM p e c t i n s and r a p i d s e t p e c t i n s , r e s p e c t i v e l y ( 4 7 ) . J e l l y Test C o n d i t i o n s . Other f a c t o r s a f f e c t i n g the i n t e r n a l g e l s t r e n g t h a r e t h e r a t e and d u r a t i o n o f b o i l i n g a t e s t j e l l y , s t a g e o f a c i d a d d i t i o n , r a t e o f c o o l i n g , time o f a g i n g , t y p e o f sugar used ( 5 3 , 5 4 ) , and the use o f b u f f e r s a l t s o r s y n t h e t i c f r u i t j u i c e s r a t h e r than water ( 4 3 ) . III.

Instrumentation

S e v e r a l good r e v i e w s hav measure p e c t i n ' s g e l s t r e n g t h ( 4 9 ) , Beltman and P i l n i k ( 5 5 ) , M i t c h e l l (51_, 5 2 ) , and Sherman ( 5 6 ) . Both M i t c h e l l (51) and Sherman (56) have c o n c i s e T a b l e s w h i c h l i s t about 20 i n s t r u m e n t s and g i v e t y p e s o f measurements w h i c h c a n be made. T a b l e I I g i v e s examples o f i n s t r u m e n t s used t o e v a l u a t e p e c t i n g e l s which w i l l be covered i n t h i s r e v i e w . I d e a l l y , i n s t r u m e n t a t i o n t o measure t h e i n t e r n a l g e l s t r e n g t h o f a p e c t i n g e l s h o u l d emulate t h e p e r c e p t i o n o f g e l s t r u c t u r e by the consumer. T h i s p e r c e p t i o n o f g e l s t r u c t u r e by the consumer i s an i n t e r a c t i o n o f s i g h t , t a s t e , and t e x t u r e . V i s u a l o b s e r v a t i o n s a r e combined w i t h the s e n s a t i o n o f t o u c h when a j e l l y i s c u t from a j a r and s p r e a d . I n the mouth, the g e l i s compressed, f r a c t u r e d , and t h e r u p t u r e d p a r t s s l i d e past each o t h e r ( 5 6 ) . Most p o l y s a c c h a r i d e g e l s are composed o f d i f f e r e n t t e x t u r a l p r o f i l e s . T h e r e f o r e , when a s e r i e s o f g e l s i s r a n k e d , the o r d e r c a n v a r y g r e a t l y depending on the weight the consumer p l a c e s on each i n d i v i d u a l a t t r i b u t e ( 5 7 ) . A s e n s o r y e v a l u a t i o n r a t i n g s c a l e has been developed f o r s e v e r a l g e l t e x t u r e a t t r i b u t e s ( 5 8 ) . Models have a l s o been developed t o r e l a t e s p r e a d a b i l i t y as b e i n g the i n v e r s e o f t h e f o r c e ( s h e a r s t r e s s ) e x e r t e d on the s u r f a c e o f a k n i f e ( 5 9 ) . G i v e n t h a t consumers use a wide range o f e v a l u a t i o n methods t o determine t h e t e x t u r e o f a g e l , i t i s e x t r e m e l y d i f f i c u l t t o r e l a t e a l l o f the p e r c e i v e d t e x t u r a l c h a r a c t e r i s t i c s t o a s i n g l e instrument. Sherman (56) and M i t c h e l l (51_) have p o i n t e d o u t t h a t d e s p i t e t h e v a s t range o f t e x t u r a l - i n s t r u m e n t s , s a t i s f a c t o r y i n s t r u m e n t s are n o t yet a v a i l a b l e t o measure a l l t e x t u r a l p r o p e r t i e s . The e l a s t i c modulus i s d e f i n e d as the r a t i o o f s t r e s s t o s t r a i n . Stress i s the f o r c e p e r u n i t a r e a p r o d u c i n g the change i n shape and s t r a i n i s t h e change i n shape o r change i n l e n g h p e r u n i t l e n g t h o f t h e sample (3). I f the s t r a i n (displacement) i s s m a l l , i t i s e s s e n t i a l l y l i n e a r t o s t r e s s ( a p p l i e d f o r c e ) and the g e l w i l l resume i t s o r i g i n a l shape a f t e r t h e f o r c e i s removed. The e l a s t i c l i m i t o f t h e g e l i s t h e l a r g e s t amount o f d e f o r m a t i o n t h a t an e l a s t i c body c a n e x p e r i e n c e and s t i l l r e g a i n i t s o r i g i n a l shape a f t e r removing t h e force. Creep c o m p l i a n c e i s s i m i l a r t o e l a s t i c i t y except a c o n s t a n t f o r c e ( s t r e s s ) i s a p p l i e d t o the g e l and the d e f o r m a t i o n ( s t r a i n ) i s f o l l o w e d over t i m e . A l t e r n a t i v e l y , s t r e s s r e l a x a t i o n c a n be used

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

CRANDALL AND WICKER

Table

II.

Examples o f i n s t r u m e n t s Stress applied to g e l

Type o f instrument I.

Non-destructive

A.

IFT-SAG

B.

Wageningen

sag

95

Pectin Internal Gel Strength

used t o examine p e c t i n g e l s Parameter measured

Price range

force of gravity

percent sag

inexpensive

force of gravity

amount o f sag m a g n i f i e d 10 f o l d

inexpensive

inexpensive

C.

F.I.R.A.

torquing blade immersed i l

amount o f wate t mak

D.

Concentric cylinders

torquing corrugated cylinders

constant inexpensive stress/strain to c r e e p compliance expensive stress relaxation

E.

Parallel plates

sliding corrugated plates

strain c r e e p compliance

inexpensive to expensive

F.

Dynamic

oscillator

change i n velocity of waves

inexpensive to expensive

II. Destructive A.

Finger

p r e s s u r e between thumb and forefinger

relative force t o break g e l

inexpensive

B.

Tarr-Baker

syringe piston p r e s s i n g on gel's surface

relative amount o f heavy l i q u i d to rupture g e l

inexpensive

C.

LuersLochmuller

pulling figure from a g e l

weight t o rupture g e l

inexpensive

D.

HerbstreithPektinometer

pulling figure from a g e l

force to rupture g e l

moderately expensive

f i x t u r e s used i n either compression o r t e n s i o n mode

force peak h e i g h t

expensive

E.

Instron

References

(51, 56)

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

96

CHEMISTRY AND

FUNCTION OF PECTINS

where a c o n s t a n t s t r a i n i s a p p l i e d and the s t r e s s r e q u i r e d t o m a i n t a i n a c o n s t a n t s t r a i n o v e r time i s measured. A d d i t i o n a l background i s found i n r e p o r t s by M i t c h e l l (52, 60). Nondestructive vs. Destructive Tests. The p e c t i n grade depends somewhat on the e v a l u a t i o n method (6J_). Gel strength i n s t r u m e n t a t i o n may be n o n d e s t r u c t i v e which measures the e l a s t i c p r o p e r t i e s of a g e l , o r d e s t r u c t i v e which measures the i n e l a s t i c o r b r e a k i n g s t r e n g t h o f a g e l . A major p e c t i n p r o d u c e r u n s u c c e s s f u l l y t r i e d s e v e r a l i n s t r u m e n t s and methods f o r more than 20 y e a r s t o measure b o t h e l a s t i c i t y and r u p t u r e s t r e n g t h w i t h a s i n g l e t e s t

(62). N o n d e s t r u c t i v e t e s t s have c e r t a i n advantages i n c l u d i n g the a b i l i t y t o measure the e l a s t i c i t y of the g e l on p r o d u c t s such as j e l l i e s c o n t a i n i n g p a r t i c u l a t e s . N o n d e s t r u c t i v e t e s t s can be used t o b l e n d raw p e c t i n s t o a c o n s i s t e n t j e l l y g r a d e . A l s o , some r e p o r t s have found n o n d e s t r u c t i v and more r e l i a b l e (63). d e f o r m a t i o n s o u t s i d e the l i n e a r r e g i o n of a s t r e s s v s . s t r a i n c u r v e a r e more d i f f i c u l t to i n t e r p r e t and more d i f f i c u l t t o measure t h a n s m a l l d e f o r m a t i o n s because r u p t u r e o c c u r s a t a d e f e c t i n the g e l and l a r g e d e f o r m a t i o n s are not as r e p r o d u c i b l e . C o n v e r s e l y , d e s t r u c t i v e t e s t s have advantages o v e r n o n d e s t r u c t i v e t e s t s because t h e y a r e c l o s e r to a consumer's p e r c e p t i o n of s p r e a d i n g a j e l l y . The f o r c e r e q u i r e d t o r u p t u r e a g e l c o r r e l a t e d b e s t w i t h g e l s t r e n g t h a s s e s s e d i n the mouth (57). K u i p e r (64) r e p o r t e d t h a t n o n d e s t r u c t i v e measurements c o r r e l a t e d w e l l w i t h the R i d g e l i m e t e r r e a d i n g , whereas d e s t r u c t i v e measurements c o r r e l a t e d b e t t e r with sensory analyses. Nondestructive Tests IFT-SAG. In the IFT-SAG method, a s t a n d a r d i z e d amount o f sugar i s cooked w i t h a t e s t amount o f p e c t i n . The m i x t u r e i s poured i n t o a 7.94 cm h e i g h t j e l l y g l a s s which c o n t a i n s an e x c e s s o f a c i d and a l l o w e d t o g e l f o r 18 t o 24 h o u r s . The j e l l y i s demolded and the amount of sag under the f o r c e o f g r a v i t y i s measured w i t h a s p e c i a l micrometer c a l l e d a R i d g e l i m e t e r . The

advantages o f the IFT-SAG method have been p r e s e n t e d The method e s t a b l i s h e s t e s t c o n d i t i o n s f o r r e p r o d u c i b l y measuring j e l l y f i r m n e s s . At a pH near 2.2, m i n i m a l e f f e c t o f pH on g e l s t r e n g t h i s o b s e r v e d (36). The e f f e c t s of temperature and a g i n g are a l s o n e g l i g i b l e a t t h i s pH (36, 5θ, 62). The IFT-SAG method uses s i m p l e and i n e x p e n s i v e l a b equipment. It i s p r e c i s e , r e p r o d u c i b l e , and s u b j e c t t o m i n i m a l o p e r a t o r e r r o r . T h i s method i s the s t a n d a r d on which comparisons o f p r i c e o f p e c t i n s and p r e d i c t i o n s of the j e l l y i n g c a p a b i l i t i e s have been made. Thus, raw p e c t i n can be c u t t o a 150 grade o r a m i x t u r e of p e c t i n s of known j e l l y g r a d e s can be c a l c u l a t e d .

(62-65).

The IFT-SAG method d i d not win unanimous s u p p o r t a t i t s i n c e p t i o n because t e s t c o n d i t i o n s do not s t i m u l a t e how p e c t i n s are used i n commercial p r a c t i c e . The t e s t pH o f 2.2 i s t e n times more a c i d t h a n the commercial j e l l y pH of 3.2 and i s w e l l below the maximum g e l s t r e n g t h pH (_7, 36, 53, 62)· The t e s t i s based on a water j e l l y which does not account f o r the n a t u r a l l y o c c u r r i n g b u f f e r s and s a l t s i n f r u i t j u i c e . The low g r a v i t y c o m p r e s s i o n r a t e

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

8.

CRANDALL AND

WICKER

Pectin Internal Gel Strength

97

of the IFT-SAG t e s t does not a d e q u a t e l y p r e d i c t the g e l performance under a c t u a l use or o t h e r h i g h compression r a t e s (56)* The t e s t r e q u i r e s 18 t o 24 h o u r s so i t i s not u s e f u l i n r e a l time m o d i f i c a t i o n during j e l l y manufacture. F i n a l l y , t h i s method o f e v a l u a t i o n u n d e r e s t i m a t e s the j e l l y grade of h i g h m o l e c u l a r w e i g h t , h i g h l y polymerized p e c t i n (49). Wageningen Sag. Doesburg (66) d e v e l o p e d a p r o c e d u r e i n which s u g a r - a c i d g e l s were c a s t i n t o a two p i e c e c y l i n d e r . The g e l was cut i n h a l f and the sag of the g e l was measured by a p i n a t t a c h e d t o a p i v i t o l p o i n t e r and a s c a l e . T h i s arrangement a m p l i f i e d the amount o f sag t e n t i m e s . A good c o r r e l a t i o n between the o r g a n o l e p t i c q u a l i t y o f h i g h sugar g e l s was found w i t h the Wageningen sag measurements· B.A.R.—F.I.R.A. J e l l y T e s t e r T h i s i n s t r u m e n t ' s measurements a r e based on a fundamenta measured by the t o r q u e o i s a p p l i e d by water f l o w i n g i n t o a bucket and a measurement i s made on the amount o f water needed t o produce a 30° t u r n o f the b l a d e — w h i c h i s w i t h i n the e l a s t i c l i m i t s o f the g e l 03, 67). M o d i f i c a t i o n s have been made on t h i s i n s t r u m e n t u s i n g an e l e c t r i c motor connected t o a t o r s i o n w i r e to t u r n the b l a d e . Wires w i t h known t o r s i o n a l moments a r e used as s t a n d a r d s . S t u d i e s have shown a s t r o n g c o r r e l a t i o n between R i d g e l i m e t e r and F.I.R.A. g r a d i n g . Other r e s e a r c h e r s have m o d i f i e d t h i s j e l l y t e s t e r to measure a 15° t u r n ( e l a s t i c i t y ) , f o l l o w e d by measurement of the b r e a k i n g s t r e n g t h o f g e l s and have found good c o r r e l a t i o n t o s e n s o r y e v a l u a t i o n s ( 5 5 ) . Concentric C y l i n d e r Instruments. S a v e r b o r n (68) d e v e l o p e d an i n s t r u m e n t t h a t r e q u i r e s the p e c t i n m i x t u r e be poured between two c o n c e n t r i c , c o r r u g a t e d c y l i n d e r s and a l l o w e d t o s e t . The i n n e r c y l i n d e r i s t w i s t e d by a t o r s i o n w i r e and the e x t e n t o f t o r s i o n caused i n the g e l i s measured. The c o r r u g a t i o n s p r e v e n t s l i p p a g e o f the g e l . K e r t e s z (3) c i t e s t h i s as one o f the f i n e s t i n s t r u m e n t s d e v i s e d f o r j e l l y s t r e n g t h measurements. Other i n s t r u m e n t s which use c o n c e n t r i c c y l i n d e r s i n c l u d e t h o s e d e s c r i b e d i n (69-72) and were r e v i e w e d by M i t c h e l l ( 5 1 ) . These i n s t r u m e n t s can be used f o r fundamental measurements on g e l s l i k e c r e e p c o m p l i a n c e , s t r e s s r e l a x a t i o n , and r i g i d i t y modulus. Parallel Plate. P l a s h c h i n a e t a l . (73) s t u d i e d the c r e e p o f HM p e c t i n g e l s p l a c e d between two c o r r u g a t e d p a r a l l e l p l a t e s . Creep compliance c u r v e s were o b t a i n e d f o r 0.5 t o 2.5% p e c t i n a t temperatures from 25 t o 55°C. R e v e r s i b l e and i r r e v e r s i b l e s t r a i n components were s e p a r a t e d . P e c t i n macromolecules were c h a r a c t e r i z e d as b e i n g v e r y s t i f f and o n l y a s l i g h t d e c r e a s e i n e n t r o p y was r e q u i r e d to form a p e c t i n g e l ( 7 3 ) . M i t c h e l l and B l a n s h a r d (50) used an automated p a r a l l e l p l a t e v i s c o e l a s t o m e t e r t o s t u d y the c r e e p compliance on low methoxyl p e c t i n s . The v a l u e o f t h e s e e x p e r i m e n t s was t h a t a c o n t i n u o u s r e s p o n s e was o b t a i n e d from the g e l r a t h e r than a s i n g l e p o i n t measurement. Dynamic T e s t i n g . G e l s can be c h a r a c t e r i z e d by dynamic t e s t i n which an o s c i l l a t o r y s t r e s s i s a p p l i e d t o the g e l and the phase

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

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a n g l e of the s t r a i n measured. Most g e l s show a phase d i f f e r e n c e l e s s than 90° out of phase w i t h the imposed s t r e s s . Gross (74) and Gross e t a l . (75) reviewed dynamic t e s t i n g and used i t t o s t u d y the t e x t u r e s of LM p e c t i n g e l s . They determined the dynamic modulus and phase a n g l e s and found t h a t a t 100 HZ, the g e l s t h a t appeared t o be the most f i r m were the weakest a t 200 HZ. However, they found some good c o r r e l a t i o n s between some of the dynamic t e s t s and sensory evaluations. D e s t r u c t i v e Tests F i n g e r T e s t . The f i r s t d e s t r u c t i v e t e s t s on a p e c t i n g e l were performed by s q u e e z i n g a s l i c e o f j e l l y between the thumb and f o r e f i n g e r u n t i l the g e l b r o k e . T h i s was made i n t o a f o r m a l i z e d j e l l y e v a l u a t i o n t e s t c a l l e d the F i n g e r T e s t . When the a n a l y s t s had adequate e x p e r i e n c e , the r e s u l t s were r e p r o d u c i b l e and d i f f e r e n c e s of 5% o r g r e a t e r between the t e s t j e l l y and a ' s t a n d a r d ' j e l l y c o u l d be d e t e c t e d ( 3 ) . A s i d e a ' s t a n d a r d ' j e l l y must p e c t i n w h i c h i s kept r e f r i g e r a t e d t o m i n i m i z e changes i n the s t a n d a r d . The c h a r a c t e r i s t i c s o f the ' s t a n d a r d ' j e l l y a r e not specified (49). Tarr-Baker. The T a r r - B a k e r (Delaware J e l l y S t r e n g t h T e s t e r ) i s based on the work of T a r r ( 7 6 ) , Baker ( 7 7 ) , and Baker and Woodmansee (78). A f o r c e i s a p p l i e d t o the g e l ' s s u r f a c e by a s y r i n g e p i s t o n powered by compressed a i r . Measurements can be i n f l u e n c e d by a ' s k i n ' on the j e l l y ' s s u r f a c e o r uneven a p p l i c a t i o n of p r e s s u r e ( 3 ) . Swenson e t a l . (48) m o d i f i e d the T a r r - B a k e r apparatus t o produce a b a l a n c e - p l u n g e r type i n s t r u m e n t . A l t h o u g h they found a l i n e a r s t r e s s - s t r a i n r e g i o n w i t h i n the e l a s t i c l i m i t s of the g e l ; they a l s o found e l a s t i c i t y was somewhat dependent on the r a t e o f l o a d i n g . T h e i r i n s t r u m e n t was c a p a b l e of s e v e r a l e l a s t i c and b r e a k i n g t e s t s on one g e l . There was l e s s than a 2% e r r o r between the t r u e and assumed grades u s i n g b r e a k i n g s t r e n g t h . C h r i s t e n s e n (49) determined a r a t i o between a m o d i f i e d 'sag' grade t o the b r e a k i n g s t r e n g t h as measured by the T a r r - B a k e r a p p a r a t u s f o r 22 p e c t i n s o f v a r y i n g MW. The b r e a k i n g / s a g r a t i o ranged from 1.38 t o 0.85, but heat o r enzyme treatment f u r t h e r reduced the r a t i o t o 0.39. The r e l a t i o n s h i p between sag grade and b r e a k i n g s t r e n g t h was r e p o r t e d t o be dependent on the MW of p e c t i n ( 4 9 ) . Doesburg (66) showed lower pH v a l u e s (2.0 to 2.3) caused s h o r t s e t t i n g times and reduced the i n t e r n a l g e l s t r e n g t h compared t o pH 3.1 as measured by the T a r r - B a k e r i n s t r u m e n t . He d i d not observe a good c o r r e l a t i o n o f o r g a n o l e p t i c q u a l i t y t o T a r r - B a k e r measurements. Luers-Lochmuller Pektinometer. The Pektinometer was developed i n Germany (79) and measures the amount o f f o r c e n e c e s s a r y t o p u l l a metal f i g u r e out a f t e r b e i n g c a s t i n s i d e a p e c t i n g e l . A hot j e l l y i s poured i n t o a s p e c i a l c o n t a i n e r w i t h c o r r u g a t e d s i d e s c o n t a i n i n g the m e t a l f i g u r e . The c o r r u g a t e d s i d e s prevent s l i p p a g e of the g e l as the amount of f o r c e n e c e s s a r y t o break the g e l i s measured. S t e i n h a u s e r e t a l . (80) used the L u e r s - P e k t i n o m e t e r i n h i s comparison of f o u r methods f o r d e t e r m i n i n g g e l s t r e n g t h and found the r e p r o d u c i b i l i t y was l i n e a r . Uhlenbrock (81) adopted t h i s i n s t r u m e n t t o determine the b r e a k i n g s t r e n g t h of s u g a r - a c i d g e l s .

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

8.

CRANDALL AND

WICKER

99

Pectin Internal Gel Strength

The b r e a k i n g s t r e n g t h v a r i e d from 500 t o 600 g and the of v a r i a t i o n was 1 t o 2%.

coefficient

1

Herbstreith-Pektinometer. L u e r s - L o c h m u l l e r s method has been f u r t h e r r e f i n e d i n the H e r b s t r e i t h - P e k t i n o m e t e r which uses a s p e c i a l l y d e s i g n e d c l e a r , c o r r u g a t e d cup and p l a s t i c f i g u r e . One hundred grams of the p e c t i n m i x t u r e from a l a b o r a t o r y o r commercial b a t c h i s poured i n t o the cup w i t h a s e l f c e n t e r i n g f i g u r e . The g e l i s h e l d f o r 2 hours a t 20°C. The cup i s l o c k e d i n p l a c e and a hook i s a t t a c h e d t o the c e n t r a l f i g u r e . An e l e c t r i c motor p u l l s up on the f i g u r e r u p t u r i n g the g e l and the peak f o r c e r e q u i r e d i s measured on a 1 kg s t r a i n gauge. The f i n a l v a l u e i s p r i n t e d out i n Pektinometer u n i t s . I n s t r o n . The I n s t r o n U n i v e r s a l T e s t i n g Machine can be used i n compression and t e n s i o n experiments where the f o r c e on a l o a d c e l l i s measured w h i l e movin In e v a l u a t i n g g e l s t r u c t u r e b r i t t l e n e s s , h a r d n e s s , and e l a s t i c i t y can be q u a n t i t a t i v e l y measured and r e l a t e d t o s e n s o r y a t t r i b u t e s such as chewiness and gumminess. Sherman (56) found sample d i m e n s i o n and c r o s s head speed a f f e c t these readings. Gels were not l i n e a r i n t h e i r force-compression behavior. Slow c r o s s head speeds can l e a d t o s t r e s s r e l a x a t i o n , so from low compression t e s t r a t e s , i t was i m p o s s i b l e t o p r e d i c t how a g e l would behave a t h i g h compression r a t e s as i n the mouth. O a k e n f u l l and S c o t t (32) used an I n s t r o n t o measure apparent shear m o d u l i and r u p t u r e s t r e n g t h s . A l t h o u g h no s a t i s f a c t o r y t h e o r y c o u l d be proposed t o r e l a t e r u p t u r e s t r e n g t h and d i s r u p t i o n of i n t e r m o l e c u l a r f o r c e s , they d i d f i n d that hydrophobic i n t e r a c t i o n s play a role i n g e l formation. M i t c h e l l (52) r e v i e w s s e v e r a l i n v e s t i g a t i o n s u s i n g I n s t r o n measurements on s e v e r a l t y p e s o f g e l s . Gels were t e s t e d i n compression and the apparent modulus was c a l c u l a t e d from the i n i t i a l s l o p e of the curve and the r u p t u r e s t r e n g t h was c a l c u l a t e d from the peak f o r c e . Conclusions T h i s r e v i e w p o i n t s out why a p e c t i n g e l ' s i n t e r n a l s t r e n g t h measurements are i m p o r t a n t . Some o f the important q u a l i t i e s f o r t e s t methods and i n s t r u m e n t a t i o n t o measure i n t e r n a l s t r e n g t h a r e given. I t a l s o r e l a t e s the c u r r e n t t h e o r y of the f o r m a t i o n of a HM p e c t i n g e l and the f a c t o r s a f f e c t i n g g e l s t r e n g t h . There a r e two g e n e r a l c a t e g o r i e s of i n s t r u m e n t a t i o n — n o n d e s t r u c t i v e and d e s t r u c t i v e . Examples and advantages o f each c a t e g o r y a r e a l s o given. I n t e r n a l g e l s t r e n g t h measurements a r e c u r r e n t l y under r e v i e w . In the next few y e a r s , new methods and m o d i f i c a t i o n s o f e x i s t i n g t e c h n i q u e s and i n s t r u m e n t a t i o n w i l l be d e v e l o p e d . These t e s t s w i l l more f u l l y c h a r a c t e r i z e the p r a c t i c a l a s p e c t s o f a p e c t i n ' s i n t e r n a l gel strength. Acknowledgments The a u t h o r s w i s h t o acknowledge the a s s i s t a n c e o f K a t h r y n C. i n the p r e p a r a t i o n o f the m a n u s c r i p t . F l o r i d a A g r i c u l t u r a l Experiment S t a t i o n J o u r n a l S e r i e s No. 7184.

