Chemistry and Function of Pectins - American Chemical Society

1 An Introduction to Pectins: Structure and Properties James N. BeMiller

Downloaded by COLORADO COLLEGE on February 18, 2015 | http://pubs.acs.org Publication Date: June 5, 1986 | doi: 10.1021/bk-1986-0310.ch001

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 units joined inα-D≡(1->4)glycosidic linkages; the polymer chains are esterified to 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 n (DA) i n d i c a t e s t h e p e r c e n t o f c a r b o x y l groups i n t h e amide form (See s e c t i o n on Chemical 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


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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) t h a t a p p l e p e c t i n c o n s i s t s of "smooth" regions [poly(a-D-galactopyranosyluronic a c i d ) r e g i o n s ] and " 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 ^C·4)-linked poly(α-D-galactop y r a n o s y l u r o n i c a c i d ) segments o f p e c t i c a c i d and~"(1 -•MJ-linked p o l y ( a - L - g u l o - p y r a n o s y l u r o n i c a c i d ) segments o f a l g i n i c a c i d s , segments t h a t a r e m i r r o r images e x c e p t f o r the c o n f i g u r a t i o n at C-3. From c i r c u l a r d i c h r o i s m and e q u i l i b r i u m d i a l y s i s s t u d i e s , i t has been c o n c l u d e d t h a t i n t e r c h a i n a s s o c i a t i o n of h y d r a t e d p e c t i n i c a c i d m o l e c u l e s , i n the presence o f swamping l e v e l s o f monovalent c o u n t e r i o n s , i s l i m i t e d t o the f o r m a t i o n of dimers o f c h a i n s o f 2y h e l i c a l symmetry w i t h s p e c i f i c s i t e - b i n d i n g of c a l c i u m i o n s a l o n g one f a c e of each p a r t i c i p a t i n g c h a i n ( 1 8 , 1 9 ; see a l s o r e f e r e n c e 20). When C a i s the s o l e or p r i n c i p a l c o u n t e r i o n , t h e s e dimers f u r t h e r aggregate w i t h o u t rearrangement, l e a d i n g t o an a p p r o x i m a t e d o u b l i n g o f the amount o f C a bound c o o p e r a t i v e l y (J_8,J_9). Based on a v a i l a b l e i n f o r m a t i o n , the U n i l e v e r R e s e a r c h group (J_9) has concluded t h a t d r y i n g of a c a l c i u m p e c t i n a t e g e l e f f e c t s a p o l y m o r p h i c phase t r a n s i t i o n i n which a s s o c i a t e d , r e g u l a r , b u c k l e d c h a i n s w i t h t w o - f o l d symmetry ("egg box") as found i n L - g u l u r o n o g l y c a n c h a i n segments a r e c o n v e r t e d i n t o a s s o c i a t e d c h a i n s w i t h t h r e e - f o l d symmetry as found i n s o l i d s t a t e c a l c i u m p e c t i n a t e (12-15). I t s h o u l d be n o t e d t h a t the a x i a l - a x i a l l i n k a g e s i n a c h a i n o f a l d o h e x o p y r a n o s y l u n i t s l i n k e d 1+4 g i v e s a b u c k l e d c o n f o r m a t i o n n a t u r a l l y ( F i g u r e 1) and t h a t t h e g e l s t r u c t u r e ( s ) i s ( a r e ) as yet not w e l l u n d e r s t o o d . 2 +