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20, 1985

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

9 Characterization of Pectins 1

1

2

2

P. Beach , E. Davis , P. Ikkala , and M . Lundbye 1

Grindsted Products, Inc., Industrial Airport, KS 66031 Grindsted Products A/S, Brabrand, Denmark

2

Breaking strengt have shown that differen SAG will differ significantly in breaking strength depending on the peel source. Direct correlations exist between the viscosity of a pectin solution and the breaking strength value. The use of breaking strength values as a means of grading pectin is proposed. More t h a n 50% o f t h e w o r l d ' s p e c t i n p r o d u c t i o n i s u s e d i n making jellies, jams, marmalades and c o n f e c t i o n e r y products, and t h e a b i l i t y o f t h e p e c t i n t o form j e l l i e s and g e l s i s t h e r e f o r e a most important property. Consequently i t i s the j e l l y forming ability of pectin expressed as t h e J e l l y Grade w h i c h determines the commercial v a l u e o f p e c t i n s . The j e l l y grade h a s been d e t e r m i n e d f o r more t h a n 30 y e a r s by the I F T SAG Method and commercial pectins a r e today standardized according to this method. Commercial pectins are usually s t a n d a r d i z e d t o 150 j e l l y grade o r US SAG. When one a n a l y t i c a l method p l a y s such an i m p o r t a n t r o l e , i t i s r e l e v a n t to assure that the results from this test really give the best possible information regarding t h e u s e v a l u e o f t h a t p a r t i c u l a r p e c t i n and due c r e d i t i s g i v e n t o d i f f e r e n c e s arising i n pectins of different o r i g i n . A l s o , one would e x p e c t t h a t t h e r e s u l t s a r e i n d i c a t i v e o f the f i n a l c h a r a c t e r i s t i c s o f t h e p r o d u c t c o n t a i n i n g p e c t i n . New methods have been d e v e l o p e d f o r t e x t u r e a n a l y s i s o f jams and j e l l i e s . These i n d i c a t e that t h e SAG t e s t i s u n r e l i a b l e f o r p r e d i c t i n g t h e e f f e c t o f p e c t i n on t h e f i n a l p r o d u c t . I s t h e SAG method the p e c t i n analysis f o r the future, or should i t be complemented b y o t h e r a n a l y s e s t o g i v e a more complete p i c t u r e o f any p e c t i n ? Jelly

Grade/SAG Method

It was r e a l i z e d v e r y e a r l y that jelly grade i s of outstanding importance t o t h e p e c t i n p r o d u c e r and u s e r a l i k e ( 1 ) . Nevertheless

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i t t o o k a number of years t o d e t e r m i n e a common method t h a t would give the best possible c h a r a c t e r i z a t i o n of this very important f u n c t i o n o f p e c t i n s . I n 1926 j e l l y grade was d e f i n e d f o r the f i r s t time by W i l s o n (2). We d e f i n e j e l l y grade as the amount o f sugar that will j e l l i f y one p a r t o f p e c t i n under certain prescribed c o n d i t i o n s to a s t a n d a r d f i r m n e s s . A f t e r many y e a r s o f d i s c u s s i o n o v e r the s t a n d a r d j e l l y f o r the j e l l y grade d e t e r m i n a t i o n , work on a s t a n d a r d method was u n d e r t a k e n by a committee o f the IFT. The committee worked f o r s e v e r a l y e a r s on p e c t i n s t a n d a r d i z a t i o n . They s e l e c t e d the Exchange R i d g e l i m e t e r as the standard i n s t r u m e n t t o measure j e l l y SAG because it was compact, i n e x p e n s i v e , easy t o use, and i t a l s o gave r e p r o d u c i b l e results. J e l l i e s f o r the t e s t s a r e p r e p a r e d i n special conical glasses o f d e f i n e d d i m e n s i o n s and w i t h s i d e b o a r d s . After a 24 hour s e t t i n g time the s i d e b o a r d s a r e removed, a c l e a n c u t s u r f a c e i s made, and the j e l l y turned on t m i c r o m e t e r screw mounte measures the SAG o r the c o l l a p s e o f the j e l l y . P r o b a b l y the pH o f the t e s t jelly was d i s c u s s e d most by the Committee. Most jams and j e l l i e s a r e p r o d u c e d i n the pH range of 3.0-3.2. U n f o r t u n a t e l y test results at t h e s e pH v a l u e s a r e more erratic. F u r t h e r m o r e , a t pH 3.0-3.2, the s t r e n g t h o f the jellies increased more upon s t a n d i n g t h a n j e l l i e s h a v i n g a pH below 3.0. Moreover, soon t h e r e was s t r o n g e v i d e n c e that the "acid in the g l a s s " procedure a t pH below 3.0 would g i v e more r e l i a b l e r e s u l t s i n a s h o r t e r time t h a n the same p r o c e d u r e a t h i g h e r pH v a l u e s . An agreement was r e a c h e d on a pH o f 2.3-2.4. The method, d e s i g n a t e d Method 5-54, was p u b l i s h e d i n 1959 ( 3 ) . P a r a l l e l t o the work i n the U.S., r e s e a r c h e r s i n England a l s o were w o r k i n g on a method t o grade p e c t i n . S i m u l t a n e o u s l y a method was p u b l i s h e d w h i c h s p e c i f i e d s o l u b l e s o l i d s i n the t e s t j e l l y of 70.5% and a pH o f 3.10 ( 4 ) . In s p i t e of the work done i n England, the IFT SAG Method (sometimes c a l l e d US SAG) o v e r the y e a r s has become the most w i d e l y accepted method f o r the g r a d i n g o f commercial p e c t i n a l l o v e r the world. Present

S t a t u s o f the SAG

Method

From the b e g i n n i n g the IFT Committee r e a l i z e d t h a t t h e i r SAG Method was a compromise between c o n f l i c t i n g interests. F u r t h e r m o r e the Committee r e a l i z e d t h a t no one t e s t c o u l d measure a l l p a r a m e t e r s o f p e c t i n performance i n a j e l l y . There are s e v e r a l n e g a t i v e aspects or shortcomings of the SAG test. The jelly u s e d f o r the SAG t e s t i s n o t r e p r e s e n t a t i v e o f a j e l l y as the consumer r e c e i v e s i t , and this i s p a r t i c u l a r l y true w i t h r e g a r d t o the v e r y low pH, a t which the SAG t e s t i s p e r f o r m e d . An e x p e r i m e n t was c o n d u c t e d i n which the pH o f SAG j e l l i e s was varied. The o b j e c t i v e was t o d e t e r m i n e the e f f e c t pH has on SAG grade. J e l l i e s were p r e p a r e d a t pH's between 2.3 and 3.1. The pH was v a r i e d by changing the amount o f t a r t a r i c a c i d used. Pectin dosage was h e l d c o n s t a n t a t .433g. For comparative p u r p o s e s two different p e c t i n s were used, one which i s known t o be a p u r e lime peel c i t r u s p e c t i n , and another citrus p e c t i n with mixed peel

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

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origin. As shown i n F i g u r e 1, a sharp drop i n the SAG v a l u e i s o b s e r v e d as the pH i s i n c r e a s e d . As the pH approaches 3.1, the SAG value as measured by t h e R i d g e l i m e t e r drops o f f dramatically and eventually a pH i s r e a c h e d where the t e s t j e l l i e s become so s o f t that i t i s impossible to obtain a SAG value using the Ridgelimeter. S t r a n g e l y , the t e s t f o r g r a d i n g the p r o d u c t f a i l s t o r e g i s t e r o r t o grade as c o n d i t i o n s approach t h o s e o f a c t u a l usage. F i g u r e 1 a l s o shows t h a t two p e c t i n s , w h i c h have s i m i l a r SAG v a l u e s a t pH 2.3, w i l l grade q u i t e d i f f e r e n t l y as the pH i s i n c r e a s e d . As the j e l l y sags, the micrometer screw i s u s e d t o measure the e l a s t i c i t y o f the j e l l y . However, the consumer would much p r e f e r a spreadable jelly, and i s certainly n o t i n t e r e s t e d i n an e l a s t i c jelly. Or e x p r e s s e d i n o t h e r words, the SAG test does n o t g i v e a v e r y good e x p r e s s i o n o f the j e l l y c h a r a c t e r i s t i c s as d e s i r e d by the consumer. Furthermore the SAG test cannot be made on a commercial production jelly as i shown by F i g u r e 1, the p SAG v a l u e has no meaning. From d i s u s s i o n s w i t h j e l l y , jam, and marmalade p r o d u c e r s in d i f f e r e n t p a r t s o f the w o r l d , i t was l e a r n e d t h a t shortcomings of the SAG t e s t w i t h r e g a r d t o f i n a l p r o d u c t characteristics, induced jam and j e l l y p r o d u c e r s t o d e v e l o p o t h e r methods t o d e t e r m i n e the dosage o f a p a r t i c u l a r pectin required to o b t a i n a s a t i s f a c t o r y end-product ( 5 ) . I n s t r u m e n t a l Methods The R i d g e l i m e t e r u s e d f o r the IFT SAG Method i s e x c e l l e n t from the point of s i m p l i c i t y and s t u r d i n e s s . Other methods, however, have been developed with a h i g h e r degree o f s o p h i s t i c a t i o n , b u t a t the same time, t h e y a r e a l s o more c o m p l i c a t e d and s u b j e c t t o m e c h a n i c a l errors. The f i r s t i n s t r u m e n t was d e v e l o p e d by Luers i n Germany as e a r l y as 1927 and i t i s the s o - c a l l e d P e c t i n o m e t e r ( F i g . 2) (.6) . The e a r l y version of the Luers/Lochmuller P e c t i n o m e t e r was b a s e d on a s p e c i a l c o r r u g a t e d sample cup. A p l a t e i s p l a c e d a t the bottom of the cup b e f o r e the j e l l y i s poured. A f t e r a l l o w i n g the j e l l y to set f o r a s t a n d a r d amount o f time, w e i g h t s a r e p l a c e d on the pan t o withdraw the plate from the j e l l y . The P e c t i n o m e t e r measures the w e i g h t n e c e s s a r y t o b r e a k the j e l l y w i t h the bottom plate. This instrument has gone through several stages of development, and i s today r e p r e s e n t e d by a s o p h i s t i c a t e d e l e c t r o n i c v e r s i o n w h i c h was d e v e l o p e d by the German company, H e r b s t r e i t h , and i s c a l l e d the H e r b s t r e i t h P e c t i n o m e t e r ( F i g u r e 3). The basic principle of operation i s s t i l l the same. The i n s t r u m e n t c o n s i s t s o f a c o r r u g a t e d sample cup w i t h an open p l a s t i c i n s e r t p l a c e d a s h o r t d i s t a n c e o v e r the bottom of the sample cup. The p l a s t i c i n s e r t i s p u l l e d o u t o f the j e l l y by means o f a motor and a pulley, on w h i c h i s mounted a load cell. Loads a r e registered on a digital display. A t the end o f a t e s t c y c l e the peak v a l u e i n Breaking Strength Units w i l l be printed out automatically. I n c r e a s i n g l y , t h i s instrument i s gaining popularity as the basis o f a f a s t and r e l i a b l e method f o r m e a s u r i n g B r e a k i n g S t r e n g t h

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

Figure 2 - The Luers and Lochmuller Pectinometer

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

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Figure 3.

The Herbstreith Pectinometer.

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of p e c t i n s . The pH o f the t e s t j e l l y i s 3.05 and i s o b t a i n e d by using a buffered pectin solution. Measurements a r e made a f t e r o n l y two h o u r s o f s e t t i n g , which makes i t one of the q u i c k e s t methods available. T h i s method i s a l s o recommended f o r use on samples s t r a i g h t o f f the p r o d u c t i o n l i n e . As f a r as i s known, t h i s i s the o n l y t e s t method w h i c h measures B r e a k i n g S t r e n g t h from i n s i d e the jelly and thereby e l i m i n a t e s any problems w i t h s k i n f o r m a t i o n on the s u r f a c e o f the t e s t j e l l y . In r e c e n t y e a r s v a r i o u s t e s t i n s t r u m e n t s have been d e v e l o p e d to measure t e x t u r e o f a number o f f o o d p r o d u c t s . Some o f these instruments also have been u s e d t o t e s t j e l l i e s . One o f t h e s e i s the V o l a n d Stevens T e x t u r e A n a l y z e r ( F i g . 4 ) . The i n s t r u m e n t i s programmed to lower a p l u n g e r i n t o a g e l a t a s p e c i f i c speed and t o a s p e c i f i c d e p t h . A load cell measures resistance which c a n be o b t a i n e d from a digital d i s p l a y or a connected recorder. When the i n s t r u m e n of 0.5 mm/sec. and a p e n e t r a t i o Figure 5 i s obtained. The p e n e t r a t i o n i s c h o s e n t o exceed the elastic limit of the j e l l y . Above the elastic limit a sharp i n c r e a s e i n the f o r c e or the l o a d i s seen u n t i l the j e l l y b r e a k s , and t h e n a sharp d r o p - o f f i s o b s e r v e d f o l l o w e d by some c o m p r e s s i o n effects. Comparison o f V a r i o u s Methods f o r P e c t i n G r a d i n g Limitations of the IFT SAG method w i t h r e s p e c t t o pH have been demonstrated. Similar limitations have been r e p o r t e d by other laboratories (7). Since the Herbstreith Pectinometer and the Voland Stevens Texture A n a l y s e r have b e e n d e v e l o p e d r e c e n t l y , i t c o u l d be o f i n t e r e s t t o see how the j e l l y c h a r a c t e r i s t i c s measured by these instruments correspond to the official results as e x p r e s s e d by IFT SAG. S t a n d a r d a p p l e j e l l i e s were p r e p a r e d h a v i n g a soluble solids of 65% and a pH of 3.15. Five commercial citrus pectins standardized to 150° SAG were u s e d i n the s t u d y . P e c t i n dosage was v a r i e d between 0.1% and 0.5%. The r e s u l t i n g j e l l i e s were t e s t e d f o r B r e a k i n g S t r e n g t h o r i n t e r n a l s t r e n g t h on the V o l a n d Stevens Tester. The r e s u l t s a r e shown i n F i g u r e 6. Internal s t r e n g t h was p l o t t e d a g a i n s t p e c t i n dosage. Four of the p e c t i n s a r e relatively close t o g e t h e r whereas one i s at a constantly higher level. A t a p e c t i n dosage o f 0.35%, w h i c h would be c o n s i d e r e d f a i r l y normal i n the i n d u s t r y , an i n t e r n a l s t r e n g t h o f a p p r o x i m a t e l y 55 g. i s o b t a i n e d w h i c h c o u l d be c o n s i d e r e d as an average v a l u e as t h i s i s g r o s s l y the v a l u e r e p r e s e n t e d by four of the five pectins i n the test. For pectin No. 1 an i n t e r n a l s t r e n g t h e q u a l t o the o t h e r f o u r p e c t i n s i s o b t a i n e d w i t h a dosage of only 0.27-0.28%. By the SAG t e s t , a l l f i v e p e c t i n s were j u d g e d to be o f the same q u a l i t y . Four o f the p e c t i n s i n the t e s t were commercial c i t r u s p e c t i n s o f mixed p e e l o r i g i n , whereas sample No. 1 w i t h the c o n s t a n t l y h i g h e r i n t e r n a l s t r e n g t h r e a d i n g s i s a p e c t i n known t o have been made from pure l i m e p e e l s . These s u r p r i s i n g r e s u l t s on f i v e p e c t i n s w h i c h were supposed to be e q u a l l e d t o f u r t h e r t e s t i n g where o t h e r p e c t i n samples were t e s t e d u s i n g the P e c t i n o m e t e r i n addition to the V o l a n d Stevens

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

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F i g u r e 4 - The V o l a n d

Stevens

Texture

Recording of Internal Strength Measurements on Pectin with the — Stevens Texture Analyser

Figure 5 - Internal Texture Analyzer

Analyzer

g

S t r e n g t h Curves O b t a i n e d w i t h V o l a n d

Stevens

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Tester. S e v e r a l t e s t s were c o n d u c t e d a t the Food R. A. in England (8) using f o u r d i f f e r e n t commercial p e c t i n samples, o f w h i c h t h r e e are c i t r u s p e c t i n s of mixed p e e l o r i g i n and one sample i s a p u r e lime p e c t i n . The f i r s t t e s t was made on a Stevens Texture Analyser using a plunger slightly d i f f e r e n t from the one u s e d i n our l a b o r a t o r i e s , b u t a l s o made on an a p p l e j e l l y with pectin dosages r a n g i n g from 0.1% to 0.5%. The b r e a k l o a d l e v e l i s s l i g h t l y d i f f e r e n t from t h o s e o b t a i n e d i n our l a b o r a t o r i e s , b u t the t r e n d i s the same. Once more, 150° SAG p e c t i n s , supposedly of the same s t r e n g t h , when measured by a T e x t u r e Analyser under usage c o n d i t i o n s appear t o be s i g n i f i c a n t l y d i f f e r e n t ( F i g . 7). The degree o f m e t h o x y l a t i o n (DM) f o r a l l f o u r p e c t i n s was 65+2. The same t e s t was r e p e a t e d u s i n g the H e r b s t r e i t h P e c t i n o m e t e r . The same p e c t i n s , j e l l y c o m p o s i t i o n , and pectin doages were u s e d . The pectinometer gives Breaking Strengths at a t o t a l l y d i f f e r e n t level from those obtaine a g a i n a marked d i f f e r e n c observed ( F i g 8). Even i n the v e r y e a r l y days o f the commercial pectin industry, molecular weight o f the p e c t i n m o l e c u l e was b e l i e v e d to play an i m p o r t a n t p a r t i n the gel formation, and i n p a r t i c u l a r i n the g e l strength. As e a r l y as 1953 one o f the major p e c t i n producers (£) stated that the r e l a t i o n s h i p between SAG Grade V a l u e and B r e a k i n g Strength Value of p e c t i n i s not constant, but depends on the molecular weight of the pectin. I t was f u r t h e r s t a t e d t h a t i t would be impossible to alter the e x i s t i n g methods of grade d e t e r m i n a t i o n so t h a t the SAG method would g i v e r e s u l t s w h i c h would coincide with Breaking Strength values. An i n v e s t i g a t i o n was c o n d u c t e d t o d e t e r m i n e t o what e x t e n t the differences o b s e r v e d between the four 150° SAG p e c t i n s u s e d i n the breaking s t r e n g t h s t u d y c o u l d be a t t r i b u t e d t o d i f f e r e n c e s in molecular weight. G e l P e r m e a t i o n Chromatography studies were c o n d u c t e d by the Food R. A. on the four pectins used i n the B r e a k i n g S t r e n g t h study. U s i n g the r e s u l t s o f d u p l i c a t e runs f o r the four samples, the d i s t r i b u t i o n c o e f f i c i e n t s (K) were calculated f o r each run g i v i n g the f o l l o w i n g r e s u l t s :

RESULTS FROM Sample CP MP SS200 CP 7384 CP 7978 CP 5725

GPC Κ 0.31, 0.36, 0.40, 0.45,

value 0.29 0.38 0.42 0.45

While i t was not possible to determine a c t u a l m o l e c u l a r weights from t h i s GPC method a t the time o f t e s t i n g , i t i s p o s s i b l e t o make the f o l l o w i n g a s s u m p t i o n s : The Κ values are inversely related to average molecular w e i g h t , and the r e s u l t s s u g g e s t t h a t sample CP MP SS 200 has a

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

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I.S. Grams

0.10

0.15

0.20

025

030

0.35

0.40

0.45

0 50

F i g u r e 6 - I n t e r n a l S t r e n g t h v s . P e c t i n Dosage U s i n g t h e V o l a n d Stevens Texture A n a l y z e r

F i g u r e 7 - I n t e r n a l S t r e n g t h v s . P e c t i n Dosage U s i n g t h e V o l a n d Stevens T e x t u r e A n a l y z e r

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

CHEMISTRY AND FUNCTION OF PECTINS

0.1

0.2

0.3

0.4 0.5

Added pectin (%)

F i g u r e 8 - I n t e r n a l S t r e n g t h v s . P e c t i n Dosage U s i n g t h e H e r b s t r e i t h Pectinometer

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significantly h i g h e r average m o l e c u l a r w e i g h t than the other samples. The four pectin samples used f o r t h e GPC were further investigated b y t h e N o r t h E a s t Wales Institute (10), using a d i f f e r e n t t e c h n i q u e , which i s size exclusion chromatography, t o give an i n d e x o f t h e r e l a t i v e m o l e c u l a r w e i g h t s and t h e m o l e c u l a r w e i g h t d i s t r i b u t i o n s o f t h e s e samples. The elution p r o f i l e s f o r t h e f o u r samples a r e shown i n F i g u r e 9. I n each c a s e t h e b r o a d peak r e p r e s e n t s t h e u r o n i c a c i d , which corresponds to the p e c t i n being e l u t e d . The h i g h e r m o l e c u l a r w e i g h t samples w i l l have peaks n e a r VO. On t h e s i d e o f t h e c u r v e s we have t h e r e s u l t i n g w e i g h t average m o l e c u l a r w e i g h t , MW, and number average m o l e c u l a r w e i g h t , H^. The range f o r the weight average m o l e c u l a r w e i g h t i s from 65,000 up t o 150,000, which is a range c o n f i r m i n g the r e s u l t s from t h e GPC method. I n the middle row we have t h e i n d e x e d v a l u e s o f MW. The r e s u l t s from t h studies together with measured i n o u r l a b o r a t o r i e s have c l e a r l y d e m o n s t r a t e d t h a t on a c o n s t a n t and c o n s i s t e n t b a s i s c i t r u s p e c t i n s d e r i v e d o n l y from l i m e p e e l s have a m o l e c u l a r w e i g h t s u p e r i o r t o c i t r u s pectins o f normal mixed peel origin. A citrus pectin o f mixed p e e l o r i g i n c a n c o n s i s t o f any p r o p o r t i o n o f p e e l s from orange, grape f r u i t , lemon, and l i m e . The differences that were o b s e r v e d i n b r e a k i n g s t r e n g t h o r i n t e r n a l s t r e n g t h between t h e f o u r p e c t i n samples, a l l o f w h i c h h a d a 150 SAG and a degree o f m e t h o x y l a t i o n (DM) o f 65, c a n be attributed to differences i n molecular weight. Most i n v e s t i g a t i o n s so f a r have worked on slow s e t p e c t i n s , which on t h e U. S. market i s by f a r t h e most w i d e l y u s e d p e c t i n t y p e . However, i f we l o o k a t a wide range o f p e c t i n types starting w i t h an e x t r a slow set pectin w i t h a low degree o f m e t h o x y l a t i o n and p r o g r e s s t h r o u g h t h e v a r i o u s i n t e r m e d i a t e t y p e s up t o a r a p i d s e t p e c t i n w i t h a h i g h degree o f m e t h o x y l a t i o n , a s h a r p i n c r e a s e i n b r e a k i n g s t r e n g t h i s observed ( F i g . 10). Viscosity v a l u e s o f t h e same p e c t i n s a l s o were measured where the v a l u e s r e p r e s e n t s c a l e units o f a Haake V i s c o s i m e t e r on a two SAG pectin solution. Also h e r e we see a l i n e a r relationship between DM v a l u e and v i s c o s i t y . Viscosity measurements a r e made on s o l u t i o n s w h i c h a r e c o n s i d e r a b l y e a s i e r t o make t h a n t e s t j e l l i e s , and when w o r k i n g under w e l l d e f i n e d c o n d i t i o n s we have f o u n d that viscosimetry i s a g r e a t advantage when s t u d y i n g p e c t i n s . Working w i t h v i s c o s i m e t r y we have t h e added advantage o f h a v i n g a method w i t h d i r e c t i n f l u e n c e from d i f f e r e n c e s i n m o l e c u l a r w e i g h t s . A good correlation between b r e a k i n g s t r e n g t h and two SAG v i s c o s i t y was f o u n d . A r e g r e s s i o n c o f f i c i e n t o f 0.90 was o b t a i n e d . With better standardization o f t h e c o o k i n g p r o c e d u r e , we expect test j e l l i e s t o g i v e an even h i g h e r c o r r e l a t i o n between b r e a k i n g s t r e n g t h and v i s c o s i t y . We conclude that p e c t i n molecular weight i s p r o p o r t i o n a l to degree o f m e t h o x y l a t i o n i n t h a t a r a p i d set pectin with a high degree of methoxylation w i l l require t h e most gentle type o f e x t r a c t i o n whereas lower levels o f m e t h o x y l a t i o n a r e exposed t o more a d v e r s e e x t r a c t i o n c o n d i t i o n s . In demethoxylating the p e c t i n molecule, t h e p e c t i n m o l e c u l e i s a l s o degraded t o a c e r t a i n degree

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Figure 9 - E l u t i o n P r o f i l e s Studies o f Pectins

from S i z e E x c l u s i o n

Chromatography

BSU 900 Extra Slow 800 Set

Slow Set

Rapid Set

700 600 500 400 300

14 16 18 20 22 24 26 28 30 32 34 36 38 40 42

Vise. units

F i g u r e 10 - B r e a k i n g S t r e n g t h o f P e c t i n J e l l i e s and V i s c o s i t y o f P e c t i n S o l u t i o n s P r e p a r e d w i t h P e c t i n s o f V a r y i n g Degrees o f Methoxylation

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

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resulting in a decreasing molecular weight. We t h e o r i z e t h a t the c o r r e l a t i o n between b r e a k i n g strength and v i s c o s i t y a t a s p e c i f i c degree o f m e t h o x y l a t i o n i s the r e s u l t o f the molecular weight of the p e c t i n . A t the time o f the i n t r o d u c t i o n o f the SAG method around 1950, apple p e c t i n s played an important part of the over-all pectin consumption i n the United States, and a t the time i t was asked whether the SAG method would be f a i r t o apple p e c t i n s , which o f t e n gave a stronger j e l l y than a corresponding c i t r u s pectin. Since t h e n the p e c t i n market has changed, and t o d a y the predominant type on the U.S. market i s c i t r u s p e c t i n , w h i c h a l s o has been the b a s i s f o r t h i s present study, although a differentiation has been made between c i t r u s p e c t i n o f mixed p e e l o r i g i n and c i t r u s p e c t i n based on l i m e p e e l s only. However, h i g h q u a l i t y a p p l e p e c t i n s a r e s t i l l b e i n g p r o d u c e d and a r e u s e d around the w o r l d . A v a i l a b l e data i n d i c a t e s possible to produce appl s i m i l a r to t h a t of lime pectinometer i n i t s present p e c t i n producer.

that

form

by

has

careful

processing

been d e v e l o p e d by

it

an

is

apple

Conclusion The SAG method f o r j e l l y grade d e t e r m i n a t i o n has served everyone concerned with pectin f o r more t h a n 30 y e a r s , b u t we b e l i e v e t h a t we may have come t o the time when a s p e c t s other t h a n j e l l y grade are t a k e n i n t o c o n s i d e r a t i o n when e v a l u a t i n g p e c t i n s . Everything i s becoming more and more consumer o r i e n t e d , and i t would t h e r e f o r e be a l o g i c a l consequence that the f i n a l c h a r a c t e r i s t i c s o f the jam as the consumer r e c e i v e s i t a r e t a k e n into consideration i n pectin grading. One o f the most i m p o r t a n t c h a r a c t e r i s t i c s o f a consumer j e l l y i s s p r e a d a b i l i t y w h i c h e s s e n t i a l l y i s a r u p t u r e o f the jelly, and this i s one very i m p o r t a n t c h a r a c t e r i s t i c t h a t the SAG t e s t w i l l n o t d e s c r i b e , whereas the s p r e a d a b i l i t y c a n be e x p r e s s e d t o a certain e x t e n t t h r o u g h the use o f internal strength or breaking strength. We have demonstrated that pectins of different origin will differ i n b r e a k i n g s t r e n g t h and t h e r e b y g i v e j e l l i e s of differing firmness. However today, the determination of breaking s t r e n g t h has reached a h i g h degree o f a c c u r a c y and can be q u a n t i t a t i v e . Thus, p e c t i n s can be standardized to a constant i n t e r n a l s t r e n g t h or b r e a k i n g s t r e n g t h , as i s done today w i t h the SAG method. I t would be technically possible to produce commercial pectins with standardized internal strength o f the same l e v e l from one b a t c h to the n e x t . T h i s s t a n d a r d i z a t i o n to internal s t r e n g t h can e i t h e r be made on a b a s i s where the p r e s e n t SAG v a l u e i s f o r g o t t e n , or a commercial p e c t i n c o u l d be s t a n d a r d i z e d t o 150° SAG as w e l l as to a well defined i n t e r n a l strength value. The most advanced j e l l y and jam companies have for several years been w o r k i n g w i t h methods whereby t h e y d e t e r m i n e the i n t e r n a l strength of each individual pectin l o t i n o r d e r t o a d j u s t the dosage accordingly. This to us demonstrates the need for the pectin industry to s t a n d a r d i z e to constant internal strength so t h a t the j e l l y and jam maker can be a s s u r e d o f a c o n s t a n t and e q u a l p e c t i n dosage from one p e c t i n l o t t o the n e x t .

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Literature Cited 1. Kertesz, A.I., "The Pectic Substances", Interscience Publishers, New York, 1951. 2. Wilson, C.P., Am. Food J. 1926 21, 279, 313. 3. Pectin Standardization: Final Report of the IFT Committee, Food Technology 1959, 13, 496. 4. Oliver M., et al., Journal of Science, Food, and Agriculture 1957, 8, 188-196. 5. Meeting of the Executive Technical Committee of the International Jelly and Preserves Association. June 11, 1985, Atlanta, GA. 6. Luers, H. & Lochmuller, Κ., Kolloid-Z. 1927 42, 154. 7. Fox, G., Confructa Studien 1984 2, 174. 8. Confidential Project, Food R. Α., Leatherhead, UK, DK/HAE/221/85. (Permission for publication has been given). 9. Christensen, P.E. 10. Confidential Project (Permission for publication has been given). RECEIVED November 19, 1985

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

10 Synergistic Gelation of Alginates and Pectins 1

2

2

Knut Toft , Hans Grasdalen , and Olav Smidsrød 1

Protan A/S, P.O. Box 420, N-3001 Drammen, Norway Institute of Marine Biochemistry, The Norwegian Institute of Technology, University of Trondheim, N-7034 Trondheim-NTH, Norway

2

Mixtures of alginate and high methoxy pectins formed thermoreversibl gel belo pH 3.8 withou that D-glucono-deltalacton (GDL) a slow a c i d i f i e r in the cold. The gelling occurred at conditions where neither alginate nor the high methoxy pectin gelled alone. The strength and melting points of these synergistic gels increase with decreasing pH and i n creasing content of L-guluronic acid r e s i dues in the alginates. The gelling effect was correlated to the sequential distribution of the two monomers, D-mannuronic (M) and L - g u l uronic acid (G) residues, in the alginate chain. The results indicated that "blocks" of at least four contiguous G-units were necessary for gelling to occur. The results were discussed in view of existing molecular theories for synergistic gelation. In a search f o r thermoreversible g e l l i n g systems t h a t could be used i n low-sugar, l o w - c a l o r i e jams and j e l l i e s , T o f t discovered that mixtures of high methoxy p e c t i n s and a l g i n a t e s with a high content of ^ - g u l u r o n i c acid residues formed g e l s under c o n d i t i o n s where n e i t h e r a l g i n a t e nor p e c t i n s g e l l e d alone ( J J .These warm-setting g e l s were formed below pH 3.8, and could be made with a sugar content as low as 20%. Later T o f t reported (2^) that thermoreversible g e l s could be formed without any requirement f o r sugar, by using D-glucono-deltalactone (GDL) i n the c o l d as a slow a c i d i f i e r . Since the number of g e l forming polysaccharides allowed f o r use i n the food and pharmaceutical i n d u s t r i e s i s l i m i t e d , the discovery of new s y n e r g i s t i c g e l l i n g systems may have potential commercial value, as have the well-known 0097-6156/86/0310-0117$06.00/0 © 1986 American Chemical Society

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

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s y n e r g i s t i c i n t e r a c t i o n s of c e r t a i n galactomannans with xanthan, agar and kappa-carrageenan (_3) · Since our molecular understanding of s y n e r g i s t i c gel-formation i s very l i m i t e d ( , the p e c t i n - a l g i n a t e system o f f e r s an opportunity t o get a general i n s i g h t i n t o the mechanism of s y n e r g i s t i c g e l a t i o n . This was the object of Thorn e t a l . , who used c i r c u l a r d i c r o i s m spectroscopy t o study the warm s e t t i n g a l g i n a t e - p e c t i n g e l s ( 5» ) . They proposed a model f o r the j u n c t i o n zones i n the mixed g e l s i n which "blocks" of e s t e r i f i e d α - g - g a l a c t u r o n i c a c i d residues i n the pectin chain interacted with "blocks" of α-^-guluronic a c i d residues i n the a l g i n a t e chain. This i n t e r a c t i o n could occur when the uronic a c i d s were mainly i n t h e i r protonated form, and was s t e r i c a l l y favoured because both types of "blocks" contain glycosidic linkages between a x i a l s u b s t i t u e n t s ( 0 - 1 and 0 - 4 ) thereby giving the same se s i m i l a r i l y ordered chai we use a l g i n a t e of d i f f e r e n t monomer sequence t o t e s t t h i s idea. A l g i n a t e s may now be c h a r a c t e r i z e d by both diad and t r i a d - f r e q u e n c i e s , due t o the development of powerful NMR-methods ( 6 ^ 7 ) which o f f e r the opportunity t o study the sequence requirements i n the a l g i n a t e chain f o r the i n t e r a c t i o n with p e c t i n t o occur. For l a c k of s i m i l a r methods f o r studying the d i s t r i b u t i o n of methoxy groups along the p e c t i n chain, only one p e c t i n sample with a high degree of methoxylation ( 7 0 % ) i s used. Although one paper has r e c e n t l y been published d e a l i n g with cold s e t t i n g a l g i n a t e - p e c t i n gels ($_), i t i s necessary here t o include some r e s u l t s on the e f f e c t of pH and calcium ions, and on varying a l g i n a t e - p e c t i n ratios, before a l g i n a t e s of d i f f e r e n t sequences are tested.

MATERIALS AND METHODS Only one sample of p e c t i n was used throughout t h i s study, a commercial sample provided by Grindsted Products A/S and l a b e l l e d Mexpectin RS 4 0 0 . The degree of methylation of the carboxylgroups as measured by the s u p p l i e r , was 70%. No information about the sequence of the e s t e r groups or the content of n e u t r a l sugar was obtained, but according t o the s u p p l i e r the p e c t i n had been produced from limes ( C i t r u s a u r a n t i f o l i a ) . Seven d i f f e r e n t a l g i n a t e samples were used. Some were supplied by Protan A/S, Drammen, Norway, and others were prepared i n the l a b o r a t o r y by standard procedures (9^) . They a l l had a weight average degree of polymeri­ z a t i o n above 5 0 0 , the lower l i m i t a t which chain length a f f e c t s g e l strength i n calcium a l g i n a t e gels (J_0 ) . They were analyzed f o r t h e i r f r a c t i o n a l content of L-guluronic a c i d , ( F ) , and g-mannuronic a c i d , ( F = 1 - F ) , and t h e i r f r a c t i o n s of the four diads, F j ^ r GG' MG GM G

M

F

Q

F

f

a

n

d

F

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

b

y

10.

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Synergistic Gelation of Alginates and Pectins

the NMR-method of Grasdalen et a l . (JM_) . F ^ , F e t c . are the weight f r a c t i o n s of the sequence MM, GG e t c . such that F + F + F + F = 1.The G-centered t r i a d s were measured according to t h e method of Grasdalen (2) . There are eight p o s s i b l e t r i a d s i n a l i n e a r binary heteropolysaccharide with weight f r a c t i o n s F ^ j ^ ^MMG ^ e

F

ι»ψ. Data f o r Table 1.

ρ

t

C

e

+

s u c

F(

t « -d%M = eG:

% κ ; ® ί ; Β > the seven a l g i n a t e samples

Preparation

G

are

summarized

in

of gels

P e c t i n - a l g i n a t e s o l u t i o n s were prepared by d i s s o l v i n g 1.65 g of the polymer mixture s l i g h t l y moistened with 5 ml ethanol i n 125 ml d i s t i l l e d water Appropriate amounts of GDL were and, a f t e r complete 25 ml of a l g i n a t e - p e c t i n s o l u t i o n . A p o r t i o n of these s o l u t i o n s was poured i n t o c y l i n d r i c a l perspex tubes, covered with d i a l y s i s membranes at both ends (J_2) and allowed to g e l . C y l i n d r i c a l p e l l e t s of g e l s with a height of 15 mm and a diameter of 14 mm were then obtained. The remaining p o r t i o n s of the s o l u t i o n s were allowed to g e l i n the beakers, and used f o r measurements of pH. Determination of mechanical p r o p e r t i e s of gels The gels were t e s t e d i n a Stevens^LFRA Texture Analyzer. The modulus of r i g i d i t y , E,Pa(N/m ), was c a l c u l a t e d from the load causing compression of one or two m i l l i m e t e r s , i n which range the s t r e s s - s t r a i n curve was l i n e a r . The breaking load ( i n grams) was a l s o determinecl. The standard e r r o r i s both types of measurements was - 10%. Melting points were determined by heating the g e l s i n a thermostat at a rate of 0.5 °C per min. Several small g l a s s spheres (diameter ~3 mm) were placed on the surface of the g e l s , and the melting point was taken as the temperature a^t which the spheres s t a r t e d to s i n k . The accuracy was - 5°C. RESULTS AND

DISCUSSION

For warm-setting p e c t i n - a l g i n a t e gels T o f t reported (J_) that calcium l e v e l s above 5 mM, weakened the g e l s . In those experiments, the calcium was present i n the water before the p e c t i n s and a l g i n a t e s were d i s s o l v e d . In s i m i l a r experiments with gels prepared by the GDL-method, we a l s o found that the gels weakened when the calcium concentration was 4 mM or higher. I t was observed, however, that during the preparation of the p e c t i n - a l g i n a t e s o l u t i o n p r i o r to mixing with GDL, the s o l u t i o n s contained small i s l a n d s of gels (microgel). It

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

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CHEMISTRY AND FUNCTION OF PECTINS

TABLE 1.

C h a r a c t e r i s t i c s of a l g i n a t e

samples.

F Type of a l g i n a t e PROTANAL LF 250 Laminaria hyperborea, 0. 70 stipe PROTANAL LF 120 DL, Laminaria hyperborea, 0. 57 whole plant + l e a f Lab.prep, from Laminaria hyperborea 0. 50 leaf Lab.prep, from Laminaria d i g i t a t a , 0. 43 leaf PROTANAL LF 200 MA Ascophyllum nodosum + 0. 41 Laminaria hyperb, l e a f Lab.prep, from Ascophyllum nodosum lower part Lab.prep, from Ascophyllum nodosum f r u i t i n g bodies

0.19 0. 11 0 .59 0.55 0. 04

0. 07

0.28 0. 14 0 .43 0.39 0. 04

0. 10

0.32 0. 18 0 .32 0.26 0. 06

0. 12

0.41 0. 16 0 .27 0.22 0. 05

0. 11

0.41 0. 18 0 .23 0.18 0. 05

0. 13

0. 43 0.34 0. 23 0 .20 0.12 0. 08

0. 15

0. 02

0

0

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was t h e r e f o r e p o s s i b l e that the l i m i t e d s o l u b i l i t y of alginate i n the calcium-containing water caused the diminished r e a c t i o n with p e c t i n , and that calcium was not antagonistic per se to the formation of mixed p e c t i n - a l g i n a t e g e l s . To t e s t t h i s idea, some g e l s were made with calcium-free water i n the standard way, and thereafter soaked i n calcium c h l o r i d e s o l u t i o n s of d i f f e r e n t strength. The i n t r o d u c t i o n of calcium ions i n t h i s way increased the g e l strength compared t o the calcium-free g e l s . In f a c t , some gels more than t r i p l e d i n strength as measured by the breaking load, and they even regained the o r i g i n a l form a f t e r compression above the breaking load. This was obviously an i n t e r e s t i n g new way of preparing mixed a c i d - c a l c i u m p e c t i n - a l g i n a t e g e l s , but no f u r t h e r s t u d i e s were performed. In the r e s t of t h i s work only calcium-free systems are considered. A l g i n a t e sample 70 and 40% L-guluroni t e s t the e f f e c t of varying the concentration of GDL i n t h i s system. The weight r a t i o between a l g i n a t e and p e c t i n was kept 1:1 i n the s e r i e s . The r e s u l t s (Fig.1) c l e a r l y demonstrate that i n c r e a s i n g amounts of GDL increase the g e l strength as measured by the modulus of r i g i d i t y . In Fig.2 the same r e s u l t s are p l o t t e d against the pH i n the gels. The curves are drawn t o zero around pH 3.8, the upper l i m i t of pH at which s e l f - s u p p o r t i n g g e l s were formed. Since the pK values of the three uronic a c i d residues i n a l g i n a t e p e c t i n are between 3.4 and 3.7, the r e s u l t s i n d i c a t e that the polyuronides have to be p a r t l y protonated f o r g e l a t i o n to occur. The r e s u l t s of the breaking load measurements on the same g e l s are given i n Fig.3. The data are q u a l i t a t i v e l y very s i m i l a r to the r e s u l t s i n Fig.1 and 2, and show that the breaking load and modulus vary i n a h i g h l y c o r r e l a t e d manner. In a s i m i l a r s e r i e s of experiments the melting p o i n t s of the gels were measured. The r e s u l t s (Fig.4) show that the melting p o i n t a l s o increases i n a systematic manner with i n c r e a s i n g concentration of GDL i n the system. The r e s u l t s Figs.1-4 a l l demonstrate that the s t a b i l i t y of the g e l s , whether c h a r a c t e r i z e d by the modulus, the breaking load or the melting temperature, i s higher f o r the a l g i n a t e with the higher f r a c t i o n of ^ - g u l u r o n i c a c i d residues. This p o i n t s to a key r o l e of ^ - g u l u r o n i c a c i d residues i n developing the network i n harmony with earlier suggestions (J5 ). Before pursuing this point f u r t h e r , some data on varying the p e c t i n a l g i n a t e r a t i o w i l l be given. The weight r a t i o between a l g i n a t e and p e c t i n was v a r i e d i n steps between 0.1 and 0.9. Since the b u f f e r c a p a c i t y of the n o n - e s t e r i f i e d a l g i n a t e i s higher than that of the p e c t i n sample, a s e r i e s of GDL-concentrations i n the range from 0.7 to 1.8 g/100 ml was used f o r each polymer mixture, g i v i n g d i f f e r e n t pH-values i n g e l s . This

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CHEMISTRY AND FUNCTION OF PECTINS

1

1

1

I

ι

r

5 -

It

ι

10 GDL,

I

U

2.0 %( /v) w

Figure 1. Modulus of r i g i d i t y , E, f o r d i f f e r e n t concen­ t r a t i o n of D-glucono-delta-lactone, GDL. T o t a l polymer concentration i s 1.2% (w/v) i n a 1:1 mixture of Mexpect i n RS 400 and the two a l g i n a t e s shown on the curves.

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

10.

TOFT ET AL.

Synergistic Gelation of Alginates and Pectins

F i g u r e 2. The the g e l s .

d a t a f r o m F i g . 1 p l o t t e d a g a i n s t pH o f

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124

CHEMISTRY AND FUNCTION OF PECTINS

F i g u r e 3. B r e a k i n g l o a d s f o r d i f f e r e n t c o n c e n t r a t i o n s o f GDL a n d t h e r e s u l t a n t pH's o f t h e g e l s . T o t a l p o l y mer c o n c e n t r a t i o n i s 1.2% (w/v) i n 1:1 m i x t u r e s o f p e c t i n a n d t h e two a l g i n a t e s shown on t h e c u r v e s .

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

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Synergistic Gelation of Alginates and Pectins

1

1

1

Γ

Figure 4. Melting p o i n t f o r d i f f e r e n t concentrations of GDL. T o t a l polymer concentration i s 1.2%(w/v) i n 1:1 mixtures of p e c t i n and the two a l g i n a t e s shown on the curves.

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

126

CHEMISTRY AND FUNCTION OF PECTINS

allowed interpolation or e x t r a p o l a t i o n of the g e l strength t o a s i n g l e pH value of 3.1 f o r comparison. Only the a l g i n a t e sample No.1 was used i n t h i s study. The r e s u l t s f o r the moduli and the breaking loads are given i n Figs.5 and 6, r e s p e c t i v e l y . Both sets of data i n d i c a t e maximum g e l strength a t r a t i o s of a l g i n a t e t o p e c t i n of 1 t o 1. Since the a l g i n a t e sample contains 70% ^-guluronic a c i d and the p e c t i n 70% methyl e s t e r groups, these r e s u l t s are i n harmony with the g e l l i n g mechanism discussed earlier, although not representing any independent evidence. Before presenting the results for different a l g i n a t e s , some b a s i c features of the monomer sequence i n a l g i n a t e s have t o be d i s c u s s e d . In a s e r i e s of papers, Haug, Larsen, P a i n t e r and Smidsrod ( f o r an e x c e l l e n t review see T. P a i n t e r (V3)) showed t h a t a l g i n a t e s were not physical mixture polyguluronic a c i d , tha repeating u n i t , and that t h e i r monomer sequence could not be described by a random (Bernoullian) d i s t r i b u t i o n of residues along the c h a i n . Fractionation of p a r t l y hydrolyzed a l g i n a t e s suggested a block-wise d i s t r i b u t i o n of monomer residues with blocks of homopolymeric sequence of both types ("MM-blocks" and "GG-blocks") and blocks enriched i n a l t e r n a t i n g sequences. Attempts were a l s o made to describe the sequence with Markow chain s t a t i s t i c s of d i f f e r e n t order, i ^ e . , with the p r o b a b i l i t y of f i n d i n g a c e r t a i n monomer next t o another depending on the nature of the preceeding units. With such a complicated sequence, a population of a l g i n a t e chains i s not f u l l y c h a r a c t e r i z e d by measurement of the average composition (F and F ) only. To i l l u s t r a t e t n i s p o i n t further,we may consider three d i f f e r e n t types of a l g i n a t e s with w e l l - d e f i n e d s t r u c t u r e s , a l l having F =0.5, and c a l c u l a t e the d i a d and t r i a d frequencies F ™ and F.^. f o r each. The f i r s t example i s an a l g i n a t e witn a s t r i c t l y alternating structure. In t h i s case, F = F ^ . = 0. At the other extreme i s an a l g i n a t e witn i n f i n i t e l y long blocks of M-units and G-units, which i s s t a t i s t i c a l l y not d i f f e r e n t from a p h y s i c a l mixture of the two homopolymers. In t h i s ' MG ' MM MMM · · example i s a Bernoullian d i s t r i b u t i o n . Here the monomer i s completely randomly d i s t r i b u t e d , which means t h a t the p r o b a b i l i t y of f i n d i n g (and hence the f r a c t i o n a l content of) a given d i a d or t r i a d i s j u s t the product of the p r o b a b i l i t y of f i n d i n g t h e i r constituent-monomers. Thus, i n our example, F==F *F =0.25 and F = F * F ' F = 0.125. We see from thes¥ e x i l e s t h a t a¥^fna^es with widely d i f f e r e n t monomer sequence (and p h y s i c a l p r o p e r t i e s ) may have the same average chemical composition. Since a l g i n a t e s i n general do not f o l l o w any of the s t a t i s t i c a l r u l e s of the three examples above, they have t o be c a s e

F

=0

a

n

d

F

=

F

=

0

5

T

h

e

t

h

i

r

M M

d

M

M M

M

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

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Synergistic Gelation of Alginates and Pectins

δ·

2

08

0.4 0.6 —Pectin

0.6 04

0.8 02

1.0 0

Figure 5. Modulus of r i g i d i t y , E, f o r d i f f e r e n t mixing r a t i o s of p e c t i n and a l g i n a t e (Sample No.1). T o t a l polymer concentration i s 1.2% (w/v) and the pH i s 3.1.