2 +

Physical Properties P e c t i n s are s o l u b l e i n pure w a t e r , but they a r e i n s o l u b l e i n aqueous s o l u t i o n s i n w h i c h they would g e l a t the same t e m p e r a t u r e i f d i s s o l v e d a t a h i g h e r t e m p e r a t u r e . Monovalent c a t i o n ( a l k a l i m e t a l ) s a l t s of p e c t i n i c and p e c t i c a c i d s a r e u s u a l l y s o l u b l e i n water; d i - and t r i v a l e n t c a t i o n s a l t s are weakly s o l u b l e or insoluble. A l t h o u g h p e c t i n s a r e not employed as t h i c k e n i n g a g e n t s , p e c t i n s o l u t i o n s e x h i b i t the non-Newtonian, p s e u d o p l a s t i c b e h a v i o r c h a r a c t e r i s t i c o f most p o l y s a c c h a r i d e s . As w i t h s o l u b i l i t y , the v i s c o s i t y of a p e c t i n s o l u t i o n i s r e l a t e d t o t h e m o l e c u l a r w e i g h t , DE, and c o n c e n t r a t i o n o f the p r e p a r a t i o n and the pH and p r e s e n c e



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CHEMISTRY A N D

F i g u r e 1. Alkaline a c i d o f DE 25.

FUNCTION

OF

PECTINS

d e p o l y m e r i z a t i o n o f a sequence of a p e c t i n i c



1.

BE M I L L E R


of c o u n t e r i o n s i n the s o l u t i o n . For example, a d d i t i o n of monovalent c a t i o n s e f f e c t s a r e d u c t i o n i n v i s c o s i t y , the degree of which i s g r e a t e r w i t h d e c r e a s i n g DE. A d d i t i o n of s a l t s o f d i - and t r i v a l e n t c a t i o n s has an o p p o s i t e e f f e c t ( 2 0 ) . I n g e n e r a l , v i s c o s i t y , s o l u b i l i t y , and g e l a t i o n a r e r e l a t e d , i . e . , f a c t o r s t h a t i n c r e a s e g e l s t r e n g t h , f o r example, w i l l i n c r e a s e the tendency t o g e l , decrease s o l u b i l i t y , and i n c r e a s e v i s c o s i t y , and vice versa. These p h y s i c a l p r o p e r t i e s o f p e c t i n s are a f u n c t i o n o f t h e i r s t r u c t u r e which i s t h a t of a l i n e a r p o l y a n i o n ( p o l y c a r b o x y l a t e ) . As s u c h , monovalent c a t i o n s a l t s o f p e c t i n s are h i g h l y i o n i z e d i n s o l u t i o n , and the d i s t r i b u t i o n of i o n i c charges a l o n g the m o l e c u l e tends t o keep i t i n an extended form by r e a s o n o f coulombic r e p u l s i o n ( 2Λ). F u r t h e r m o r e , t h i s same columbic r e p u l s i o n between the c a r b o x y l a t e anions p r e v e n t s a g g r e g a t i o n o f the polymer c h a i n s . (The number of n e g a t i v e charges i s , of c o u r s e , determined by the DE.) I n a d d i t i o n , each p o l y s a c c h a r i d e c h a i n , and e s p e c i a l l y each c a r b o x y l a t e group, w i l l be h i g h l y h y d r a t e d . S o l u t i o n s of monovalent s a l t s of p e c t i n s e x h i b i t s t a b l e v i s c o s i t y because each polymer c h a i n i s h y d r a t e d , extended, and independent. Because the commercial importance of p e c t i n i s p r e d o m i n a t e l y the r e s u l t of i t s unique a b i l i t y t o form s p r e a d a b l e g e l s i n the presence of a d e h y d r a t i n g agent (sugar) a t a pH a t or near 3 o r i n the presence of c a l c i u m i o n (jams, j e l l i e s , and marmalades made from f r u i t j u i c e s o r whole f r u i t ) , t h a t i s the p r o p e r t y most o f t e n s t u d i e d and f o c u s e d upon. F a c t o r s t h a t determine whether g e l a t i o n can occur and t h a t i n f l u e n c e g e l c h a r a c t e r i s t i c s are pH, c o n c e n t r a t i o n o f c o s o l u t e s ( s u g a r s ) , c o n c e n t r a t i o n and type of c a t i o n s , t e m p e r a t u r e , and p e c t i n c o n c e n t r a t i o n . The ways i n w h i c h these f a c t o r s i n f l u e n c e g e l a t i o n a r e dependent upon the f o l l o w i n g m o l e c u l a r p r o p e r t i e s of the s p e c i f i c p e c t i n : m o l e c u l a r weight (Jp, degree o f e s t e r i f i c a t i o n (DE), degree of a m i d a t i o n (DA), presence of a c e t a t e e s t e r s , and h e t e r o g e n e i t y . A l l these parameters are i n t e r d e p e n d e n t . I n g e n e r a l , under s i m i l a r c o n d i t i o n s , the degree of g e l a t i o n , the g e l l i n g t e m p e r a t u r e , and the g e l s t r e n g t h a r e g e n e r a l l y p r o p o r t i o n a l t o each o t h e r and each p r o p e r t y i s g e n e r a l l y p r o p o r t i o n a l t o the m o l e c u l a r weight and i n v e r s e l y p r o p o r t i o n a l t o the DE. As the pH i s l o w e r e d , i . e . , as the hydrogen i o n c o n c e n t r a t i o n of the s o l u t i o n i s i n c r e a s e d , i o n i z a t i o n o f the c a r b o x y l a t e groups i s r e p r e s s e d , i . e . , the h i g h l y h y d r a t e d c a r b o x y l a t e groups a r e c o n v e r t e d i n t o o n l y s l i g h t l y h y d r a t e d c a r b o x y l i c a c i d groups. As a r e s u l t of l o s i n g some of t h e i r c h a r g e , the p o l y s a c c h a r i d e m o l e c u l e s no l o n g e r r e p e l each o t h e r over t h e i r e n t i r e l e n g t h ; and as a r e s u l t o f l o s i n g some of the water o f h y d r a t i o n , they can a s s o c i a t e 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 g e l . Apparent pK v a l u e s (pH a t 50% d i s s o c i a t i o n ) v a r y w i t h the DE of the p e c t i n (22); a 65% DE p e c t i n has an apparent pK of 3.55, w h i l e a 0ί DE p e c t i c a c i d has an apparent pK of 4.10. However, p e c t i n s w i t h i n c r e a s i n g l y g r e a t e r degrees of m e t h y l a t i o n w i l l g e l a t somewhat h i g h e r pH, undoubtedly because they have fewer c a r b o x y l a t e a n i o n s at any g i v e n pH (See l a t e r p a r a g r a p h ) . pH a f f e c t s g e l t e x t u r e more t h a n g e l s t r e n g t h .