1.0

0.2 0.8

0.4 0.6 — - Pectin

0.6 0.4

0.8 0.2

1.0 0

Figure 6. Breaking load f o r d i f f e r e n t mixing r a t i o s of p e c t i n and a l g i n a t e . The g e l s are the same as i n F i g . 5 .

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

CHEMISTRY AND FUNCTION OF PECTINS

128

c h a r a c t e r i z e d by a s many measured d i a d t r i a d a n d h i g h e r order frequencies as p o s s i b l e . I n a s e r i e s o f p a p e r s G r a s d a l e n e t a l . (.6*2' J J _ / 1 4 ) showed t h a t t h e f o u r d i a d f r e q u e n c i e s f F ^ , MG' G M GG < 3 MMG MMG -, MGM/ GMM' MGG/ GGM' GMG GGG measured W l « l d i r r e r e n t TSiMR-^techniques. Sucn measurements h a v e , on t h e one hand, made i t p o s s i b l e t o t e s t what t y p e o f statistics best explain t h e sequence i n different a l g i n a t e s a m p l e s , a n d , on t h e o t h e r , t o c a l c u l a t e t h e average l e n g t h o f t h e d i f f e r e n t blocks i n a l g i n a t e s . I n t h e p r e s e n t work, where ^ - g u l u r o n i c a c i d r e s i d u e s o b v i o u s l y p l a y a dominant r o l e i n t h e i n t e r a c t i o n w i t h p e c t i n , t h e a l g i n a t e s have been c h a r a c t e r i z e d w i t h o n l y the four G-centered triads i n addition t o diad frequencies. These v a l u e s a l l o w _the c a l c u l a t i o n o f t h e average block l e n g t a l l the G-units l y i n l e n g t h o f G - b l o c k s l o n g e r t h a n one u n i t , ) : f

F

P

)

a

n

F

d

t

F

h

e

e

i

g

F

h

t

t

r

i

a

F

d

a

F

n

d

R

E

U

F

)

E

N

C

c

o

I

E

u

(F

S

l

d

b

f

F

a

n

d

f

e

m a

N

F

N

F

F

F

GGG + GGM + MGG

G

>

F

G - MGM

G>1 F

F

GGM

GGM

In Table 2 a r e given the results of g e l strength measurements o f 1 : 1 m i x t u r e s o f a l g i n a t e a n d p e c t i n a t pH 2.9. J u s t some r e l e v a n t s t r u c t u r a l c h a r a c t e r i s t i c s o f t h e a l g i n a t e s , namely F^, F ^ , F„„„ a n d N _ are given i n the Table. The g e n e r a l t e n d e n c y shown by t h e d a t a i n T a b l e 2 i s that increasing the f r a c t i o n of L-guluronic acid residues results i n increasing the g e l strength. However, t h e r e are c l e a r l y exceptions t o t h i s general r u l e . The s a m p l e s 4 , 5 a n d 6 have a l m o s t i d e n t i c a l F - v a l u e s , b u t o n l y t w o o f them ( 4 a n d 5 ) g i v e a g e l o f m e a s u r a b l e g e l s t r e n g t h . T h i s i s most p r o b a b l y , c a u s e d by t h e l o w e r v a l u e s o f F , and, i n p a r t i c u l a r , F o f sample N o . 6 . T h i s s a m p l e must o b v i o u s l y c o n t a i n T e w a n d s h o r t " G G - b l o c k s " compared t o t h e o t h e r two s a m p l e s o f s i m i l a r a v e r a g e c o m p o s i t i o n . The d a t a f o r t h e modulus measurements a r e p l o t t e d against F , F and F i nFig. 7. These p l o t s c l e a r l y indicate that the correlation with F i s the best, p o i n t i n g t o t h e importance o f t h e "Gu^blocks" i n t h e interaction with pectin. I n F i g . 8 t h e same g e l s t r e n g t h d a t a a r e p l o t t e d a g a i n s t t h e average l e n g t h s o f t h e "GG-blocks", f i g u r e i n d i c a t e s t h a t t h e a v e r a g e number o f G - u n i t s i n t h e b l o c k s must e x c e e d 4 f o r the interaction with p e c t i n t o occur. One must, o f c o u r s e , remember t h a t N i s an a v e r a g e v a l u e , a n d t h a t t h e d i s t r i b u t i o n o f b l o c k lengths i s probably broad i n a l l a l g i n a t e samples. I n t h e p h y s i c a l s i t u a t i o n i t may w e l l be t h a t m a i n l y t h e v 1

G

G

G

G

G

G

G

>

1

Q

f î G G

N

T

n

e

G > 1

p

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

Synergistic Gelation of Alginates and Pectins

10. TOFT ET AL.

129

TABLE 2. Gel strengt tions o t i n RS400 a t pH 2.9. T o t a l polymer conc e n t r a t i o n i s 1.2% (v/w).

F

F

G

F

N

G>1

Breaking load (g)

4

Ex10 , P a

GG

GGG

0.70

0.59

0.55

15.8

1000

0.57

0.43

0.39

1 1.8

830

3.9

0.50

0.32

0.26

6.3

460

3.0

0.53

0.27

0.22

6.4

230

2.5

0.41

0.23

0.18

5.6

250

1.9

0.43

0.20

0.12

3.5

0

0

0.02

0

0

2

0

0

4.85

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

CHEMISTRY AND FUNCTION OF PECTINS

130 1

1

•

1

'

1

1

r

08

Figure 7. Modulus of r i g i d i t y against F and f o r d i f f e r e n t a l g i n a t e samples. T o t a l polymer concent r a t i o n i s 1.2% (w/v) i n 1:1 mixtures of p e c t i n and a l g i n a t e s . ç /

ExKT

4

Average b l o c k lenght, N (» 1} G

F i g u r e 8. Data from F i g . 7 p l o t t e d against average block length of G-units excluding the s i n g l e t G-units, N . Q > 1

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

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131

t a i l of the d i s t r i b u t i o n with higher block lengths than 4 takes part i n the i n t e r a c t i o n with p e c t i n . However, the importance of the "GG-blocks" f o r the i n t e r a c t i o n t o occur i s evident. The data i n t h i s paper taken together give full support t o the idea of Thorn e t a l . r e f e r r e d t o e a r l i e r , t h a t f u l l y or p a r t l y protonated^GG-blocks" may i n t e r a c t with e s t e r i f i e d p e c t i n chains g i v i n g j u n c t i o n s i n the g e l network. To exclude the p o s s i b i l i t y t h a t n o n - e s t e r i f i e d p e c t i n chains take p a r t i n the i n t e r a c t i o n , some s t u d i e s with p e c t i n samples b e t t e r c h a r a c t e r i z e d with respect t o the d i s t r i b u t i o n of methoxy groups should be c a r r i e d out. The d e t a i l s of the i n t e r a c t i o n are not known, but s i n c e the chains of α-Q-galacturonic a c i d and of a - L - g u l u r o n i c a c i d are mirror images, apart from the c o n f i g u r a t i o n around C-3, there should be a number of r e g u l a r l y spaced atomic groups along for a strong, cooperative ACKNOWLEDGMENT We thank Dr.Terence J . P a i n t e r f o r v a l u a b l e d i s c u s s i o n s , and f o r r e v i s i n g the E n g l i s h of the manuscript.

Literature Cited 1. 2.

Toft, K, in "Prog. Food. Nutr. Sci.", 1982, 6, 89-96 Toft, K. "Gel Formation in Alginate-Pectin Systems Using Slow Acidifier" presented at the European Science Fundation Workshop on Specific Interactions in Polysaccharide Systems, Uppsala, Sweden, April 26-27, 1983. 3. Dea, I.C.M.; Morrison, A. Adv. Carbohydr. Chem. Biochem., 1975, 31, 241-312. 4. Miles, M.J.; Morris, V . J . ; Carrol, V. Macromolecules, 1984, 17, 2443-2445. 5. Thorn, D; Dea, I.C.M; Morris, R.E.; Powell, D.A. in "Prog. Food Nutr. Sci.", 1982, 6, 97-108. 6. Grasdalen, H.; Larsen, B.; Smidsrød, O. Carbohydr, Res. 1981, 89, 179-191. 7. Grasdalen, H. Carbohydr. Res., 1983, 118, 255-260. 8. Morris, V.J.; Chilvers, G.R. J . Sci. Food Agric., 1984, 35, 1370-1376. 9. Haug, A. "Composition and Properties of Alginates", Report No. 30, Norwegian Institute of Seaweed Research, Trondheim 1964. 10. Smidsrød, O.; Haug, A. Acta Chem. Scand. 1972, 26, 79-88. 11. Grasdalen, H.; Larsen, B.; Smidsrød, O. Carbohydr. Res., 1979, 68, 23-31. 12. Smidsrød, O.; Haug, Α.; Lian, B. Acta Cem. Scand. 1972, 26, 71-78.

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

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13. Painter. T . J . in "The Polysaccharides", Vol. 2, G.O. Aspinall, Ed., Academic Press, New York, 1983, 196-285. 14. Grasdalen, H.; Larsen, B.; Smidsrød, O. Carbohydr. Res. 1977, 56, C11-C15. RECEIVED December 20,

1985

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

11

C o n t r o l o f Pectin Synthesis a n d D e p o s i t i o n during Plant C e l l W a l l G r o w t h D. H. Northcote Department of Biochemistry, University of Cambridge, Cambridge, CB2 1QW England The assembly of the cell wall outside the protoplasts is controlled at 3 levels: a) at the synthase systems which use the nucleosid diphosphat donor b) at the operation of material into syste ) regulation of the directed movement and fusion of vesicles derived from the endomembranes. Pectin is deposited in the wall during primary growth only. The nucleoside diphosphate sugars for pectin synthesis arise by epimerases from the corresponding nucleotides of the glucose series. The epimerases are active during secondary growth so that little control is exerted at these steps. Assembly of pectin occurs in the Golgi apparatus by synthases which probably do not use lipid intermediates. These synthases are controlled and are shut down during secondary growth. The sugar nucleotides are specifically transported into the Golgi cisternae and regulation can occur at these sites. Vesicles, containing pectin, are moved to the plasma-membrane probably directed by microtubules. Fusion of these vesicles with the cell membrane is partly controlled by the level of Ca at the points of fusion. 2+

Pectin is a mixture of complex polymers (1-3). These are formed during primary wall formation while the cell wall is expanding in surface area. At these early stages of growth the pectin polymers contribute in a major way to the texture of the wall especially to its ability to expand and stretch. The wall is much more a fluid structure at this time and water is an extremely important constituent. The primary wall when the matrix is non-lignified can be considered as a fluid plastic structure so that any load applied to the wall is transmitted to the microfibrils by the viscous drag of the plastic deformation of the matrix. This can alter very much with the composition and physical state of the matrix materials, especially with that of the pectin complex (3). The pectin polymers are constituents of the cell plate during cell division and they are the first set of polysaccharides to be 0097-6156/86/0310-0134$06.00/0 © 1986 American Chemical Society

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

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s e c r e t e d by p r o t o p l a s t s d u r i n g the f o r m a t i o n of a new w a l l (4,5). The c o m p o s i t i o n o f the p e c t i n polymers can be a l t e r e d by the a p p l i c a t i o n o f p l a n t growth f a c t o r s (6). The t e x t u r e of f r u i t and the r i p e n i n g p r o c e s s depends upon the a l t e r a t i o n of the p e c t i n components of the c e l l w a l l s o f f r u i t t i s s u e . I t i s apparent t h a t the p e c t i n polymers are a v a r i a b l e and i m p o r t a n t f e a t u r e of the young, growing wall. Numerous s l i m e s and m u c i l a g e s s e c r e t e d by p l a n t c e l l s have c h e m i c a l c o m p o s i t i o n s w h i c h a r e r e l a t e d t o the p e c t i n s (polymers c o n t a i n i n g a r a b i n o s e , g a l a c t o s e and g a l a c t u r o n i c a c i d ) and these m a t e r i a l s are formed t o l u b r i c a t e such organs as r o o t s and t o r e t a i n water w i t h i n a g e l s t r u c t u r e t o keep the organs m o i s t a t the v a r i o u s stages of growth of the p l a n t ( 7 , 8 ) . The f o r m a t i o n of p e c t i n and i t s assembly f o r f i n a l e x p o r t a c r o s s the plasmamembrane w i l l be c o n s i d e r e d here as a p r o d u c t i o n l i n e t o i n d i c a t e the v a r i o u s l i m i t i n g s t e p s w h i c h a r e c o n t r o l l e d t o m o n i t o r i t s p r o d u c t i o n . The v a r i o u w i l l be d e s c r i b e d s e p a r a t e l assessed. The c h a n n e l s w h i c h o p e r a t e f o r p r o d u c t i o n and movement of the polymers w i t h i n the c y t o p l a s m and endomembrane system o f the c e l l s a r e : - Channel 1. P r o d u c t i o n and movement of n u c l e o t i d e sugar d o n o r s . Channel 2. S y n t h e s i s and c o m p a r t m e n t a l i z a t i o n of the p e c t i n polymers w i t h i n the endomembrane system. Channel 3. Movement of v e s i c l e s and f u s i o n w i t h the plasmamembrane f o r assembly and deposi t i o n w i t h i n the w a l l . D u r i n g f r u i t r i p e n i n g the p e c t i n i s degraded and t h i s i s a l s o a c o n t r o l l e d p r o c e s s brought about by the i n d u c t i o n of d e g r a d a t i o n enzymes such as p o l y g a l a c t u r o n a s e ( 9 ) . D e g r a d a t i o n or t u r n o v e r may go on t o a l i m i t e d e x t e n t i n the normal growing and t h i c k e n i n g w a l l d u r i n g d i f f e r e n t i a t i o n . However, t h e r e i s e v i d e n c e t h a t the p e c t i n m a t e r i a l once d e p o s i t e d i n the w a l l remains t h e r e (10) so t h a t the main c o n t r o l s o f the amount p r e s e n t d u r i n g d i f f e r e n t i a t i o n a r e those w h i c h operate f o r i t s s y n t h e s i s and d e p o s i t i o n . Channel 1.

P r o d u c t i o n and Movement of N u c l e o t i d e Sugar Donors

The n u c l e o t i d e sugar donors a r e formed o u t s i d e the endomembrane system i n the c y t o s o l . Those f o r p e c t i n a r i s e by epimerase r e a c t i o n s from the c o r r e s p o n d i n g n u c l e o t i d e sugar compounds of the g l u c o s e s e r i e s , F i g u r e 1. The epimerases a r e a c t i v e throughout growth. Thus the p o t e n t i a l f o r the p r o d u c t i o n o f UDPGal, UDPGalAc and UDPAra, w h i c h are the donors o f the p e c t i c m a t e r i a l , i s p r e s e n t even d u r i n g secondary t h i c k e n i n g when no p e c t i n i s d e p o s i t e d i n the w a l l (11,12). The epimerase r e a c t i o n s are t h e r e f o r e not s i t e s a t w h i c h c o n t r o l of the p o l y s a c c h a r i d e s y n t h e s i s i s e x e r t e d . The n u c l e o t i d e sugar compounds a r e h y d r o p h i l i c and i t i s n e c e s s a r y t o t r a n s f e r them t o the s i t e s o f the synthases w i t h i n the endomembrane system. T h e i r t r a n s p o r t a c r o s s the membrane i s an o b v i o u s c o n t r o l p o i n t . Not o n l y i s the r a t e of p o l y s a c c h a r i d e s y n t h e s i s c o n t r o l l e d by t h i s mechanism but i n a d d i t i o n the q u a l i t a t i v e n a t u r e of the polymer found i n the membrane compartment may be d e t e r m i n e d . Thus a l t h o u g h the synthases may be p r e s e n t i n a p a r t i c u l a r p a r t of the membrane system e.g. endoplasmic r e t i c u l u m , the assembly of the p o l y s a c c h a r i d e does not o c c u r a t t h i s l e v e l i f the n u c l e o t i d e sugar cannot e n t e r the lumen of the compartment ( 1 3 ) . The main assembly o f the p o l y s a c c h a r i d e s o c c u r s a t the G o l g i a p p a r a t u s , F i g u r e 1.

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Epinerases a c t i v e throughout growth

UDPGal

Donor Synthesis Cytosol

-

UDPGlcA

UDPXyl

NT Control of transport i n t o G o l g i apparatus

Endomembrane System

Channel 2 Synthases control of activity

NX Control f o r sorting and Packaging i n t o vesicles

Channel 3 Movement t o plasmamembrane

C o n t r o l o f F u s i o n and deposition i n wall. Modification of material i n wall

Wall

F i g u r e 1. Epimerase r e a c t i o n s sugar d o n o r s .

f o r the

f o r m a t i o n of

nucleotide

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The assembly o f the p e c t i c p o l y s a c c h a r i d e s , l i k e most of the c e l l w a l l p o l y s a c c h a r i d e s , w i t h the p o s s i b l e e x c e p t i o n of c e l l u l o s e , appears to take p l a c e by t r a n s f e r of the g l y c o s y l group from the n u c l e o t i d e - s u g a r compound t o the growing polymer w i t h o u t the n e c e s s i t y f o r a d o l i c h o l phosphate i n t e r m e d i a t e a c c e p t o r . T h i s has consequences f o r the mechanism o f the method o f t r a n s p o r t of the sugars a c r o s s the membrane. F o r the assembly o f the sugar p o r t i o n of Nl i n k e d g l y c o p r o t e i n s w h i c h o c c u r s i n the endoplasmic r e t i c u l u m , i s o p r e n o i d phosphates a c t as i n t e r m e d i a t e a c c e p t o r s and s e v e r a l mechanisms of t r a n s p o r t i n v o l v i n g these l i p i d sugar compounds have been suggested ( 1 4 ) . I t i s a l s o c l e a r t h a t when the g l y c o s y l a t i o n s o c c u r w i t h i n the G o l g i a p p a r a t u s these methods of t r a n s p o r t are not i n v o l v e d and d i r e c t e n t r y of the n u c l e o t i d e - s u g a r compounds by s p e c i f i c t r a n s p o r t e r s may take p l a c e ( 1 5 ) . Channel 2. S y n t h e s i s and C o m p a r t m e n t a l i z a t i o n w i t h i n the Endomembrane

o f the P e c t i n Polymers

The s y n t h a s e s or t r a n s g l y c o l y s e s w h i c h w i l l t r a n s f e r the g a l a c t o s e , g a l a c t u r o n i c a c i d and a r a b i n o s e to the p e c t i c polymers are the p r i n c i p a l enzymic p o i n t s a t w h i c h c o n t r o l i s m a i n t a i n e d f o r the s y n t h e s i s of the p e c t i n (13^, 16-20). The a c t i v i t y o f these enzymes d e c r e a s e s d u r i n g secondary t h i c k e n i n g of the w a l l when p e c t i n depos i t i o n c e a s e s . By the c a r e f u l use o f i n h i b i t o r s o f t r a n s c r i p t i o n and t r a n s l a t i o n and by the use o f a m o n o c l o n a l a n t i b o d y i t was shown t h a t the changes i n a c t i v i t y o f enzymes were c o r r e l a t e d w i t h changes i n the amount o f enzyme p r o t e i n p r e s e n t a t the membrane (19,21). Thus t h i s c o n t r o l i s o p e r a t e d a t the l e v e l o f the genome and the changes i n the c e l l w a l l a r e p a r t of a d e v e l o p m e n t a l p r o c e s s . One of the main c o n t r o l p o i n t s of p o l y s a c c h a r i d e s y n t h e s i s , t h e r e f o r e , i s the amount and c o n s e q u e n t l y the a c t i v i t y of the t r a n s g l y c o s y l a s e s o r s y n t h a s e s w h i c h o p e r a t e s f o r the p a r t i c u l a r n u c l e o t i d e sugar a t the lumen o f the membrane. However t h e r e i s a f u r t h e r c o m p l i c a t i o n w h i c h a r i s e s w i t h complex polymers such as those i n p e c t i n . These are h e t e r o p o l y m e r s e.g., the rhamnogalacturonan, the a r a b i n o g a l a c t a n and the r h a m n o g a l a c t u r o n a n - a r a b i n o g a l a c t a n . There a r e a l s o some, l i k e the a r a b i n o g a l a c t a n w h i c h a r e h i g h l y branched p o l y s a c c h a r i d e s (3,22) and, i n a d d i t i o n , minor sugars such as fucose and x y l o s e o c c u r i n these complex p o l y s a c c h a r i d e s . I f the sugars are added d i r e c t l y to the growing polymer one a t a t i m e , then the s y n t h e s i s of the main c h a i n s may depend t o some e x t e n t on the i n c o r p o r a t i o n a t v a r i o u s stages of a d i f f e r e n t sugar o r b r a n c h p o i n t so t h a t the a c t i v i t y o f the t r a n s g l y c o s y l a s e f o r m i n g the c h a i n i s maint a i n e d . A l t e r n a t i v e l y s m a l l e r s u b u n i t s o f the main polymer may be preformed on i n t e r m e d i a t e c a r r i e r s , such a l i p i d o r p r o t e i n , and then i n c o r p o r a t e d as b l o c k s i n t o the polymer ( 2 3 ) . T h i s method o f s y n t h e s i s would resemble t h a t f o r the f o r m a t i o n of some g l y c o p r o t e i n s i n a n i m a l s and b a c t e r i a . I t would r e s u l t i n a c e r t a i n amount of r e g u l a r i t y i n the c h e m i c a l c o m p o s i t i o n of the p o l y s a c c h a r i d e t h a t i s formed and i t would i n t r o d u c e secondary stages a t w h i c h c o n t r o l o f the assembly c o u l d be e x e r t e d . I n a d d i t i o n t o the c o n t r o l o f the amount o f s y n t h a s e w h i c h d e f i n e s the t o t a l a c t i v i t y o f the enzyme, the a c t i v i t y of some synthases are modulated by the energy s t a t u s of the c e l l s i n c e they a r e i n h i b i t e d by l e v e l s of n u c l e o s i d e mono and d i p h o s p h a t e s ( 1 7 ) .