1


8

CHEMISTRY AND FUNCTION OF PECTINS

I t i s g e n e r a l l y regarded t h a t g e l s form when p e c t i n m o l e c u l e s , n o r m a l l y s t r o n g l y h y d r a t e d by water m o l e c u l e s , l o s e some water o f h y d r a t i o n owing t o c o m p e t i t i v e h y d r a t i o n o f c o s o l u t e molecules. Reduced h y d r a t i o n r e s u l t s i n g r e a t e r c o n t a c t between p e c t i n c h a i n s , f o r m i n g j u n c t i o n zones ( p r i m a r i l y by means o f hydrogen bonding) and r e s u l t i n g i n a network o f polymer c h a i n s t h a t e n t r a p s water and s o l u t e m o l e c u l e s (^3). I n most jams, j e l l i e s , and marmalades, t h e c o s o l u t e i s sugar ( s u c r o s e ) . HM-pectins w i l l g e l o n l y i n t h e presence o f l a r g e c o n c e n t r a t i o n s (at l e a s t 55% w/w) o f sugar. L M - p e c t i n w i l l g e l i n t h e absence o f sugar ( i f a d i v a l e n t c a t i o n i s p r e s e n t ) , but i n c r e a s i n g t h e s o l u b l e s o l i d s w i l l r a i s e t h e g e l l i n g temperature and g e l strength. L M - p e c t i n w i l l g e l o n l y i n t h e presence o f d i v a l e n t c a t i o n s . Increasing the concentration of d i v a l e n t c a t i o n s (only calcium i o n i s used i n food a p p l i c a t i o n s ) i n c r e a s e s t h e g e l l i n g temperature and g e l s t r e n g t h . D i v a l e n t c a t i o n s a r e unnecessary f o r t h e f o r m a t i o n o f a HM-pectin g e l because o f t h e low number o f c a r b o x y l a t e groups t h a t need t o be b r i d g e d and because o f t h e f o r m a t i o n o f h y d r o p h o b i c a r e a s p a r a l l e l t o t h e h e l i x axes by a columnar s t a c k i n g o f methyl e s t e r groups ( 1 5 ) . Any system c o n t a i n i n g p e c t i n a t p o t e n t i a l g e l l i n g c o n d i t i o n s ( i . e . , n e c e s s a r y c o n c e n t r a t i o n o f an a p p r o p r i a t e p e c t i n , pH, c o n c e n t r a t i o n o f c o s o l u t e s , and c o n c e n t r a t i o n of d i v a l e n t c a t i o n s ) must be prepared a t a temperature above t h e g e l l i n g t e m p e r a t u r e . Then, as t h e hot p e c t i n s o l u t i o n i s c o o l e d , t h e thermal energy o f the m o l e c u l e s d e c r e a s e s and t h e i r tendency t o form j u n c t i o n zones upon c o l l i s i o n i n c r e a s e s . The temperature a t which g e l a t i o n o c c u r s i s t h e g e l l i n g t e m p e r a t u r e . Gels made w i t h L M - p e c t i n form r a p i d l y ; those made w i t h HM-pectin form s l o w l y . L M - p e c t i n g e l s are t h e r m o r e v e r s i b l e ; HM-pectin g e l s a r e n o t . The c o n c e n t r a t i o n o f p e c t i n r e q u i r e d f o r g e l f o r m a t i o n i s i n v e r s e l y r e l a t e d to the concentration of s o l u b l e s o l i d s , f o r i n general, i n c r e a s i n g the concentration of c o s o l u t e s , i . e . , d e c r e a s i n g the water a c t i v i t y , i n c r e a s e s t h e s i z e and number o f j u n c t i o n zones. When o t h e r f a c t o r s a r e h e l d c o n s t a n t , i n c r e a s i n g the c o n c e n t r a t i o n o f p e c t i n i n c r e a s e s t h e g e l s t r e n g t h because i t i n c r e a s e s t h e number o f j u n c t i o n zones. At c o n s t a n t pH, g e l s t r e n g t h o f HM-pectin g e l s i n c r e a s e s w i t h i n c r e a s i n g DE, as does t h e r a t e o f g e l a t i o n . As t h e DE o f a HM-pectin i s l o w e r e d , a lower pH i s r e q u i r e d f o r g e l a t i o n . A g a i n , t h i s can be e x p l a i n e d by t h e f a c t t h a t , as t h e DE i s l o w e r e d , even though t h e pH r e q u i r e d t o produce a g i v e n percentage o f c a r b o x y l groups i n t h e c a r b o x y l i c a c i d form i n c r e a s e s owing t o an i n c r e a s e i n apparent pK (22), t h e a b s o l u t e number o f c o n t i g u o u s c a r b o x y l a t e groups t h a t must be c o n v e r t e d i n t o u n i o n i z e d c a r b o x y l i c a c i d groups b e f o r e c h a i n a s s o c i a t i o n can occur a l s o i n c r e a s e s . Thus, as t h e DE o f a HM-pectin i s l o w e r e d , a g r e a t e r and g r e a t e r percentage of c a r b o x y l a t e groups must be c o n v e r t e d i n t o f r e e c a r b o x y l i c a c i d groups t o e f f e c t g e l a t i o n . Among the commercial L M - p e c t i n s , those w i t h t h e l o w e s t DE v a l u e s have t h e h i g h e s t g e l l i n g temperatures and t h e g r e a t e s t requirement f o r d i v a l e n t c a t i o n s ( f o r c r o s s b r i d g i n g ) .


1.

Β ΕMIL L Ε R


Ami d a t i o n r e s u l t s i n a h i g h e r g e l l i n g temperature and a decreased need f o r a d i v a l e n t c a t i o n . The d i s t r i b u t i o n of c a r b o x y l / c a r b o x y l a t e groups a l s o a f f e c t s g e l a t i o n . P e c t i n s w i t h b l o c k s of m e t h y l e s t e r and c a r b o x y l groups (as opposed t o a random d i s t r i b u t i o n ) g e n e r a l l y produce weaker g e l s and have a g r e a t e r r e q u i r e m e n t f o r d i v a l e n t c a t i o n s .


Chemical P r o p e r t i e s D i s s o l v e d p e c t i n s undergo d e e s t e r i f i c a t i o n and d e p o l y m e r i z a t i o n i n aqueous systems. The pH of g r e a t e s t s t a b i l i t y i s about 4. A t pH v a l u e s b o t h above and below 4, d e e s t e r i f i c a t i o n and d e p o l y m e r i z a t i o n occur c o n c u r r e n t l y , w i t h the r a t e of d e e s t e r i f i c a t i o n b e i n g g r e a t e r t h a n the r a t e o f d e p o l y m e r i z a t i o n . The presence of s o l u t e s , which l o w e r s water a c t i v i t y , reduces the r a t e s of both r e a c t i o n s . D e e s t e r i f i c a t i o n o c c u r s by normal a c i d - and b a s e - c a t a l y z e d mechanisms of e s t e r h y d r o l y s i s . D e p o l y m e r i z a t i o n a t low pH v a l u e s o c c u r s by means of a c i d - c a t a l y z e d h y d r o l y s i s of g l y c o s i d i c bonds (24). A c i d - c a t a l y z e d h y d r o l y s i s occurs p r e f e r e n t i a l l y at the L-rhamnopyranosyl g l y c o s i d i c bonds. H y d r o l y s i s of these l i n k a g e s produces g a l a c t u r o n o g l y c a n c h a i n s w i t h a degree of p o l y m e r i z a t i o n of about 25 (6,9). S i d e c h a i n s , p a r t i c u l a r l y those c o n t a i n i n g L - a r a b i n o f u r a n o s y l u n i t s , s h o u l d a l s o be p r e f e r e n t i a l l y removed by " h y d r o l y s i s because of the i n h e r e n t s t a b i l i t y t o a c i d - c a t a l y z e d h y d r o l y s i s of g l y c u r o n o s y l g l y c o s i d i c bonds and the i n h e r e n t l a b i l i t y of f u r a n o s y l g l y c o s i d i c bonds (24). However, i f the s i d e c h a i n s are a t t a c h e d t o rhamnogalacturonan sequences (8), i t s h o u l d not be p o s s i b l e t o c o n v e r t " h a i r y " r e g i o n s t o "smooth" r e g i o n s by treatment w i t h a c i d because the l a b i l i t y of the L-rhamnopyranosyl bonds would r e s u l t i n c o n c u r r e n t d e p o l y m e r i z a t i o n of the main chain. At pH v a l u e s o f 5-6 p e c t i n s o l u t i o n s are s t a b l e o n l y a t room t e m p e r a t u r e . As the temperature i s r a i s e d , p e c t i n c h a i n s c l e a v e by a b e t a - e l i m i n a t i o n r e a c t i o n (25~39) ( F i g u r e 1), a r e a c t i o n which i s s t i m u l a t e d by o r g a n i c anions (40). D e e s t e r i f i c a t i o n of p e c t i n proceeds s i m u l t a n e o u s l y w i t h the b e t a - e l i m i n a t i o n d e p o l y m e r i z a t i o n r e a c t i o n , which o c c u r s o n l y a t monosaccharide u n i t s t h a t are e s t e r i f i e d . A t pH v a l u e s above 6, d e e s t e r i f i c a t i o n and d e p o l y m e r i z a t i o n a r e r a p i d r e a c t i o n s even at room t e m p e r a t u r e , the r a t e o f each r e a c t i o n i n c r e a s i n g w i t h i n c r e a s i n g pH. H y d r o x y l - g r o u p r e a c t i o n s , such as e t h e r i f i c a t i o n , a c e t a l a t i o n (4j_), e s t e r i f i c a t i o n (42-51 ), and o x i d a t i o n , can be done i n the same manner as they are on o t h e r p o l y s a c c h a r i d e s . Esterifications (47) o f c a r b o x y l groups and i n t e r a c t i o n s w i t h c a t i o n s , i n c l u d i n g p o l y c a t i o n s such as p r o t e i n s below t h e i r i s o e l e c t r i c pH, o c c u r a s they do w i t h o t h e r g l y c u r o n o g l y c a n s . R e d u c t i o n o f c a r b o x y l groups t o hydroxyraethyl groups has been done w i t h diborane (52, see a l s o 53) and by b o r o h y d r i d e treatment o f methyl and h y d r o x y e t h y l e s t e r s (54). R e d u c t i o n of c a r b o x y l groups which have been a c t i v a t e d w i t h a w a t e r - s o l u b l e c a r b o d i i m i d e s h o u l d be s t r a i g h t f o r w a r d (55,56). When ammonia (57-61) i s used t o p r e p a r e L M - p e c t i n from HM-pectin, some o f the methyl c a r b o x y l a t e groups a r e c o n v e r t e d i n t o carboxaraide groups, p r o d u c i n g "amidated p e c t i n " . The