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As the p o l y s a c c h a r i d e i s b e i n g formed i t must be t r a n s f e r r e d to p a r t i c u l a r p a r t s o f the membrane system and f i n a l l y packaged f o r e x p o r t to d e f i n i t e r e g i o n s o f the p e r i p h e r y of the c e l l , F i g u r e 1. Very l i t t l e i s known about the c o n t r o l of t h i s i m p o r t a n t p a r t of the assembly system. Some i d e a s f o r the mechanisms o f the s o r t i n g and t r a n s p o r t p r o c e s s e s may be o b t a i n e d from the l i m i t e d e v i d e n c e known f o r the s y n t h e s i s and e x p o r t o f some g l y c o p r o t e i n s from the G o l g i a p p a r a t u s and t h e i r c a r r i a g e between the endoplasmic r e t i c u l u m and G o l g i a p p a r a t u s . I t has been shown f o r i n s t a n c e t h a t the c i s t e r n a e of the G o l g i a p p a r a t u s can be d i v i d e d i n t o c i s , m e d i a l and t r a n s r e g i o n s and t h a t d i f f e r e n t t r a n s g l y c o s y l a s e s are l o c a t e d a t d i f f e r e n t r e g i o n s (24,25). I t has a l s o been shown f o r a few polymers t h a t d u r i n g t h e i r p a c k a g i n g and e x p o r t from one membrane compartment to a n o t h e r , they are s e p a r a t e d from o t h e r s by s e q u e s t e r i n g them t o p a r t i c u l a r r e g i o n s or c i s t e r n a e . T h i s i s done by the p r o v i s i o n of s p e c i f i c r e c o g n i t i o n markers on the polymer such as removal of the terminal glucose u n i t s t from endoplasmic r e t i c u l u 6-phosphate a t the n o n - r e d u c i n g end of the o l i g o s a c c h a r i 4 e ( f o r t r a n s p o r t from G o l g i a p p a r a t u s to lysosome) (14,25-29). The enzymic r e a c t i o n s w h i c h e n a b l e the marker to be developed upon the o l i g o s a c c h a r i d g e o f the g l y c o p r o t e i n do however r e c o g n i s e the p r o t e i n p o r t i o n o f the polymer i n a d d i t i o n to the o l i g o s a c c h a r i d e . I n t h i s way, f o r example, d i s t i n c t i o n s can be made between g l y c o p r o t e i n s t h a t c a r r y mannose s i n c e o n l y some o f these w i l l be t r a n s p o r t e d t o the lysosome. Channel 3. Movement o f V e s i c l e s and F u s i o n w i t h the Plasmamembrane f o r Assembly and D e p o s i t i o n w i t h i n the W a l l V e s i c l e s c o n t a i n i n g the p e c t i n p o l y s a c c h a r i d e a r i s e from the G o l g i a p p a r a t u s . The membranes o f the c i s t e r n a e o f the G o l g i a p p a r a t u s , where d i s p e r s a l o c c u r s , become m o d i f i e d so t h a t the membranes o f the s e c r e t o r y v e s i c l e s r e s e m b l e , b o t h i n t h e i r c h e m i c a l c o m p o s i t i o n and u l t r a s t r u c t u r a l appearance, the plasmamembrane. The v e s i c l e s are t r a n s p o r t e d t o s p e c i f i c a r e a s of the c e l l s u r f a c e when the c e l l i s expanding i n s u r f a c e a r e a . I n some c e l l s i t can be seen t h a t v e s i c l e f u s i o n depends upon a c h a r a c t e r i s t i c d i s t r i b u t i o n o f p r o t e i n p a r t i c l e s w i t h i n the membrane (30,32). There i s thus an e l a b o r a t i o n of the membranes b o t h c h e m i c a l l y and by u l t r a s t r u c t u r a l changes so t h a t f u s i o n i s p o s s i b l e a t the r e q u i r e d s i t e . The v e s i c l e s produced from the membrane system may be d i r e c t e d , i n p a r t , to p a r t i c u l a r c e l l s u r f a c e s by m i c r o t u b u l e s . A l t h o u g h t h i s i s not so apparent a t the e a r l y s t a g e s o f growth, when p e c t i n i s b e i n g d e p o s i t e d i n the w a l l , as i t i s d u r i n g secondary w a l l f o r m a t i o n . D u r i n g the development o f xylem e l e m e n t s , f o r i n s t a n c e , when a s p i r a l or r e t i c u l a t e secondary w a l l i s formed, the m i c r o t u b u l e s are d i s t r i b u t e d j u s t under the plasmamembrane a t the s i t e s o f secondary t h i c k e n i n g (33,34). However a t t h i s stage no p e c t i n i s b e i n g accumulated i n the w a l l and the d i s t r i b u t i o n o f the m i c r o t u b u l e s r e f l e c t s the a d d i t i o n s o f hemic e l l u l o s e and c e l l u l o s e w i t h i n the t h i c k e n i n g . D u r i n g the f o r m a t i o n of the p r i m a r y w a l l the m i c r o t u b u l e s a r e d i s t r i b u t e d j u s t under the plasmamembrane but they a r e not so o b v i o u s l y l o c a l i z e d . They o c c u r i n groups o f t h r e e or f o u r randomly d i s t r i b u t e d i n a c i r c u l a r o r

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

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helical arrangement around the cell. It may be that the pectin is deposited fairly evenly over the wall at this time. Pectin-like material is secreted from suspension tissue culture cells of sycamore and the rate of secretion of these polysaccharides can be measured using radioactive arabinose which is incorporated into arabinogalactans. The addition of Ca increased the steady state rate of secretion within seconds. The stimulation was brought about by an action on the normal mechanism of cell-wall polysaccharide export from the cytoplasm. It seemed therefore that the fusion of the vesicles with the plasmamembrane was a rate-limiting step and is probably a control point. The action of the Ca was to increase the rate of fusion at the cell membrane so that there was an immediate increase in the amount of polysaccharide secreted (35). The control could be established if at any one time there was at the membrane more vesicles than those that were fusing. However in order to sustain the increase in secretion it would be necessary to transmit the signal back to the syntheti Golgi apparatus so that would produce the requisite number of vesicles containing the polysaccharides necessary to maintain the new rate of fusion and secretion. The turnover of the Golgi apparatus can be very fast in plant cells and times of 5-40 minutes have been calculated (36). In vitro experiments which involved isolation of membrane fractions from maizeroot cells also showed that Ca^ was necessary for membrane fusion (37). Analysis of the membranes indicated that the Ca dependence involved membrane proteins and one of these was a Ca and Mg^ dependent ATPase (38). Once deposited in the wall the pectin may undergo further chemical modification by transglycosylases which join particular polymers such as the arabinogalactans to polygalacturonans (39,40). Even ester linkages with diferulic acid may occur to give bridges between different polysaccharides of the wall (41). It is well known that Ca form ionic bonds between the acidic groups of the pectin polymers. However, regardless of the presence of these intermolecular covalent and ionic bonds, in the primary wall when it is unlignified, the main cohesion between the constituents must be the hydrogen bonding that allows a flexibility to the structure of the wall which depends on its most variable feature the water content especially in its relationship to pectin (3). When lignification occurs during secondary thickening the polysaccharides become enclosed in a crosslinked polymer cage and bridges between the polysaccharides and the phenylpropanoid radicals are formed. The wall thus develops great tensile strength because of the microfibrils and it becomes a rigid structure because of the lignified matrix in which the hydrophobic lignin has replaced the water. The pectin at this time represents only a minor constituent of the wall and contributes very little to its texture or properties. 2+

2+

+

2+

+

2+

Literature Cited 1. 2.

Ericson, M.C.; Elbein, A.D. In "Biosynthesis of cell wall polysaccharides and glycoproteins"; Preiss, J . Ed. Academic Press: New York, 1980; vol. 3, Chap. 16; p. 589-616. Dey, P.M.; Brinson, K. Advanc. Carb. Chem. Biochem. 1984, 42, 265-382.

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

140 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40.

chemistry and function of pectins Northcote, D.H. Ann. Rev. Pl. Physiol. 1972, 23, 113-132. Northcote, D.H. In "The synthesis, Assembly and turnover of Cell Surface Components"; Poste, G.; Nicolson, G.L., Eds.; NorthHolland: Amsterdam, 1977; Chap. 10; p. 717-739. Hanke, D.E.; Northcote, D.H. J . Cell Sci. 1974, 14, 29-50. Rubery, P.H.; Northcote, D.H. Biochim. Biophys. Acta 1971, 222, 95-108. Wright, K.; Northcote, D.H. Biochem. J . 1974, 139, 525-534. Gould, J.; Northcote, D.H. In "Bacterial Adhesion: Mechanisms and Physiological Significance"; Fletch, M.M.; Savage, D.W.; Eds.; Plenum Press: New York, 1985; Chap. 5; p. 89-109. Grierson, D.; Slater, Α.; Spiers, J.; Tucker, G.A. Planta 1985, 163, 263-271. Thornber, J.; Northcote, D.H. Biochem. J . 1961, 81, 455-464. Dalessandro, G.; Northcote, D.H. Biochem. J . 1977, 162, 267-279. Dalessandro, G.; Northcote, D.H. Biochem. J . 1977, 162, 281-288. Bolwell, G.P.; Northcote Kornfeld, R.; Kornfeld Dixon, W.T.; Northcote, D.H. In "Developments in Cell Biology: Secretory processes"; Deane, R.T.; Stahl, P. Eds.; Butterworths: London, 1985, p. 77-98. Dalessandro, G.; Northcote, D.H. Planta 1981, 151, 53-60. Dalessandro, G.; Northcote, D.H. Planta 1981, 151, 61-67. Bolwell, G.P.; Northcote, D.H. Planta 1981, 152, 225-233. Bolwell, G.P.; Northcote, D.H. Biochem. J . 1983, 210, 509-515. Bolwell, G.P.: Northcote, D.H. Phytochem. 1985, 24, 699-702. Bolwell, G.P.; Northcote, D.H. Planta 1985, 162, 139-146. McNeil, M.; Darvill, A.G.; Fry, S.C.; Albersheim, P. Ann. Rev. Biochem. 1984, 53, 625-663. Northcote, D.H. Phil. Trans. Roy. Soc. Β 1982, 300, 195-2o6. Roth, J.; Berger, E.G. J . Cell Biol. 1982, 93, 223-229. Dunphy,W.G.;Brands, R.; Rothman, J.E. Cell 1985, 40, 463-472. Lodish, H.F.; Kong, N. J . Cell Biol. 1984, 98, 1720-1729. Kornfeld, S.; Reitman, M.L.; Varki, Α.; Goldberg, D.; Gabel, C.A. In "Membrane Recycling"; Evered, D.; Collins, G.M. Eds.; Pitman: London, 1982, p. 138-156. Shepherd, V.; Schlesinger, P.; Stahl, P. Current Topics in Membranes and Transport 1983, 1, 317-338. Sly, W.S.; Fischer, H.D. J . Cell Biochem. 1982, 18, 67-85. Satir, B.; Schooley, C.; Satir, P. J . Cell Biochem, 1973, 56, 153-156. Burwen, S.J.; Satir, B.H. J . Cell Biol. 1977, 73, 660-671. Da Silva, P.P.; Nogueira, L. J . Cell Biol. 1977, 73, 161-181. Wooding, F.B.P.: Northcote, D.H. J . Cell. Biol. 1964, 23, 327-337. Pickett-Heaps, J.D.; Northcote, D.H. J . Exptl. Bot. 1966, 17, 20-26. Morris, M.R.; Northcote, D.H. Biochem. J . 1977, 166, 603-618. Robinson, D.G.; Kristen, N. Int. Rev. Cytol. 1982, 7, 89-127. Baydoun, E.A.H.; Northcote, D.H. J . Cell Sci. 1980, 45, 169-186. Baydoun, E.A.H.; Northcote, D.H. Biochem. J . 1981, 193, 781-792. Stoddart, R.W.; Northcote, D.H. Biochem. J . 1967, 105, 45-59. Rubery, P.H.; Northcote, D.H. Biochim. Biophys. Acta 1970, 222, 95-108.

41. Fry, S.C. Planta 1983, 157, 111-123. Received December 12, 1985 In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

12

C o m p a r a t i v e Analysis o f Pectins f r o m Pericarp a n d L o c u l a r G e l i n D e v e l o p i n g T o m a t o Fruit Donald J. Huber and James H. Lee Vegetable Crops Department, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL 32611

Pectins from tomat pericar d locula l examined to ascertai polysaccharides change accompany development of the respective tissue types. Ripening of pericarp was marked by increased quantities and depolymerization of soluble pectins. Soluble pectins from ripe fruit exhibited a lower degree of esterification, a lower average molecular size and a decreased neutral sugar content compared to pectins from unripe fruit. Pectins from gel showed little evidence of depolymerization during the active period of gel autolysis, consistent with the observation that only trace levels of endo-D-galacturonanase were found in this tissue. Degree of esterification of gel pectins was high and showed no change during development. Gel pectins were rich in arabinose, galactose, and xylose, all of which decreased during early gel formation. The ripening of many fruit types includes as a major event extensive modifications in texture, often attributed to the action of specific cell-wall hydrolases, notably the D-galacturonanases (1,2). The temporal relationship between softening, pectin solubilization and depolymerization, and the appearance of endo-D-galacturonanase (EDG, E.C. 3.2.1.15) activity together strongly underscore the dominant role of these enzymes (3-11). It is becoming increasingly evident that processes other than those involving EDG are operative in the cell-wall metabolism of ripening fruit. The cell-wall neutral sugars galactose and/or arabinose decrease during ripening of many fruit types (8,12-14) including rm tomato fruit (15,16), which has no EDG (17,18). In a species survey, Gross and Sams (19) reported losses of cell-wall galactose and arabinose during ripening in fourteen of seventeen fruit types examined. In both tomato (11) and strawberry fruit (20), ripening was accompanied by decreases in the molecular size 0097-6156/86/0310-O141$06.00/0 © 1986 American Chemical Society

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o f h e m i c e l l u l o s e s and the absence o f EDG i n s t r a w b e r r y (20-22) i n d i c a t e s t h a t t h e a l t e r e d a l k a l i - s o l u b l e polymers d i d not a r i s e as an i n d i r e c t r e s u l t o f p e c t i n d e g r a d a t i o n . Of equal i n t e r e s t i n s t r a w b e r r y f r u i t was the o b s e r v a t i o n t h a t l e v e l s of s o l u b l e p e c t i n s i n c r e a s e d i n t h e apparent absence o f EDG. S o l u b l e p e c t i n s showed no e v i d e n c e o f t h e p r o g r e s s i v e d e p o l y m e r i z a t i o n e x h i b i t e d by s o l u b l e p e c t i n s from tomato (U_), watermelon (23) and peach ( 4 ) , a l l of which c o n t a i n EDG. In a d d i t i o n t o s t r a w b e r r y , plum (2T), hot pepper (25) and muskmelon ( 3 , 2 6 ) r e p r e s e n t o t h e r f r u i t s which show i n c r e a s e d l e v e l s of s o l u b l e p e c t i n s d u r i n g r i p e n i n g y e t a p p a r e n t l y c o n t a i n no EDG. I t i s apparent from t h e s e s t u d i e s t h a t i n c r e a s e d p e c t i n s o l u b i l i z a t i o n d u r i n g r i p e n i n g i s not i n i t s e l f s u f f i c i e n t e v i d e n c e f o r i n f e r r i n g t h a t EDG i s r e s p o n s i b l e . Tomato f r u i t appeared t o r e p r e s e n t a unique system f o r i n v e s t i g a t i n g p o s s i b l e v a r i a b i l i t y i n the f u n c t i o n s of p e c t i n s i n t e x t u r e m o d i f i c a t i o n s . F i r s t l y , t h e tomato f r u i t i s composed o f d i s t i n c t tissue types, includin e x h i b i t markedly d i f f e r e n gel d e v e l o p s p r i o r t o r i p e n i n g o f p e r i c a r p and e v e n t u a l l y , toward the t e r m i n a l s t a g e of r i p e n i n g , e x h i b i t s an almost l i q u i d n a t u r e . T h i s developmental s c e n a r i o i s i n some r e s p e c t s s i m i l a r t o t h e e t h y l e n e - i n d u c e d m a c e r a t i o n o f watermelon f r u i t ( 2 7 ) , a process i n v o l v i n g g r e a t l y i n c r e a s e d l e v e l s o f EDG and e x t e n s i v e s o l u b i l i z a t i o n and d e p o l y m e r i z a t i o n o f p e c t i n s ( 2 3 ) . In c o n t r a s t , tomato l o c u l a r gel has been r e p o r t e d t o c o n t a i n l i t t l e (b) o r no EDG a c t i v i t y ( 2 8 ) . The o b j e c t i v e o f t h i s study was t o examine some of t h e s t r u c t u r a l f e a t u r e s o f p e c t i n s from tomato p e r i c a r p and g e l , and t o r e l a t e t h i s i n f o r m a t i o n t o t h e t e x t u r a l c h a r a c t e r i s t i c s o f these t i s s u e s . M a t e r i a l s and Methods P l a n t M a t e r i a l . Tomato ( L y c o p e r s i c o n e s c u l e n t u m , M i l l , var Sunny) f r u i t were h a r v e s t e d from p l a n t s grown at t h e IFAS H o r t i c u l t u r a l Farm near G a i n e s v i l l e , F L . F r u i t were s e l e c t e d a t f i v e s t a g e s o f development: immature green (undeveloped f r u i t , gel t i s s u e f i r m ) , mature-green (gel f o r m a t i o n i n i t i a t e d ) , t u r n i n g , p i n k and r i p e (red). F r u i t were s u r f a c e s t e r i l i z e d w i t h 0.05% N a - h y p o c h l o r i t e and r i n s e d . F r u i t were q u a r t e r e d , the p e r i c a r p and l o c u l a r gel s e p a r a t e d , and both s t o r e d a t - 2 0 ° C . Preparation of Ethanol-Insoluble S o l i d s . P e r i c a r p o r gel (100 g) w h i l e f r o z e n was p l a c e d i n 400 ml c o l d ( 4 ° C ) ethanol and p a r t i a l l y thawed. The t i s s u e was homogenized i n a S o r v a l l Omnimixer f o r 1 min and then r e f l u x e d at 84°C f o r 30 min t o i n a c t i v a t e p e c t i n hydrolase a c t i v i t y . This technique i s e f f e c t i v e at i n a c t i v a t i n g w a l l - b o u n d p e c t i n - h y d r o l y z i n g a c t i v i t y (29) and r e s u l t s i n no apparent changes i n the c h a r a c t e r i s t i c s o f p e c t i n s ( 3 0 ) . After r e f l u x i n g , t h e suspension was r i n s e d through M i r a c l o t f i and washed, i n s u c c e s s i o n , w i t h 80% e t h a n o l ( 1 1 ) , 80% acetone ( 1 1 ) , and 100% acetone ( 1 1 ) . The r e s u l t i n g powders were a i r - d r i e d at 40°C and s t o r e d covered a t room t e m p e r a t u r e .

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Measurement of T o t a l and S o l u b l e P e c t i n s . T o t a l p e c t i n s i n e t h a n o l - i n s o l u b l e powders were measured u s i n g t h e procedure d e s c r i b e d by Ahmed and L a b a v i t c h ( 3 1 ) . P e c t i n s were assayed c o l o r i m e t r i c a l l y u s i n g t h e method "67 Blumenkrantz and Asboe-Hansen ( 3 2 ) . For measurement o f s o l u b l e p e c t i n s , 20 mg e t h a n o l - i n s o l u b l e powder were p l a c e d i n 7 ml of e i t h e r d i s t i l l e d water o r N a - a c e t a t e b u f f e r (40 mM, pH 5.0) c o n t a i n i n g 20 mM EDTA and i n c u b a t e d w i t h s h a k i n g a t room temperature f o r 6 h r . A l i q u o t s ( 0 . 5 ml) were f i l t e r e d t h r o u g h g l a s s f i b e r f i l t e r s and a n a l y z e d f o r s o l u b l e pectins. Gel F i l t r a t i o n and Ion-Exchange Chromatography. P e c t i n s f o r gel f i l t r a t i o n and ion-exchange chromatography were s o l u b l i z e d from powders u s i n g t h e Na-acetate-EDTA b u f f e r a t room t e m p e r a t u r e . This e x t r a c t i o n procedure was found t o maximize the y i e l d o f p e c t i n s ( r e l a t i v e t o water e x t r a c t i o n s ) and produced no apparent modifications in pecti two v a r i a b l e s o f major concer gel f i l t r a t i o n and ion-exchange chromatography o f p e c t i n s have been described (20). N e u t r a l Sugar A n a l y s i s . A n a l y s e s o f n e u t r a l sugars a s s o c i a t e d w i t h tomato p e c t i n s were performed u s i n g polymers r e c o v e r e d from ion-exchange p r o f i l e s . G r a d i e n t f r a c t i o n s were combined, c o n c e n t r a t e d t o a volume of 10 ml and d i a l y z e d ( S p e c t r a p o r 2,000) a g a i n s t d i s t i l l e d hLO f o r 36 hr at 4 ° C . Samples were a i r - d r i e d under a stream of f i l t e r e d a i r , h y d r o l y z e d and a c e t y l a t e d ( 3 3 ) , and examined u s i n g gas chromatography on a column o f GP 3% SP-23T0 ( S u p e l c o ) at 2 2 5 ° C . N e u t r a l sugar a n a l y s e s were performed on p e c t i n s p r e p a r e d from t h r e e s e p a r a t e tomato f r u i t i s o l a t e s . Percent E s t e r i f i c a t i o n . E t h a n o l - i n s o l u b l e powder (200 mg) was suspended i n 10 ml 0 . 5 N NaOH c o n t a i n i n g e t h a n o l as i n t e r n a l s t a n d a r d . A f t e r 1 h r , the s u s p e n s i o n s were f i l t e r e d , d i s t i l l e d under vacuum u n t i l 5 ml were c o l l e c t e d , and t h e d i s t i l l a t e a n a l y z e d f o r methanol u s i n g gas chromatography on a column o f Super Q ( A l l t e c h ) operated at 160°C. H e m i c e l l u l o s e E x t r a c t i o n and A n a l y s i s . H e m i c e l l u l o s e s were prepared from a - a m y l a s e - t r e a t e d e t h a n o l - i n s o l u b l e powders i n the manner p r e v i o u s l y d e s c r i b e d ( 1 1 ) . A p p r o x i m a t e l y 4 mg h e m i c e l l u l o s e i n 2 ml N a - c i t r a t e - p h o s p h a t e b u f f e r were f r a c t i o n a t e d on a column (1.5 cm x 60 cm) o f U l t r o g e l AcA 34 e l u t e d w i t h N a - c i t r a t e phosphate b u f f e r (20mM, pH 5.5) c o n t a i n i n g 50 mM NaCl ( 1 1 ) . F r a c t i o n s o f 0 . 5 ml were a n a l y z e d f o r t o t a l sugar u s i n g t h e p h e n o l - s u l f u r i c a c i d procedure ( 3 4 ) , and f o r x y l o g l u c a n u s i n g Kooiman's i o d i n e s t a i n i n g t e c h n i q u e (35) as d e s c r i b e d by N i s h i t a n i and Masuda ( 3 6 ) . Enzyme A s s a y s . E n d o - D - g a l a c t u r o n a n a s e a c t i v i t y i n tomato p e r i c a r p and gel was e x t r a c t e d and assayed as p r e v i o u s l y d e s c r i b e d f o r p e r i c a r p t i s s u e ( 1 1 ) . P r o t e i n was measured u s i n g t h e method of B r a d f o r d (37J employing bovine serum albumin as s t a n d a r d .

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Results I n i t i a l experiments employing aqueous (water o r b u f f e r ) e x t r a c t i o n procedures f o r p r e p a r i n g w a l l i s o l a t e s from tomato p e r i c a r p and gel d i s c l o s e d t h a t s i g n i f i c a n t q u a n t i t i e s o f p e c t i n s were l o s t d u r i n g washing of c e l l w a l l . T h i s problem was most s e r i o u s i n p e r i c a r p at advanced s t a g e s o f r i p e n i n g and w i t h gel a t a l l developmental stages examined. For t h i s r e a s o n , e t h a n o l - i n s o l u b l e powders were used as s t a r t i n g m a t e r i a l f o r a l l p e c t i n a n a l y s e s . As another advantage, i n a c t i v a t i o n of endogenous enzymes which remain bound and a u t o l y t i c a l l y a c t i v e i n c e l l - w a l l i s o l a t e s (29) i s more r e a d i l y a c c o m p l i s h e d i n ethanol homogenates. Heat t r e a t m e n t of aqueous e x t r a c t s promotes g - e l i m i n a t i v e degradation of pectins (38), whereas we have noted no apparent e f f e c t of b o i l i n g 80% ethanol on the s o l u b i l i t y , m o l e c u l a r s i z e o r n e u t r a l sugar c o n t e n t o f p e c t i n s s o l u b i l i z e d from powders so p r e p a r e d ( 3 0 ) . A n a l y s i s of t o t a l pectin o f i n c r e a s i n g p e c t i n (on p e r i c a r p (Table I ) . P e r i c a r p l e v e l s g r e a t l y exceeded t h o s e i n

Table I.

T o t a l and S o l u b l e (Acetate-EDTA B u f f e r ) P e c t i n s i n Ethanol Powders from Tomato P e r i c a r p and G e l . Average o f 3 r e p l i c a t i o n s . Numbers i n parentheses r e p r e s e n t t o t a l p e c t i n s e x p r e s s e d on t i s s u e f r e s h weight b a s i s .

Stage of development

Total

Pericarp Soluble

Gel Total

Soluble

, y g . mg powder" _ ( y g . mg f r e s h wgt" ) (6.9) 6175 143?8 (6.9) 91.9 168.2 (5.7) 105.7 196.4 (5.6) 192.2 188.5 (6.1) 201.3 211.1 1

Immature green Mature green Turning Pink Ripe

258.7 298.1 310.9 328.4 334.6

(2.7) (1.8) (1.7) (1.8) (2.2)

5 0 145.6 117.0 122.8 164.9

gel a t a l l s t a g e s o f development. I t s h o u l d be noted t h a t a l l subsequent a n a l y s e s a r e performed u s i n g o n l y t h o s e p e c t i n s s o l u b i l i z e d from powders by acetate-EDTA b u f f e r . In p e r i c a r p , t h e q u a n t i t y of p e c t i n s s o l u b i l i z e d as a percentage of t o t a l p e c t i n s ranged from 24% a t t h e immature-stage t o 60% a t the r i p e s t a g e . In g e l , g r e a t e r than 60% of t h e t o t a l p e c t i n s were r e a d i l y s o l u b l e i n b u f f e r and were n e a r l y e q u a l l y s o l u b l e i n water a l o n e (not shown). T h i s l a t t e r o b s e r v a t i o n u n d e r s c o r e s t h e importance of a v o i d i n g aqueous procedures when p r e p a r i n g i s o l a t e s o f s t r u c t u r a l p o l y s a c c h a r i d e s from g e l . P r i o r t o chromatography, e x t r a c t s c o n t a i n i n g s o l u b l e p e c t i n s were c e n t r i f u g e d and f i l t e r e d through glass f i b e r f i l t e r s . I n c l u d i n g t h e s e s t e p s d i d not a f f e c t p e c t i n l e v e l s and g r e a t l y improved column performance and l o n g e v i t y i n terms o f both f l o w r a t e and e l u t i o n c h a r a c t e r i s t i c s . It i s understood t h a t t h e b e h a v i o r o f p o l y s a c c h a r i d e s , p a r t i c u l a r l y the a n i o n i c p e c t i n s , on s i z e e x c l u s i o n g e l s s h o u l d be i n t e r p r e t e d w i t h

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c a u t i o n . The known tendency o f t h e p e c t i n s t o form i n t e r m o l e c u l a r a g g r e g a t e s , v i a hydrogen, i o n i c and/or hydrophobic i n t e r a c t i o n s ( 3 9 - 4 3 ) , i s one p o t e n t i a l p r o b l e m . Furthermore, t h e extreme s t r u c t u r a l d i v e r s i t y of the p e c t i n s (44) and c o n s e q u e n t l y the v a r i a b l e c o n f o r m a t i o n a l f e a t u r e s o f t h e s e polymers (45) a l s o i m p a i r s t h e assignment of a c c u r a t e m o l e c u l a r s i z e i n f o r m a t i o n . For t h e s e r e a s o n s , t h e gel f i l t r a t i o n d a t a p r e s e n t e d a r e c o n s i d e r e d o n l y i n terms of how e l u t i o n b e h a v i o r d i f f e r s between t i s s u e types and d u r i n g f r u i t development. Gel f i l t r a t i o n p r o f i l e s o f p e c t i n s from gel and p e r i c a r p from f r u i t at s e l e c t e d stages of development are shown i n F i g u r e 1. In undeveloped f r u i t , s o l u b l e p e c t i n s from both gel and p e r i c a r p were e x c l u d e d from U l t r o g e l AcA 3 4 . L i t t l e change was observed i n gel p e c t i n s throughout development whereas p e c t i n s from p e r i c a r p e x h i b i t e d a c l e a r t r e n d of d e p o l y m e r i z a t i o n . The d i f f e r e n t i a l f r a c t i o n a t i o n p a t t e r n s were g e n e r a l l y c o n s i s t e n t w i t h the r e l a t i v e l e v e l s o f EDG a c t i v i t y foun

Table I I .

Endo-D-Galacturonanase A c t i v i t y

Stage o f development Mature green Turning Pink Ripe

i n P e r i c a r p and Gel

Endo-D-Galacturonanase A c t i v i t y ~T (u9 r e d u c i n g equiv . mg p r o t e i n " . hr" ) Pericarp Gel 0.18 OT2 2.36 0.12 6.70 0.24 8.23 0.32

Gel t i s s u e c o n t a i n e d o n l y t r a c e l e v e l s of EDG a c t i v i t y . The f a c t t h a t e x t r a c t s from t h r e e s e p a r a t e l o t s of f r u i t e x h i b i t e d s i m i l a r a c t i v i t y i s an i n d i c a t i o n t h a t t h e presence o f the enzyme i n gel was not a consequence of h a n d l i n g - i n d u c e d c o n t a m i n a t i o n from p e r i c a r p t i s s u e . Evidence f o r jm s i t u a c t i o n o f t h e enzyme was apparent when gel p e c t i n s were examined on U l t r o g e l AcA 2 2 , a f i l t r a t i o n m a t r i x w i t h a b r o a d e r f r a c t i o n a t i o n range than t h a t o f U l t r o g e l AcA 34 ( F i g u r e 2 ) . On AcA 2 2 , p e c t i n s from gel showed evidence o f d e p o l y m e r i z a t i o n , a l b e i t l e s s e x t e n s i v e and f i r s t apparent much l a t e r i n f r u i t development ( r i p e s t a g e ) than t h a t affecting pericarp pectins. Ion-Exchange Chromatography. S o l u b l e p e c t i n s from gel and p e r i c a r p showed markedly d i f f e r e n t e l u t i o n b e h a v i o r on DEAE-Sephadex, a l t h o u g h w i t h i n each t i s s u e t y p e l i t t l e change i n e l u t i o n p a t t e r n was e v i d e n t d u r i n g development ( F i g u r e 3 ) . P e c t i n s from gel e l u t e d i n a range o f t h e g r a d i e n t c o r r e s p o n d i n g t o a c o n c e n t r a t i o n o f 200 mM NaCl whereas t h o s e from p e r i c a r p e l u t e d at h i g h e r c o n c e n t r a t i o n s , a v e r a g i n g about 500 mM. These o b s e r v a t i o n s were c o n s i s t e n t w i t h t h e range of v a l u e s of from 70-80% o b t a i n e d f o r percent e s t e r i f i c a t i o n o f gel p e c t i n s . P e r i c a r p p e c t i n s showed a lower p e r c e n t e s t e r i f i c a t i o n , t h e degree of which decreased p r o g r e s s i v e l y d u r i n g development from a high o f 40% a t the mature-green s t a g e t o 25% a t t h e r i p e s t a g e .

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

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F i g u r e 1. U l t r o g e l AcA 34 p r o f i l e s o f p e c t i n s from tomato g e l (A) and p e r i c a r p ( B ) . T i s s u e s were p r e p a r e d from mature green (top l e f t ) , p i n k ( t o p r i g h t ) , and r e d (bottom) f r u i t . Fractions were a n a l y z e d f o r a c i d s u g a r s ( A ) . q

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

12.

HUBER A N D LEE

Analysis of Pectins from Pericarp and Locular Gel

147

0.8L

Fraction

No.

F i g u r e 2 . U l t r o g e l AcA 22 p r o f i l e s o f gel p e c t i n s from tomato f r u i t a t mature-green (A) and r i p e (B) stage o f development. A c i d sugars

(A

5 2 Q

).

American Chemical Society. Library 1155 15th St., N.W.

In Chemistry and Function of Pectins; Fishman, M., et al.; Washington, D.C. Society: 20036 Washington, DC, 1986. ACS Symposium Series; American Chemical

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

F i g u r e 3 . DEAE Sephadex p r o f i l e s o f p e r i c a r p ( l e f t ) and gel ( r i g h t ) p e c t i n s from tomato f r u i t a t immature green (A) and r i p e (B) stage o f d e v e l o p m e n t . A c i d s u g a r s ( A ^ Q ) *

o * *n S

12.

HUBER AND LEE

Analysis of Pectins from Pericarp and Locular Gel

149

N e u t r a l Sugar A n a l y s i s . F i g u r e 4 i l l u s t r a t e s t h e t r e n d of n e u t r a l sugar changes i n s o l u b l e p e c t i n s from p e r i c a r p and g e l . T o t a l n e u t r a l sugars were c o n s i d e r a b l y h i g h e r i n g e l , a c c o u n t i n g f o r n e a r l y 40% (mole r a t i o b a s i s ) o f t h e p e c t i n . In p e r i c a r p p e c t i n s , n e u t r a l sugars never exceeded 12-13%. The q u a n t i t a t i v e t r e n d i n n e u t r a l sugar l e v e l s i n gel and p e r i c a r p p e c t i n s were t e m p o r a l l y d i s t i n c t , w i t h gel l e v e l s d e c r e a s i n g n e a r l y 62% d u r i n g the t r a n s i t i o n from immature-green t o m a t u r e - g r e e n , t h e most a c t i v e p e r i o d o f gel f o r m a t i o n . T h i s d e c r e a s e was due t o l o s s e s i n g a l a c t o s e (64%), a r a b i n o s e (53%) and x y l o s e (62%) ( F i g u r e 4 C ) . Of p a r t i c u l a r i n t e r e s t r e g a r d i n g t h e gel p e c t i n s was t h e presence o f high a b s o l u t e q u a n t i t i e s of x y l o s e , which a t 10% exceeded the l e v e l s o f a r a b i n o s e (8%). L i t t l e change i n n e u t r a l sugar c o n t e n t o c c u r r e d beyond the mature-green s t a g e . T o t a l n e u t r a l sugars o f p e r i c a r p p e c t i n s changed l i t t l e o r i n c r e a s e d s l i g h t l y through t h e mature-green s t a g e and d e c r e a s e d 25% d u r i n g r i p e n i n g . F i g u r e 4 B i l l u s t r a t e s t h a t the n e u t r a p e c t i n was a r e f l e c t i o n o l e s s e r e x t e n t a r a b i n o s e (13%). H e m i c e l l u l o s e A n a l y s e s . H e m i c e l l u l o s e s (4N a l k a l i - s o l u b l e ) from both gel and p e r i c a r p e x h i b i t e d a t r e n d of d e c r e a s i n g h i g h - m o l e c u l a r - w e i g h t polymers and i n c r e a s i n g q u a n t i t i e s of l o w - m o l e c u l a r - s i z e polymers ( F i g u r e 5 ) . The p r o f i l e s shown r e p r e s e n t h e m i c e l l u l o s e s from mature-green and r i p e f r u i t ; however e x a m i n a t i o n o f f r u i t at i n t e r m e d i a t e s t a g e s of r i p e n i n g d i s c l o s e d t h a t t h e change was g r a d u a l . Polymers from t i s s u e s from immature f r u i t resembled those d e r i v e d from mature-green f r u i t . The m o l e c u l a r weight s h i f t was accompanied by a change i n the e l u t i o n b e h a v i o r o f x y l o g l u c a n , as i n d i c a t e d by the Kooiman's iodine-staining test. Discussion It has f o r some time been e v i d e n t t h a t f r u i t s o f t e n i n g can not i n a l l cases be a t t r i b u t e d s o l e l y t o t h e a c t i o n of endo-Dg a l a c t u r o n a n a s e (EDG). In some f r u i t s , i n c l u d i n g muskmelon ( 3 , 2 6 ) , plum ( 2 4 ) , and s t r a w b e r r y ( 2 0 - 2 2 ) , s o f t e n i n g o c c u r s i n the apparent absence o f t h i s enzyme. Apple f r u i t contain exo-D-galacturonanase ( 4 6 ) ; however i t seems u n l i k e l y t h a t t h i s enzyme i s r e s p o n s i b l e f o r tRe s o l u b i l i z a t i o n o f p o l y m e r i c p e c t i n d u r i n g r i p e n i n g . Even i n f r u i t s known t o c o n t a i n EDG, w a l l changes a p p a r e n t l y u n r e l a t e d t o EDG a c t i o n have been observed d u r i n g r i p e n i n g and s o f t e n i n g . In some c a s e s ( d i s c u s s e d below) t h e p a r t i c i p a t i o n of w a l l polymers o t h e r than p e c t i n s has been i m p l i c a t e d . T h i s study addressed p o t e n t i a l v a r i a b i l i t y i n the c o n t r i b u t i o n of p e c t i n s i n p a r t i c u l a r t o t e x t u r e changes i n tomato p e r i c a r p and l o c u l a r t i s s u e ( g e l ) . The r e s u l t s demonstrate t h a t t h e changes a f f e c t i n g p e c t i n s i n t h e s e t i s s u e s are q u a n t i t a t i v e l y , q u a l i t a t i v e l y , and t e m p o r a l l y d i s t i n c t . In s t u d i e s w i t h tomato p e r i c a r p and o t h e r f r u i t t y p e s , p e c t i n changes, p a r t i c u l a r l y i n c r e a s e d s o l u b i l i t y and/or d e p o l y m e r i z a t i o n , have been shown t o be t e m p o r a l l y a s s o c i a t e d w i t h i n c r e a s e d EDG a c t i v i t y and w i t h r i p e n i n g i n general ( 3 - 1 1 ) . T h i s i s not the case w i t h tomato g e l . T h i s t i s s u e undergoes d r a m a t i c a n a t o m i c a l changes

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

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14. 12. o* -J

o

o.

0.2

0.4 NaCl

F i g u r e 3.

0.8

0.6

CONCENTRATION

(M)

E f f e c t o f N a C l c o n c e n t r a t i o n on t h e e x t r a c t i o n o f tomato p o l y g a l a c t u r o n a s e s a t pH 1.8. o, t o t a l PG; • , PG I ; x , PG I I . Reproduced w i t h t h e p e r m i s s i o n o f R e f e r e n c e 37.

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

1.0

13.

PRESSEY

Polygalacturonases in Higher Plants

165

PG I , t o m i n i m a a t a b o u t 0 . 1 5 M N a C l ( F i g u r e 4 ) . A possible e x p l a n a t i o n f o r t h e lower r e c o v e r y o f PG a c t i v i t y i n v o l v e s t h e r o l e o f p e c t i n e s t e r a s e (PE) which accompanied PG i n these e x t r a c t s . The h i g h l e v e l o f PE i n t h e s a l t e x t r a c t s p l u s t h e e n h a n c e m e n t o f PE a c t i v i t y by t h e c a t i o n s l e a d s t o r a p i d d e - e s t e r i f i c a t i o n o f p e c t i n i n t h e c e l l w a l l f r a c t i o n . The n e g a t i v e l y c h a r g e d p e c t a t e s t h e n a d s o r b t h e c a t i o n i c PG's a n d t h u s remove them f r o m s o l u t i o n . E x t r a c t i o n o f c e l l w a l l f r a c t i o n a t pH 6 w i t h NaCl s o l u t i o n s h i g h e r t h a n 0 . 1 5 M i n c r e a s e d t h e y i e l d s o f t o t a l PG a c t i v i t y and PG I t o maxima a t about 1.2 M NaCl ( F i g u r e 4 ) . However, t h e h i g h e s t amount o f PG I I w a s o b t a i n e d w i t h 0 . 5 M N a C l . This again i n d i c a t e s d i f f e r e n c e s i n s o l u b i l i t i e s o f PG I I and PG c o n v e r t e r i n r e l a t i o n t o N a C l c o n c e n t r a t i o n . The amount o f t o t a l PG a c t i v i t y e x t r a c t e d by 1.2 M NaCl was r e l a t i v e l y independent o f pH over t h e r a n g e o f 2 t o 9 . The y i e l d o f PG d e c r e a s e d o n l y when t h e pH was lowered t o 1, w h i c h may r e f l e c t i n s t a b i l i t y o f t h e e n z y m e a t h i g h l y a c i d conditions. Another consideratio i s t h e method f o r c o n c e n t r a t i n g t h e a c t i v i t y i n c r u d e e x t r a c t s . The m e t h o d t h a t i s commonly u s e d i s p r e c i p i t a t i o n w i t h ammonium s u l f a t e f o l l o w e d by d i a l y s i s o f t h e r e - d i s s o l v e d p r o t e i n a g a i n s t d i l u t e N a C l o r e v e n w a t e r ( 3 0 . 3 6 . 38) . A study was conducted t o d e t e r m i n e t h e r e c o v e r y o f PG a c t i v i t y by t h i s method compared w i t h u l t r a f i l t r a t i o n . An e x t r a c t o f a w a s h e d c e l l w a l l f r a c t i o n was p r e p a r e d i n 1.0 M N a C l a t pH 6 . O n e - t h i r d o f t h e e x t r a c t was u l t r a f i l t e r e d t o 20 m l a n d d i a l y z e d a g a i n s t 1.0 M N a C l . The r e m a i n i n g t w o - t h i r d s o f t h e e x t r a c t was t r e a t e d w i t h ammonium s u l f a t e a t 75% o f s a t u r a t i o n . The p r e c i p i t a t e was d i s s o l v e d i n 40 ml o f 1 .0 M N a C l , and a l i q u o t s o f t h e s o l u t i o n were d i a l y z e d a g a i n s t 0.15 M and 1.0 M N a C l . R e l a t i v e t o t h e PG a c t i v i t y i n t h e u l t r a f i l t r a t e , o n l y 43% a n d 78% o f t h e a c t i v i t y was r e c o v e r e d i n the ammonium s u l f a t e f r a c t i o n s d i a l y z e d a g a i n s t 0 . 1 5 M a n d 1.0 M N a C l , r e s p e c t i v e l y . The much l o w e r a c t i v i t y i n t h e f r a c t i o n d i a l y z e d a g a i n s t 0.15 M NaCl was due p r i m a r i l y t o t h e l o s s o f PG I . T h e s e r e s u l t s e x p l a i n t h e low l e v e l s o f PG I r e p o r t e d by Tucker e t a l . (36.). Their procedure f o r preparing extracts included p r e c i p i t a t i o n w i t h ammonium s u l f a t e f o l l o w e d by d i a l y s i s a g a i n s t 0.15 M N a C l . They found t h a t o n l y a b o u t 10% o f t h e a c t i v i t y was due t o PG I i n e x t r a c t s o f t h e f r u i t o f two c u l t i v a r s o f tomatoes. I have u s e d t h e m e t h o d o l o g y d e s c r i b e d a b o v e t o s t u d y t h e changes i n PG I , PG I I , and PG c o n v e r t e r i n r i p e n i n g tomatoes (39) * F r u i t w e r e s e l e c t e d a t s i x s t a g e s o f r i p e n e s s . The f r u i t s o f t e n e d markedly d u r i n g r i p e n i n g and t h e l e v e l o f w a t e r - s o l u b l e p e c t i n increased sharply. PG a c t i v i t y was f i r s t d e t e c t e d i n e x t r a c t s o f f r u i t a t t h e t u r n i n g s t a g e , i n agreement w i t h e a r l i e r r e p o r t s ( 2 3 . 32. 26). PG I was t h e o n l y enzyme p r e s e n t i n t h e s e e x t r a c t s . PG I I was f i r s t f o u n d i n e x t r a c t s o f f r u i t a t t h e p i n k s t a g e . On f u r t h e r r i p e n i n g , b o t h enzymes i n c r e a s e d m a r k e d l y b u t PG I r e m a i n e d t h e m a j o r isoenzyme even a t the o v e r - r i p e stage. This i s i n c o n t r a s t w i t h t h e r e s u l t s o f Tucker e t a l . (36) mentioned above and a l s o w i t h t h o s e o f M o s h r e f i and Luh (38) t h a t PG I was absent i n e x t r a c t s of r i p e tomatoes. The l o w r e c o v e r y o f PG I i n b o t h of these s t u d i e s may be a t t r i b u t e d t o p r e c i p i t a t i o n o f PG I d u r i n g d i a l y s i s o f t h e e x t r a c t s a g a i n s t 0 . 1 5 M NaCl ( 3 7 ) . Whereas t h e p o l y g a l a c t u r o n a s e s a p p e a r e d a f t e r r i p e n i n g o f

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

166

CHEMISTRY AND FUNCTION OF PECTINS

NaCl F i g u r e 4,

CONCENTRATION

(M)

E f f e c t o f N a C l c o n c e n t r a t i o n on t h e e x t r a c t i o n o f tomato p o l y g a l a c t u r o n a s e a t pH 6. o, t o t a l PG; •, PG I ; x , PG I I . Reproduced w i t h t h e p e r m i s s i o n o f R e f e r e n c e 37•

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

13. PRESSEY

Polygalacturonases in Higher Plants

167

tomatoes began, PG converter was present i n the f r u i t at the green stage. The l e v e l of PG converter remained low through the breaker s t a g e and t h e n i n c r e a s e d w i t h r i p e n i n g . The presence of the converter i n unripe tomatoes raises the p o s s i b i l i t y that PG I may be formed during extraction of ripening f r u i t . As PG II appears i n the f r u i t , during the p r e p a r a t i o n of e x t r a c t s the enzyme r e a c t s w i t h the excess of converter to form PG I. Thus free PG II i s not found i n the extracts u n t i l the enzyme exceeds the l e v e l of the converter. P o l y g a l a c t u r o n a s e s i n Peaches. Freestone peaches soften markedly d u r i n g r i p e n i n g , r e s u l t i n g i n the c h a r a c t e r i s t i c juicy texture of peaches. Accompanying peach softening i s a conversion of insoluble p e c t i n to w a t e r - s o l u b l e forms (Figure 5 ) . The s o l u b i l i z a t i o n of pectin suggest8 that degradation of pectin occurs, but PG a c t i v i t y was not detected i n peaches i n e a r l y studies (40) • The reason i t was not detected i s tha much lower than that i n i t i s necessary to use very long assay incubation period (41) or to concentrate the enzyme by u l t r a f i l t r a t i o n (42) to measure i t . But as i n tomatoes, PG a c t i v i t y appears i n peaches near the onset of ripening and then increases sharply during ripening (Figure 5 ) . The PG a c t i v i t y i n r i p e f r e e s t o n e peaches i s due to two enzymes ( 4 2 ) . In contrast to tomatoes which contain two endo-PG's, peaches c o n t a i n an endo-PG but a l s o an exo-PG ( 4 2 ) . The peach enzyme8 can be separated by chromatography on a Sephadex G-100 column. The endo-PG has a pH optimum near 4 and i s very e f f e c t i v e i n s o l u b i l i z i n g p e c t i n from peach c e l l w a l l s . The discovery of exo-PG i n peaches represented the f i r s t time that t h i s enzyme was found i n f r u i t t i s s u e . The exo-PG has a pH optimum near 5.5 and requires C a 2 for a c t i v i t y with an optimum concentration of 0.4 mM. It removes monomer units from the nonreducing ends of the substrate molecules. T h i s enzyme does not s o l u b i l i z e the p e c t i n from isolated peach c e l l walls. R i p e n i n g c l i n g s t o n e ( n o n - m e l t i n g f l e s h ) peaches s o f t e n considerably less than do freestone peaches. They also r e t a i n more i n s o l u b l e p e c t i n , suggesting that less pectin degradation occurs. An examination of a number of c u l t i v a r s of peaches (43) showed that a l l r i p e f r e e s t o n e peaches c o n t a i n approximately equal levels of endo-PG and exo-PG whereas clingstone peaches contain a high l e v e l of exo-PG but zero or very low endo-PG (Table II) . The absence of the endoenzyme i n c l i n g s t o n e peaches i s c o n s i s t e n t with the low s o l u b i l i z a t i o n of p e c t i n and retention of f r u i t firmness i n these peaches. Thus the markedly d i f f e r e n t t e x t u r a l c h a r a c t e r i s t i c s of the two types of peaches are accounted f o r by the difference i n polygalacturonase composition. +

P o l y g a l a c t u r o n a s e s i n Other F r u i t s . I t has long been known that r i p e pears contain a low l e v e l of PG ( 4 4 ) . Yamaki and Matsuda (45) f o u n d t h a t PG a c t i v i t y was present i n pears during the c e l l d i v i s i o n stage and that i t decreased during the enlargement stage before i t i n c r e a s e d markedly during ripening. We have extracted the PG a c t i v i t y from ripe D'Anjou pears and separated i t i n t o two

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

168

CHEMISTRY AND FUNCTION OF PECTINS

- 6

O)

-

4

CO CO UJ

z

1

1

•

1

•

1

• • • o w

0

1

1 1 1 1 1 1

—

O •

WATER SOLUBLE PECTIN TOTAL PECTIN

1

DAYS F i g u r e 5.

Changes i n f r u i t firmness, polygalacturonase, w a t e r - s o l u b l e and t o t a l p e c t i n i n p e a c h e s d u r i n g p o s t h a r v e s t r i p e n i n g ( 4 1 ) • Reproduced w i t h the p e r m i s s i o n o f R e f e r e n c e 41_. C o p y r i g h t 1 9 7 1 , I n s t i t u t e o f Food T e c h n o l o g i s t s .

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

13.

PRESSEY

Table I I ,

169

Polygalacturonases in Higher Plants Polygalacturonase

Variety Baby Gold 6 Mountain Gold Suncling Fay Elberta Sullivan Elberta

a c t i v i t y i n u l t r a f i l t e r e d extracts of peaches.

Stone freeness cling cling cling free free

Exo

Endo units/ml 0.2 0.2 0.2 4.6 4.0

3.0 2.6 4.2 4.8 3.8

peaks by chromatography on Sephadex G-100 (Figure 6 ) . The enzyme i n the f i r s t peak was ver p e c t a t e and thus i s a optimum of 5 . 5 , required C a 2 for a c t i v i t y , and i t s mode of action was c o n s i s t e n t w i t h that of an exo-PG (46.) . The exo-PG was the major PG in these pears. Another f r u i t that contains both endo-PG and exo-PG i s papaya (47). PG a c t i v i t y has also been found i n ripe avocados ( 4 8 ) , dates (49) , and mangoes (50) , but these enzymes were not characterized f o r t h e i r modes of a c t i o n . Bartley (51) extracted a low l e v e l of PG a c t i v i t y from ripe apples which he i d e n t i f i e d as an exo-PG. The apple enzyme degraded c o r t i c a l c e l l walls r e l e a s i n g low molecular weight u r o n i c a c i d r e s i d u e s and polyuronide. Immature cucumbers contain PG a c t i v i t y that increases during growth of the f r u i t (52). The enzyme i s an exo-PG but d i f f e r s from other exo-enzymes i n that i t i s r e a d i l y soluble i n water and i t exhibits the highest r a t e of r e a c t i o n w i t h p e c t a t e , the l a r g e s t s u b s t r a t e . McFeeters et a l . (53) have i s o l a t e d and characterized an endo-PG from ripe cucumber fruit. +

Polygalacturonases i n Other Plant Tissues. PG a c t i v i t y i s usually a s s o c i a t e d w i t h r i p e f r u i t t i s s u e s and, as we have seen, the a c t i v i t y can be due to an endo-PG, exo-PG, or to both enzymes. However, I have already mentioned the occurrence of PG a c t i v i t y i n u n r i p e tomatoes (21) , pears (45) , and cucumbers (52) . The enzymes i n g r e e n tomatoes and cucumbers have been c h a r a c t e r i z e d as exo-PG's. Exo-PG has a l s o been r e p o r t e d to be present i n carrot roots (54) and c i t r u s l e a f explants (55) . I t occurred to me that this enzyme may be w i d e l y d i s t r i b u t e d i n h i g h e r p l a n t s and an examination of various tissues showed this to be true (56). PG has been found i n s e e d l i n g s of v a r i o u s plants including beans, corn, oats, and peas (Table I I I ) . In oat c o l e o p t i l e s , the a c t i v i t y was highest near the growing t i p and i t decreased markedly as the d i s t a n c e from the t i p i n c r e a s e d . S i m i l a r l y i n pea s e e d l i n g s , PG a c t i v i t y was highest i n the plumules and hook, but the a c t i v i t y was quite high i n the 1 cm section below the hook and c o n s i d e r a b l y lower i n l o w e r s e c t i o n s of the e p i c o t y l ( 5 7 . ) . However, PG was a l s o found i n stem and l e a f t i s s u e s from these plants.

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

170

CHEMISTRY AND FUNCTION OF PECTINS

Figure 6.

S e p a r a t i o n of pear p o l y g a l a c t u r o n a s e s chromatography on Sephadex G-100.

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

by

13.

PRESSEY

171

Polygalacturonases in Higher Plants

The PG i n oat s e e d l i n g s has been p a r t i a l l y p u r i f i e d and c h a r a c t e r i z e d (56) . The molecular weight of oat PG was 6 3 , 0 0 0 , as determined by g e l f i l t r a t i o n . The enzyme was o p t i m a l l y active between pH 5 . 0 and 5 . 5 i n the absence and presence of added C a (Figure 7). The p u r i f i e d enzyme had low a c t i v i t y which was further reduced by EDTA and c i t r a t e . C a stimulated the enzyme, with an optimum concentration of 0.4 mM that produced a 7 - f o l d i n c r e a s e i n r e a c t i o n rate over the c o n t r o l . Higher concentrations of C a were i n h i b i t o r y , and the i n h i b i t i o n was g r e a t e s t f o r the l a r g e s t s u b s t r a t e , s u g g e s t i n g t h a t the e f f e c t i s due to s u b s t r a t e i n s o l u b i l i z a t i o n r a t h e r than to actual i n h i b i t i o n of the enzyme. max m w e r e d e t e r m i n e d f o r o a t PG a c t i n g on oligogalacturonides, p o l y g a l a c t u r o n a t e s and p e c t a t e (Table I V ) . The h i g h e s t r e a c t i o n r a t e s were obtained w i t h moderately long substrates, but the enzyme had the h i g h e s t a f f i n i t y f o r p e c t a t e , the l a r g e s t s u b s t r a t e I t was e s t a b l i s h e d that oat PG i s an exo-enzyme that remove the substrate molecule 2 +

2 +

2 +

v

a

n

d

Table I I I .

Tissue

K

Polygalacturonase a c t i v i t y i n a v a r i e t y of plant t i s s u e s . Polygalacturonase

Red Kidney bean hypocotyls Blue Lake bean pods Seneca Chief corn seedling Seneca Chief corn plants Alaska pea seedling Swiss Chard stalks Pokeweed stems Better Boy tomato stems Butterbar squash stalks Asparagus shoots Turnip roots Red beet roots

units/g 0.52 0.34 0.54 0.62 0.24 0.21 0.17 0.18 0.10 0.72 0.42 0.30

pH optimum

A c t i v a t i o n by 0.6 mM Ca2+

5-5.5 5 5-5.5 5-5.5 4.5-5 5-5.5 5-5.5 5.5 4.5 5.5 4.5-5 5

X 65 160 43 86 100 44 —

84 —

66 64 —

The PG i n pea s e e d l i n g s has a l s o been determined to be an exo-hydrolase (57) . Based on small e f f e c t s on the v i s c o s i t y of p e c t a t e and on a c t i v a t i o n by Ca2 + , the PG's i n the other sources l i s t e d i n Table IV appear to be exo-PG's. Endo-PG a c t i v i t y appears to be extremely low or absent i n v e g e t a t i v e and storage tissue. O t h e r w o r k e r s have found exo-PG i n potato tubers (58) and i n suspension cultures of a liverwort (59) .

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

172

CHEMISTRY A N D FUNCTION O F PECTINS

Figure 7.

E f f e c t of pH on the a c t i v i t y of oat s e e d l i n g polygalacturonase. °, i n the absence of added C a ; • •, i n the presence of 0.4 mM Ca . R e p r o d u c e d w i t h the p e r m i s s i o n of Reference 56. Copyright 1977, American Society of Plant Physiologists. 0

+ +

+ +

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

13. pressey

173

Polygalacturonases in Higher Plants

Table IV. Kinetic parameters of the hydrolysis of galacturonans by oat polygalacturonase. Substrate Digalacturonate Trigalacturonate Tetragalacturonate Pentagalacturonate Reduced Pentagalacturonate Hexagalacturonate PGA III Reduced PGA III PGA II Reduced PGA II PGA I Pectate

Km juM 290 210 180 160 130 140 74 68 46 48

Maximum Velocity Relative 0.2 0.6 1.7 1.9 2.0 2.3 3.0 2.9 3.8 4.1

Literature Cited 1. Keegstra, K., Talmadge, K. W., Bauer, W. D. and Albersheim, P. Plant Physiol. (1973) 51:188. 2. Talmadge, K. W., Keegstra, K., Bauer, W. D. and Albersheim, P., Plant Physiol. (1973) 51:158. 3. Knee, M., Phytochemistry (1978) 17:1257. 4. Ishii, S., Phytochemistry (1981) 20:2329. 5. Pressey, R. and Himmelsbach, D. S., Carbohydr. Res. (1984) 127:356. 6. Knee, M., Phytochemistry (1972) 12:637. 7. Knee, M., Fielding, A. H . , Archer, S. A. and Laborda, F., Phytochemistry (1975) 14:2213. 8. Albersheim, P . , Neukom, H. and Deuel, H . , Helv. Chim. Acta (1960) 43:1422. 9. Nagel, C. W., and Vaughn, R. H., Arch. Biochem. Biophys. (1961) 94:328. 10. Preiss, J . and Ashwell, G., J. Biol. Chem. (1963) 238:1571. 11. Albersheim, P. and K i l l i a s , U., Arch. Biochem. Biophys. (1962) 97:107. 12. Jensen, E. F. and MacDonnel, L. R., Arch. Biochem. (1945) 8:97. 13. Hobson, G. E., Biochem. J . (1963 ) 86:358. 14. Hobson, G. E., Nature (1962) 195:804. 15. Wallner, S. J . and Bloom, H. L., Plant Physiol. (1977) 60:207. 16. Gross, K. C., HortScience (1982) 17:933. 17. Nelson, N., J . Biol. Chem. (1944) 153:375. 18. Pressey, R. and Avants, J . K., J . Food Sci. (1971) 36:486. 19. Pressey, R. and Avants, J . K., Phytochemistry (1975) 14:957. 20. Kertesz, Z. I., Food Res. (1938) 3:481. 21. Pressey, R., Phytochemistry (In preparation). 22. Hobson, G. E., Biochem. J . (1964) 92:324. 23. Hobson, G. E., J. HortScience (1965) 40:66. 24. Sobotka, F. E. and Watada, A. E., Proc. W. Va. Aca. Sci. (1970) 42:141.

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chemistry and function of pectins

25. Brady, C. J., McGlasson, W. B., Pearson, J . A., Mel drum, S. K. and Kopeliovitch, E . , J. Amer. Soc. HortScience (1985) 110:254. 26. Buescher, R. W., Sistrunk, W. A., Tigchelaar, E. C. and Ng, T. Hort. Sci. (1976) 11:603. 27. Pressey, R. and Avants, J . K., HortScience (1982) 17:398. 28. Kopeliovitch, E., Mizrahi, Y . , Rabinovitch, H. D. and Kedar, N., Physiol. Plant. (1980) 48-307. 29. McColloch, R. J. and Kertesz, Z. I., Arch. Biochem. (1948) 17:197. 30. Pressey, R. and Avants, J . K . , Biochim. Biophys. Acta (1973) 309:363. 31. Pressey, R., HortScience (1984) 19:572. 32. A l i , Z. M. and Brady, C. J., Aust. J . Plant Physiol. (1982) 9:155. 33. Tucker, G. A. and Grierson, D., Planta (1982) 155:64. 34. Pressey, R., Eur. J . Biochem. (1984) 144:217. 35. Morgens, R. H., Pyle M., Plant Physiol. (1985 36. Tucker, G. A., Robertson, N. G. and Grierson, D., Eur. J. Biochem. (1980) 112:119. 37. Pressey, R., HortScience (1985). In press. 38. Moshrefi, M. and Luh, B. S., Eur. J . Biochem. (1983) 135:514. 39. Pressey, R., HortScience (1985). In press. 40. McCready, R. M. and McComb, E. A., Food Res. (1954) 19:530. 41. Pressey, R., Hinton, D. M. and Avants, J . K . , J. Food Sci. (1971) 36:1070. 42. Pressey, R. and Avants, J . K., Plant Physiol. (1973) 52:252. 43. Pressey, R. and Avants, J . K., J. Food Sci. (1978) 43:1415. 44. Weurman, C., Acta Botan. Neerl. (1954) 3:108. 45. Yamaki, S. and Matsuda, K., Plant Cell Physiol. (1977) 18:81. 46. Pressey, R. and Avants, J . K., Phytochemistry (1976) 15:1349. 47. Chan, H. T. and Tam, S. Y. T., J . Food Sci. (1982) 47:1478. 48. Reymond, D. and Phaff, H. J., J . Food Sci. (1965) 30:266. 49. Hasegawa, S., Maier, V. F., Kaszychi, H. P. and Crawford, J . K., J . Food Sci. (1969) 34:527. 50. Roe, B. and Bruemmer, J . H., J. Food Sci. (1981) 46:187. 51. Bartley, I. M., Phytochemistry (1978) 17:213. 52. Pressey, R. and Avants, J . K., J. Food Sci. (1975) 40:937. 53. McFeeters, R. F., B e l l , T. A. and Fleming, H. P., J . Food Biochem. (1980) 4:1. 54. Hatanaka, C. and Ozawa, J., Agr. Biol. Chem. (1964) 28:627. 55. Riov, J., Plant Physiol. (1974) 53:312. 56. Pressey, R. and Avants, J . K., Plant Physiol. (1977) 60:548. 57. Pressey, R., Plant Physiol. (1980) 65:S-128. 58. Puri, A., Solomos, T. and Kramer, A . , Lebensm Wiss Technol. (1981) 14:118. 59. Kormo, H., Yamasaki, Y. and Katoh, K . , Plant Physiol. (1983) 73:216. RECEIVED December 2, 1985

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

14

Paramagnetic Ion S p i n - S p i n C o u p l i n g as D i r e c t Evidence for Cooperative I o n B i n d i n g to H i g h e r P l a n t C e l l Walls 1

Peter L. Irwin, Michael D. Sevilla, James J. Shieh, and Carla L. Stoudt Agricultural Research Service, Eastern Regional Research Center, U.S. Department of Agriculture, Philadelphia, PA 19118

Electron spin resonanc periments were performed to estimate Mn2+ and Cu near neighbor distances, thereby determining if carboxylate-divalent cation complexes potentiate ion association at adjacent sites on cell wall polyuronides. Distances were estimated to be 12 and 14Å for Cu and Mn respectively. At the maximal bound ion concentration, the lattice constant {K} was ca. 2.5 indicating that approximately 6 paramagnetic ion near neighbor spin-spin interactions occur per dipole in the nearly-filled lattice. Competitive ion exchange with Ca was found to reduce the Mn spin-spin line broadening at similar total bound [Mn ]; this could only be observed if Ca competes with Mn at adjacent sites. These data offer strong support for the sequential-cooperative ion binding mechanism. Polyuronides are a major component of the primary cell wall and middle lamella of higher plant cortical tissues (1, 2). These polymers are efficient cation exchangers (3, 4) and, thus, can affect ion activity, transmembrane potential and electrolyte flux (5). In addition, the divalent salts of pectic substances 2+

2+

2+,

2+

2+

2+

2+

2+

1

Current address: Department of Chemistry, Oakland University, Rochester, MI 48063. This chapter not subject to U.S. copyright. Published 1986, American Chemical Society

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

176

CHEMISTRY A N D FUNCTION O F PECTINS

appear to play some s t r u c t u r a l role i n adjacent

cell

wall-to-wall adhesion (6-9) as w e l l as exert s t e r i c control over the a c t i v i t y of c e r t a i n h y d r o l y t i c enzymes (7, 8). Solution studies on the binding behavior of polygalacturonate and polyguluronate (10-16) i n d i c a t e that a molecular s i z e dependent cooperative ion binding takes place (12, 13).

Some

authors (11, 15) have proposed that these a c i d i c polymers bind divalent cations i n electronegative c a v i t i e s between chains l i k e "eggs i n an egg box". L i t t l e information has been a v a i l a b l e with regard to the ion binding mechanism or the polyuronide-cation aggregate structure i n s o l i d matrices (3-5, 17). In t h i s paper we w i l 19) which supports the concept that carboxylate group-divalent cation complexes potentiate ion association at adjacent binding s i t e s (the sequential binding mechanism, 18). We w i l l also present evidence that the cation-polyuronide aggregate structure i s s i m i l a r to the model proposed f o r polyuronides i n s o l u t i o n (11),

that polymer blocks e x i s t i n three dimensional

lattices

low i n methyl ester content and that subtle conformational changes can be observed with t h i s ESR technique (19).

This

method could prove useful i n the e l u c i d a t i o n of the s t r u c t u r a l mechanisms of c e r t a i n developmental changes i n plant tissues (e.g., ripening, abscission, extensive growth, etc.; 6-9, 19). Materials and Methods Intact c o r t i c a l tissues of Malus pumila {cv. Golden Delicious} f r u i t were used throughout these experiments.

Tissue f i x a t i o n

and determination of free {e.g., nonmethylated} uronic acid concentrations were described previously (18). For the ESR experiments,

i n t a c t tissues {lxlx4mm} were

equilibrated at pH 5 i n water f o r 15 min. and decanted; t h i s procedure was repeated t h r i c e .

About 10-40 mg dry weight of

c e l l w a l l complex was used f o r each treatment

combination.

Samples were e q u i l i b r a t e d {22±2°} i n 10 ml of various concentrations of M n

2+

(18, 19), M n

2+

with C a

2 +

(18) or C u

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

2 +

14. IRWIN E T A L .

(19).

Cooperative Ion Binding to Cell Walls

A f t e r ca. 24 hrs., the solutions were decanted and t i s s u e

specimens washed t h r i c e i n {pH 7} water, allowing 15 min. e q u i l i b r a t i o n i n each wash.

A f t e r an a d d i t i o n a l 1 hr. soak,

t h i s washing procedure was repeated.

Samples were dehydrated

with ethanol and c r i t i c a l point dried as described previously (18, 20). Some samples were rehydrated i n a saturated water vapor chamber (19) at 160 t o r r and 22°C. immediately

A l l samples were

loaded and sealed i n 3x120 mm quartz ESR tubes.

After each experiment, the samples were removed, washed i n methanol, vacuum dried at 35°C, weighed, and dry-ashed f o r 2+ 2+ atomic absorption spectrophotometri 2+ and/or Cu using standard procedures (18, 19). 2+ 2+ 2+ Mn and Cu ESR s p e c t r a l parameters, Mn linewidths {AHj^j (H)/dH]max^ * dimer-only nearest neighbor distance an(

parameter {d} c a l c u l a t i o n s were as previously described (18, 19).

A l l experiments i n t h i s report were run at near l i q u i d

^

temperatures {-176 to -180°C} to avoid c e r t a i n r e l a x a t i o n time 2+ phenomena which can be problematic (19).

Cu

linewidths were

calculated d i r e c t l y from the f i r s t d e r i v a t i v e gj^component (19) as the difference i n f i e l d strength {G} between the maximum and minimum amplitude.

This empirical measure of linewidth was

found to be d i r e c t l y porportional to the true linewidth as determined by an anisotropic simulation routine (21); t h i s simulation showed that the true linewidth {AH

r

1

, / „ n , t

, „ !

[dI(H)/dH]max,

Figure 3} was equal to the empirical measure, described above, divided by 1.17 (19). Results and Discussion 2+ The results i n Figure 1 demonstrate that c e l l w a l l bound Mn i s associated with an acid t i t r a t a b l e s i t e ; s i m i l a r results have 2+ been obtained with Ca . Polyuronides are the most l i k e l y i o n binding species since they are the predominant anionic component (2, 20) i n these t i s s u e s . From the standpoint of the ESR experiment, i o n i c a l l y bound paramagnetic ions can be treated as fixed points which i n t e r a c t

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

CHEMISTRY A N D FUNCTION OF PECTINS

1

5 FRACTION

10 NUMBER (15ml)

15

2+

Figure 1. T i t r a t i o n of Mn

o f f i n t a c t c e l l w a l l matrices

under m i l d l y a c i d i c conditions.

Reproduced with permission

from Ref. 18. Copyright 1984, Biochimica et Biophysica Acta.

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

IRWIN ET A L .

14.

Cooperative Ion Binding to Cell Walls

only through t h e i r couplings with each other (22).

Thus, any

i t h spin i n the midst of other paramagnetic t r a n s i t i o n ions bound w i t h i n a s o l i d matrix, such as these c e l l w a l l matrices, has a t o t a l spin i n t e r a c t i o n p r o f i l e equal to the sum of the spin-spin and exchange i n t e r a c t i o n s (22). In t h i s summation, the series converges to a value which depends only on those dipoles i n the v i c i n i t y of each i t h spin and, thus, i s the same f o r a l l the dipoles of the same type i n the l a t t i c e .

Of these terms,

spin-spin i n t e r a c t i o n s cause l i n e broadening (23) and are proportional to the inverse cube of the distance between q i n t e r a c t i n g spins (1/r 22-26) as described by the square root of the second momen

(

;

,

,

)

line: 2

1/2

4

2

= {3/5.g pV .S[S+l].I.l/r

6 i j

}

1 / 2

;

(1; units i n Hz)

whereupon g, (3 and h have t h e i r usual values and S represents 2+ the t o t a l electron spin of the i o n {S=5/2 for Mn and 1/2 f o r 2+ Cu

}.

In nonmagnetic i n s u l a t o r s (28), linewidths and -shapes

agree with basic Van Vleck theory (23).

However, as

paramagnetic ions approach one another (r< 5-10 A; 25) the linewidths are affected by exchange coupling (29). While 2 s are independent of the exchange term (2J..S.'S. = 2J..S.S.cosO, where J . , i s the "exchange i n t e g r a l " IJ I j IJ I j ' IJ and 0 i s the angle formed between the i t h - j t h spins and the !

5

6

magnetic f i e l d ; 22, 30) the lineshapes no longer remain Gaussian, become mixed (27) and l i n e narrowing r e s u l t s (23, 29, 31, 32).

Figures 2 and 3 i l l u s t r a t e that, r e l a t i v e to the

spin-spin i n t e r a c t i o n s , we have l i t t l e exchange since the linewidths of the bound dipoles only broaden as the concentration increases. 2 We (18) have s i m p l i f i e d the r e l a t i o n s h i p by making use of the f a c t that, f o r a Gaussian l i n e , the linewidth i s 2 1/2

2

1

. In t h i s r e l a t i o n s h i p

^[dlOO/dHjmax

=

1/2

2 8 6 9 0 . G A . { S [ S + l ] } . K / d ; (2; units i n G) 3

3

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

180

CHEMISTRY A N D FUNCTION O F PECTINS

2oA> 01 0

i 5

i 10 MOLES

i 15

i— 20

Mn */g x I0" 2

5

2+ Figure 2. C e l l w a l l bound Mn concentration dependency of linewidth ^ [ ^ ( u j / d H j m a x ^ ' ^ -'- l nearest neighbor


++

CDC-C00"(Ca )"00C-Pectin + Pectin-COO" The release of the stronger carboxylate groups of p e c t i n that t h i s equation produces would account for the drop i n pH that i s observed upon binding of b i l e acids to carrot AAIR. Pectin complexed wit the demonstrated a b i l i t exchange (9). These complexes are hypocholesterolemic i n rats and can i n t e r a c t with b i l e acids as w e l l as anionic l i p i d micelles (9,10). Our studies favor a role for calcium pectate i n the observed binding of b i l e acids to plant c e l l w a l l residue. I t remains to be seen i f calcium pectate w i l l have a greater hypocholesterolemic e f f e c t than has been reported f o r commercial pectins i n feeding studies (11-15). During the course of b i l e acid binding assays of carrot AAIR i t was noted that a peak emerged at the void volume during HPLC of the s o l u t i o n of b i l e acid that had been i n contact with the f i b e r . We therefore suspected that the b i l e acid s o l u t i o n was s o l u b i l i z i n g some small f r a c t i o n of the AAIR. Accordingly, carrot AAIR was then extracted with sodium deoxycholate and a p e c t i n f r a c t i o n was i s o l a t e d by alcohol p r e c i p i t a t i o n of the 13

a c i d i f i e d extract. S o l i d state CPMAS C NMR spectroscopy ( F i g . 2) revealed t h i s p e c t i n to be 75% methylated and therefore s i m i l a r to the highly methylated p e c t i n that A s p i n a l l , et a l . , (4) i s o l a t e d from carrot c e l l w a l l residue by hot water extract i o n . The p e c t i n was found to bind 6.39 ± 0.26 % CDC at pH 7.2. This p e c t i n f r a c t i o n requires further study because the data suggest that hydrophobic i n t e r a c t i o n s may have a role i n binding of b i l e acids to some highly methylated pectins. A combination of hydrophobic i n t e r a c t i o n s and calcium s a l t linkages could possibly r e s u l t i n some very strong binding between b i l e acids and highly methylated calcium pectate. Carrot AAIR can now be viewed as a dietary f i b e r with a growing l i s t of b e n e f i c i a l properties. The large water holding capacity of t h i s material (1) makes carrot f i b e r an e f f e c t i v e , gentle bulking agent (6). The binding a c t i v i t y for b i l e acids that has been experimentally demonstrated (1,2) for carrot f i b e r (AAIR) o f f e r s a dietary means of c o n t r o l l i n g blood c h o l e s t e r o l l e v e l s (6,16-17). The calcium content of carrot f i b e r may be a s i g n i f i c a n t dietary source of calcium for the senior population. In a d d i t i o n , some of the calcium of carrot AAIR may be released i n the colon with the b e n e f i c i a l e f f e c t of removing free f a t t y acids as calcium s a l t s (18). Carrot f i b e r , prepared as an

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

20.

HOAGLAND AND PFEFFER

Binding of Bile Acids to Carrot Fiber

273

ppm 13

Figure 2. CPMAS C NMR spectra f o r carrot AAIR and f o r p e c t i n extracted from AAIR with deoxycholate. Chemical s h i f t s (ppm): AAIR, 173.2 (a, carbonyl), 106.4 (b, anomeric), 73.4 (c, hydroxylated methylene), 53.7 (d, methoxy), 22.0 (e, methyl of a c e t y l ) ; p e c t i n , 172.1 ( a ) , 102.5 (b), 71.4 ( c ) , 54.0 (d) 22.0 (e); Sigma c i t r u s p e c t i n , 70% methylated (not shown), 171.9 ( a ) , 101.0 (b), 71.2 (c) 54.0 (d), no (e).

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alcohol insoluble cell wall residue, can be prepared easily and economically. Additional beneficial properties as a component of food formulations can be anticipated. Acknowledgments 13

We thank Richard T. Boswell for providing C NMR spectra and Maryann Taylor for calcium analyses. We thank Diedre Lyons and Christine Daubt for technical assistance. We thank Robert J. Carroll for the scanning electron micrograph. Literature Cited 1. 2. 3. 4.

5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

Robertson, J.A.; Eastwood, M.A.; Yeoman, M.M. J. Nutr. 1980, 110, 1130-1137. Hoagland, P.D.; Pfeffer, P.E. J. Agri. Food Chem. 1986, in press. Program available fro Aspinall, G.O.; Fanous, H.K.; Sen, A.K. In "Unconventional Sources of Dietary Fiber"; Furda, I., Ed.; ACS Symposium Series No. 214, American Chemical Society: Washington, D.C., 1983; pp. 33-48. DeMarty, M.; Monvan, C.; Thellier, M. Plant Cell Envir. 1984, 7, 441-448. Jenkins, D.J.A.; Reynolds, D.; Leeds, A.R.; Waller, A.L.; Cummings, J.H. Am. J. Clin. Nutr. 1979, 32, 2430-2435. Whatman Technical Bulletin IE2, "Advanced Ion-Exchange Celluloses" 1966; pp. 52-53, Scientifica Division, Reeve Angel, Clifton, NJ 07014. Rees, D.A. Carb. Polym. 1982, 2, 254-263. Nagyvary, J.; Bradbury, E.L. Biochem. Biophys. Res. Comm. 1977, 77, 592-598. Furda, I. In "Dietary Fibers: Chemistry and Nutrition"; Inglett, G.E.; Falkenhag, S.I., Eds.; Academic: New York, 1979; pp. 31-48. Leveille, G.A.; Sauberlich, H. J. Nutr. 1966, 88, 209-214. Kay, R.; Truswell, A.S. Am. J. Clin. Chem. 1977, 30, 171-175. Reddy, B.S.; Watanabe, K.; Sheinfil, A. J. Nutr. 1980, 110, 1247-1254. Ginter, E . ; Kohn, R.; Malovikova, A.; Ozdin, L . ; Kohnova, Z. Nutr. Rep. Int. 1983, 27, 1049-1057. Lafont, H.; Lairon, D.; Vigne, J.L.; Chanussot, F.; Chabert, C.; Portugal, H. J. Nutr. 1985, 115, 849-855. Kern, F . , Jr.; Birkner, H.J.; Ostrower, V.S.; Am. J. Clin. Nutr. 1978, 31, S175-S179. Selvendran, R.R. Chem. and Ind. 1978, 17 June, 428-430. Newmark, H.L.; Wargovich, M.J.; Bruce, W.R.; Proc. Nat. Large Bowel Cancer Project, 1983; pp. 1-15, Praeger Scientific Press.

Received February 3, 1986

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

Author Index Barford, R. A., 22 Beach, P., 103 Behall, Kay, 248 BeMiller, James N., 2 Carr, J . M., 200 Cesaro, A., 73 Ciana, A., 73 Crandall, P h i l i p G., 88 Damert, W. C , 22 Davis, E., 103 de Vries, J . A., 38 Delben, F., 73 Doner, Landis W., 13 Fishman, Marshall L. 22 Grasdalen, Hans Hoagland, Pete Huber, Donald J . , 141 Ikkala, P., 103 Irwin, Peter L., 175 Labavitch, J . M., 200 Lee, James H., 141 Lundbye, M., 103 McFeeters, Roger F., 217

Northcote, D. H., 134 P a o l e t t i , S., 73 Pepper, L., 22 P f e f f e r , P h i l i p E., 266 P h i l l i p s , J . G., 22 P i l n i k , W., 38,230 Pressey, Russel, 157 Reid, D. S., 200 Reiser, Sheldon, 248 Rinaudo, M., 61 Rombouts, F. M., 38,49 Sajjaanantakul, T., 200 Schols, H. A., 230 S e v i l l a Michael 175 Smidsr/rfd, Olav, 117 Stoudt, Carla L., 175 Thibault, J . F., 49,61 Toft, Knut, 117 Van Buren, Jerome P., 190 Voragen, A. G. J . , 38,230 Wicker, Louise, 88

Subject Index Apple juice processing—Continued sugar composition of alcohol-insoluble s o l i d s , 231-232t Alginates sugar and g l y c o s i d i c linkage features of the monomer composition of the retentate sequence, 126 f r a c t i o n , 235,237t modulus measurements vs. s t r u c t u r a l uronide content of apple c h a r a c t e r i s t i c s , 128,130f j u i c e s , 235t structural Apple pectins c h a r a c t e r i s t i c s , 126,128,129t degree of polymerization Apple juice processing d i s t r i b u t i o n , 44,46f,47f changes i n juice and pulp a f t e r d i s t r i b u t i o n methoxylated and free enzyme treatment, 233,234f carboxyl group, 44-47 degree of branching of arabinan extraction schematic, 39,40f f r a c t i o n s , 237,239t high-pressure LC, 44,46f elution p r o f i l e for press neutral sugar composition, 4l,43f j u i c e , 235,236f neutral sugar d i s t r i b u t i o n , 39-44 gel f i l t r a t i o n chromatography of the Apricot nectar processing retentate f r a c t i o n , 237,238f effects of enzymes on cloud mold enzyme degradation, 231-232 s t a b i l i t y , 24l,243t points of attack of arabinane l u t i o n patterns of pectic degrading enzymes, 239-240f material, 243,244f preparation of u l t r a f i l t r a t i o n i s o l a t i o n of pectic material, 243 permeate and retentate neutral sugars and galacturonides f r a c t i o n s , 235,237,238f released by pure role of pectinase i n increasing enzymes, 24l,243t j u i c e y i e l d s , 241 A

277

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Author Index Barford, R. A., 22 Beach, P., 103 Behall, Kay, 248 BeMiller, James N., 2 Carr, J . M., 200 Cesaro, A., 73 Ciana, A., 73 Crandall, P h i l i p G., 88 Damert, W. C , 22 Davis, E., 103 de Vries, J . A., 38 Delben, F., 73 Doner, Landis W., 13 Fishman, Marshall L. 22 Grasdalen, Hans Hoagland, Pete Huber, Donald J . , 141 Ikkala, P., 103 Irwin, Peter L., 175 Labavitch, J . M., 200 Lee, James H., 141 Lundbye, M., 103 McFeeters, Roger F., 217

Northcote, D. H., 134 P a o l e t t i , S., 73 Pepper, L., 22 P f e f f e r , P h i l i p E., 266 P h i l l i p s , J . G., 22 P i l n i k , W., 38,230 Pressey, Russel, 157 Reid, D. S., 200 Reiser, Sheldon, 248 Rinaudo, M., 61 Rombouts, F. M., 38,49 Sajjaanantakul, T., 200 Schols, H. A., 230 S e v i l l a Michael 175 Smidsr/rfd, Olav, 117 Stoudt, Carla L., 175 Thibault, J . F., 49,61 Toft, Knut, 117 Van Buren, Jerome P., 190 Voragen, A. G. J . , 38,230 Wicker, Louise, 88

Subject Index Apple juice processing—Continued sugar composition of alcohol-insoluble s o l i d s , 231-232t Alginates sugar and g l y c o s i d i c linkage features of the monomer composition of the retentate sequence, 126 f r a c t i o n , 235,237t modulus measurements vs. s t r u c t u r a l uronide content of apple c h a r a c t e r i s t i c s , 128,130f j u i c e s , 235t structural Apple pectins c h a r a c t e r i s t i c s , 126,128,129t degree of polymerization Apple juice processing d i s t r i b u t i o n , 44,46f,47f changes i n juice and pulp a f t e r d i s t r i b u t i o n methoxylated and free enzyme treatment, 233,234f carboxyl group, 44-47 degree of branching of arabinan extraction schematic, 39,40f f r a c t i o n s , 237,239t high-pressure LC, 44,46f elution p r o f i l e for press neutral sugar composition, 4l,43f j u i c e , 235,236f neutral sugar d i s t r i b u t i o n , 39-44 gel f i l t r a t i o n chromatography of the Apricot nectar processing retentate f r a c t i o n , 237,238f effects of enzymes on cloud mold enzyme degradation, 231-232 s t a b i l i t y , 24l,243t points of attack of arabinane l u t i o n patterns of pectic degrading enzymes, 239-240f material, 243,244f preparation of u l t r a f i l t r a t i o n i s o l a t i o n of pectic material, 243 permeate and retentate neutral sugars and galacturonides f r a c t i o n s , 235,237,238f released by pure role of pectinase i n increasing enzymes, 24l,243t j u i c e y i e l d s , 241 A

277

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Apricot nectar processing—Continued preparation of nectars, 24l,2M2f

B B i l e acid binding to carrot f i b e r ammonium oxalate extraction of carrot alcohol-acetone insoluble residue, 267-268,270t binding of chenodeoxycholate to calcium (carboxymethyl)c e l l u l o s e , 270,271t binding of chenodeoxycholate to calcium pectate, 267,271t,272 calcium (carboxymethyl)c e l l u l o s e , 267 calcium analysis, 267 calcium effects, 268,270 carbon-13 NMR spectroscopy of pectin, 272,273f carrot fiber preparation, 266,267 deoxycholate extraction of carrot alcohol-acetone insoluble residue, 268 NMR spectroscopy, 268 preparation of calcium pectate, 267,271 p u r i f i c a t i o n of pectin, 267 scanning electron micrograph of carrot alcohol-acetone insoluble residue, 268,269f

C Calcium binding to pectins characteristics of the pectins, 63,64f c i r c u l a r dichroisra measurements, 66,67f,68 counterion a c t i v i t y c o e f f i c i e n t s , 6M-65,67f effective charge parameter, 68,69f egg-box model, 61-62 influence of pattern of e s t e r i f i c a t i o n , 65-66,67f influence of polymer concentration on calcium a c t i v i t y c o e f f i c i e n t , 65-66 neutralization effects, 65,68,69f Calcium ion, effect on texture of f r u i t s and vegetables, 217-228 Carbazole method, galacturonic acid determination, 14 Carbon-13 NMR spectroscopy, galacturonic acid determination, 16

Carrot f i b e r b e n e f i c i a l properties, 272-273 binding of b i l e acids, 266-27M Chemical properties of pectins acid-catalyzed hydrolysis, 9 6-elimination reaction, 9 deesterification, 9 depolymerization, 9 hydroxy1-group reactions, 9-10 lyases, 10 pectinesterases, 10 polygalacturonases, 10 transeliminases, 10 C o l l o i d a l t i t r a t i o n method, galacturonic acid determination, 15 Colorimetric method, acetyl and f e r u l o y l ester determination, 18 formation of junction zones, 5 gels, 4-5 Cucumbers c e l l wall structure, 220 textural changes during processing and storage, 218-228

D Decarboxylation with hydroiodic a c i d , galacturonic acid determination, 15 Degree of amidation, d e f i n i t i o n , 3 Degree of e s t e r i f i c a t i o n , definition, 3 Degree of polymerization, number average, 32,33t,3*J Destructive tests for gel strength measurements advantages, 96 finger test, 98 Herbstreith pectinoraeter measurements, 99 Instron measurements, 99 Luers-Lochmuller pectinometer measurements, 98-99 Tarr-Baker method, 98 Dynamic testing, 97-98

E End-group t i t r a t i o n , determination of number-average molecular weight, 26 Endopectin lyases, role i n f r u i t processing, 231

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INDEX

Endopolygalacturonaese, role i n f r u i t processing, 231 Enzymic c e l l wall degradation, stages, 244,245t

F Fiber effects on humans approaches, 248 carbohydrate metabolism, 251,253-255t effect and fate i n the g a s t r o i n t e s t i n a l t r a c t , 261-262 glycemic e f f e c t s , 257-260 l i p i d e f f e c t s , 250,256 l i p i d metabolism, 250,251-252 mineral b i o a v a i l a b i l i t y vitamin b i o a v a i l a b i l i t y e f f e c t s , 260-261 Firmness influencing factors, 200 assessment—See Texture assessment Foods, pectin content, 248,249t Fractionation of apple pectin elution p r o f i l e , 39,40f enzymic degradation, 4l,42f ion-exchange chromatography, 41 neutral sugar d i s t r i b u t i o n curves, 39,4l,42f F r u i t , ripening, c e l l wall metabolism, 141-154 F r u i t juices processing, 231-245 sources, 230 tissue composition, 230-231 F r u i t nectars d e f i n i t i o n , 241 use of pectic enzymes for cloud s t a b i l i t y , 241 F r u i t processing, pectic processing, 231 F r u i t softening, effect of endo-D-galacturonanase, 149,152

H

Hairy region f r a c t i o n s , gel f i l t r a t i o n p r o f i l e s , 4l,44,45f Hemicelluloses, d e f i n i t i o n , 13 Herbstreith pectinometer i n t e r n a l strength vs. pectin dosage, 110,111f measurement of pectin breaking strength, 99,105,108 photograph, 105,107f Herbstreith pectinometer measurements, 99 High-methoxyl pectins categories, 89t definition, 3 g e l a t i n factors, 8

Human metabolism, effects of pectin, 248-262

I IFT sag method effect of pH on measured sag of j e l l i e s , 104-105,106f instrumental methods, 105,106-107f,108,109f Instron measurements, 99 Internal gel strength influencing factors j e l l y pH, 94 j e l l y test conditions, 94 molecular weight, 92 pectin type, 92,93f Internal gel strength instrumentation creep compliance, 94,96 destructive t e s t , 96,98-99 e l a s t i c l i m i t , 94 nondestructive t e s t s , 96-98 textural p r o f i l e s , 94 IR spectroscopy, galacturonic acid determination, 15-16

G Galacturonic acid analysis i n pectin chemical methods 14-15 chromatographic methods, 16-17 physical methods, 15-16 Gel strength, instruments for measurement, 94,95t Gels, d e f i n i t i o n , 91 GLC procedure galacturonic acid determination, 16 neutral sugar determination, 19 methyl ester determination, 18

J J e l l y , d e f i n i t i o n , 89 J e l l y grade, d e f i n i t i o n , 104 J e l l y grade determination IFT age method, 104-115 preparation of j e l l i e s , 104 J e l l y manufacturers needs, pectin evaluation, 89-90 J e l l y tester, 97

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Junction zones a l k a l i n e depolymerization, 5,6f formation models, 5

L

LC methods galacturonic acid determination, 17 neutral sugar determination, 19 Length, number average, 34,35t Low-methoxyl pectins definition, 3 g e l a t i n factors, 8 Luers-Lochmuller pectinometer photograph, 105,106f measurements, 98-99

M Membrane osmometry, determination of number-average molecular weight, 26 Methyl, a c e t y l , and f e r u l o y l substitution i n pectin, 17-18

N

Neutral sugar composition of pectin determination, 19 l i s t of sugars, 18 values, 4l,43f Neutral sugar residues, s i z e , 54 Nondestructive tests for gel strength measurements advantages, 96 concentric cylinder instruments, 97 description, 96-97 dynamic t e s t i n g , 97-98 IFT sag method, 96-97 j e l l y t e s t e r , 97 p a r a l l e l plate measurements, 97 Wageningen sag methods, 97 Nucleotide sugar donors formation, 135,136f transport across the membrane, 135,137

P P a r a l l e l plate measurements, 97 Partition coefficients, determination, 25-26

Pectate aggregation, 79,82 concentration determination, 84 definition, 2 dependence of osmotic pressure on concentration, 74-75,76f dependence of p r e c i p i t a t e amount on divalent ion addition, 79,81f,82 energetics of interaction with divalent ions, 75,77 enthalpy data, 77,78f interactions i n aqueous solutions, 73-86 molar r a t i o vs. e l l i p t i c i t y , 79,80f molecular weight vs. bound divalent ions, 82,83f osmometric measurements, 84,86 pH-induced conformational

rheology gels, 82,84,85f s t r u c t u r a l properties, 79 volume change on addition of divalent cations, 75,76f Pectic a c i d , d e f i n i t i o n , 2 Pectic polysaccharide, d e f i n i t i o n , 13 Pectin a l k a l i n e d e e s t e r i f i c a t i o n , 62 analysis by uronic acid assay, 201,204 apple, 38-47 c h a r a c t e r i s t i c parameters, 32,33t characterization of samples, 62-63 chemical properties, 9-10 chromatograms for methyl-esterified pectins, 28,29f c i r c u l a r dichroism, 63 composition, 157-158 conformation, 4,5,6f content i n foods, 248,249t d e f i n i t i o n , 3,14,90 enzyme degradation of ot— 1,4—galacturonan, 158 enzymic d e e s t e r i f i c a t i o n , 62 extraction from c e l l w a l l material, 201 formation during plant c e l l wall growth, 134-139 glycemic e f f e c t s , 257-260 influence on i n t e r n a l gel strength, 92,93f interactions with counterions, 61-71 l i p i d e f f e c t s , 250,256 methyl, a c e t y l , and f e r u l o y l substitution determination, 17-18 mineral b i o a v a i l a b i l i t y e f f e c t s , 260 model of types A-E, 4l,43f neutral sugar composition, 18 number-average degree of polymerization, 32,33t,34 number-average length, 34,35t

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

INDEX

281

Pectate—Continued occurrence i n plant materials, 157 physical properties, 5,7-9 potentiometry, 63 quantitative analysis of galacturonic a c i d , 14 r a d i i of gyration, 28,29t role i n binding of b i l e acids to carrot f i b e r , 266-274 sample preparation, 62-63 self-disaggregation, 23-36 structure, 3-4,38-39,90 subdivision, 3 sugar beet, 49-59 theory of gel formation, 90-91 use i n manufacture of j e l l y , 88 vitamin b i o a v a i l a b i l i t y e f f e c t s , 260-261 Pectin-alginate systems determination of mechanica properties of gels, 119 effect of calcium on gels, 120-121 effect of d i f f e r e n t r a t i o s of pectin and alginate, 121,126,127f effects of g-glucono-6-lactone on gels, 121,122-125f preparation of gels, 119 source of pectin and alginate, 118-119,120t Pectin esterases, role i n f r u i t processing, 231 Pectin function i n tomato ripening endo-^-galacturonanase a c t i v i t y , I45t enzyme assays, 143 gel f i l t r a t i o n p r o f i l e s , 143,145,146-I47f,152 hemicellulose extraction and analysis, I43,149,151f,154 ion-exchange chromatography, I43,l45,l48f measurement of t o t a l and soluble pectins, 143 neutral sugar analysis, 143,149,150f,153-154 percent e s t e r i f i c a t i o n , 143 plant material, 142 preparation of ethanol-insoluble s o l i d s , 142 t o t a l and soluble pectins i n ethanol powders, I44t,l45 Pectin gel network, 91 Pectin grading breaking strength vs. molecular weight, 110,113,H4f ,115 breaking strength vs. v i s c o s i t y , 113,1l4f,115 comparison of various methods, 108,115 Pectin polymers chemical modification i n the c e l l w a l l , 139 compartmentalization within the endomembrane system, 138

Pectin polymers—Continued fusion with the plasmamembrane, 139 movement of v e s i c l e s , 138-139 synthesis within the endomembrane system, 137 Pectinate, d e f i n i t i o n , 2 Pectinic a c i d , d e f i n i t i o n , 2 Physical s o l u b i l i t y of pectins function of structure, 7 gelation factors, 7-9 pH e f f e c t s , 7 v i s c o s i t i e s , 5,7 water s o l u b i l i t i e s , 5 Plant c e l l wall channels for pectin movement and synthesis, 135-139 formation of pectin, 134-135 Polygalacturonase mechanism of degradation, 158 Polygalacturonase converter effect on isoenzymes, 162 properties, 161-162 p u r i f i c a t i o n , 161t Polygalacturonase i n f r u i t s a c t i v i t y , I67,l69,170f isoenzymes, 169 Polygalacturonase isoenzymes a c t i v i t y , 165,167 conversion of I to I I , 159,161 converter, I6lt,162,165,167 separation, 159,l60f s i m i l a r i t i e s , 159 Polygalacturonase i n peaches a c t i v i t y , I67,l69t isoenzymes, 167 s o l u b i l i t y , I67,l68f,l69 Polygalacturonase i n plant tissues a c t i v i t y , 169,171t characterization, 171,172f,173t isoenzymes, 169,171 Polygalacturonase i n tomatoes a c t i v i t y , 165,167 effect of pH on s o l u b i l i t y , I62,l63f effect of s a l t on s o l u b i l i t y , 162,l64f,165,I66f isoenzymes, 159 l e v e l s , 159 Polysaccharides, universal c a l i b r a t i o n curves, 26,27f,28 Polyuronides properties, 175-176 sequential cooperative ion binding mechanism, 176-186

R Radii of gyration d e f i n i t i o n , 28

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Radii of gyration—Continued influencing factors, 28,30 number average, 30,31t,32 values, 28,29t Root-mean-square r a d i i of gyration, determination, 25

S Self-disaggregation of pectin experimental procedures, 23-26 van't Hoff plots, 23,27f Sequential cooperative ion binding mechanism effect of competitive exchange on broadening, 183,184-I85f,18 evidence for a divalent cation-polyuronide aggregat structure, 182-183 experimental conditions, 177 paramagnetic ion couplings, 177,179 paramagnetic ion dependency of l i n e width, !79,180-I8lf,182 preparation of tissues, 176-177 t i t r a t i o n of Mn , 177,178f Serum cholesterol, mechanism for reduction, 256-257 Softening of vegetables during heat processing effect of adding s a l t s after cooking, 193f,194-195,196f effect of adding s a l t s b e f o r e ^ cooking, corrected for Ca displacement, 194t,195 effect of 3-elimination hypothesis effect of cooking time and temperature on firmness, 192,195t,197 effect of K f e r t i l i z a t i o n on firmness, 192t effect of leaching and NaCl on firmness, 192t,194t effect of NaCl added before or a f t e r cooking on firmness, 192,194t effect of s a l t s present before cooking on cohesiveness, 191t effect of s a l t s present before cooking on firmness, 191t effect of soaking on firmness, 192,193f role of C displacement, 197,198 Structure of pectin acetyl groups, 3-4 linear chain, 3 neutral sugars, 4 types, 90 Sugar beet pectin a n a l y t i c a l methods, 50 chromatography, 51 +

Sugar beet pectin—Continued composition and properties, 52t cross-linking and gelation, 51,53 degradation l i m i t s , 53t,54 degradation studies, 51,53t,54 effect of enzymic cross-linking on v i s c o s i t y and molecular mass, 58t,59f extraction and p u r i f i c a t i o n , 50 gel permeation chromatograms, 52-53,55f,57f gelation through oxidation coupling, 56,58,59f location of acetyl ester groups, 54,56,57f neutral sugar content, 52t new applications, 58 presence and location of f e r u l o y l

yield anhydrogalacaturoni content, 51,52t Sugar beet pulp, source of pectin, 49-50 Synergistic g e l l i n g systems, pectin-alginate system, 117-131

T Tarr-Baker method, 98 Textural changes of cucumbers calcium as a firming agent, 2l8,219t,220 changes i n c e l l wall neutral sugar content, 220,221f changes i n pectin fractions, 220,222f effect of calcium concentration, 223,225f effect of pectin methylation, 223,224f enzymatic softening, 218 e s t e r i f i c a t i o n vs. calcium concentration, 226,227t fermentation, 220 firmness vs. degree of methylation, 223,226 Scatchard plot of calcium binding, 226,227f Texture assessment effect of blanching, 204,205f effect of freezing, 204,205f,206,207f elution p r o f i l e vs. storage time, 209,212-2l4f,215t extraction performance of d i f f e r e n t chelators, 206,207f instrumentation, 200-201,202f loss i n frozen storage, 209,211f

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

INDEX

Texture assessment—Continued pectic fractions before and a f t e r processing, 206,208t preparation and p u r i f i c a t i o n of pectin from c e l l wall material, 201,203f,204 tissue systems used, 204 uronide content vs. storage time, 206,210f,t Texture measurements internal strength curves, 108,109f Voland Stevens texture analyzer, 108,109f,110,111f T i t r a t i o n method, methyl ester determination, 18 Tomato f r u i t , pectin function i n texture modification, 142-154

Vegetables, softening during heat processing, 190-198 Voland Stevens texture analyzer, internal strength vs. pectin dosage, 108,109f,110,111f

Wageningen sag measurements, 97

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.