9


10

CHEMISTRY A N D FUNCTION O F

PECTINS

presence o f amide groups i n an L M - p e c t i n makes t h e m o l e c u l e s l e s s h y d r o p h i l i c , i n c r e a s i n g t h e i r tendency t o form g e l s , and l e s s s e n s i t i v e t o c a l c i u m i o n s . I n g e n e r a l , t h e g e l l i n g temperature i n c r e a s e s w i t h i n c r e a s i n g DA. P e c t i n amides can a l s o be made by r e a c t i o n o f p e c t i n s w i t h p r i m a r y and secondary a l k y l a m i n e s (62-64). There a r e s e v e r a l types o f enzymes t h a t a c t on p e c t i n molecules. Those enzymes produced by t h e h i g h e r p l a n t s t h e m s e l v e s play a s i g n i f i c a n t r o l e i n t h e processes r e s u l t i n g i n t e x t u r a l changes i n f r u i t s and v e g e t a b l e s d u r i n g r i p e n i n g , s t o r a g e , and p r o c e s s i n g ( 6 5 - 6 7 ) . F u n g a l enzyme p r e p a r a t i o n s a r e used by t h e f r u i t j u i c e i n d u s t r y t o improve t h e c l a r i t y o f j u i c e s and t h e y i e l d from p r e s s i n g ( 6 6 - 6 7 ) . C o n t r o l o f t h e i r a c t i o n i s important to the production o f p e c t i n . P e c t i n e s t e r a s e s c a t a l y z e h y d r o l y s i s o f t h e m e t h y l e s t e r group (68). Because many p e c t i n e s t e r a s e s a c t p r e f e r e n t i a l l y on a m e t h y l α-D-galactopyranosyluronate u n i t a d j a c e n t t o an n o n e s t e r i f i e d α-TJ-galactopyranosyluronic a c i d u n i t , t h e y produce p e c t i n s t h a t c o n t a i n b l o c k s , r a t h e r than a random d i s t r i b u t i o n , o f c a r b o x y l groups ( 6 8 ) and t h a t a r e g e n e r a l l y u n d e s i r a b l e f o r commercial u s e . Some f u n g a l p e c t i n e s t e r a s e s , however, produce L M - p e c t i n s t h a t a r e s i m i l a r t o p e c t i n s d e e s t e r i f i e d w i t h a c i d s o r bases w i t h r e s p e c t t o g e l l i n g a b i l i t y (69) and s e n s i t i v i t y t o c a l c i u m i o n s ( 7 0 ) . S e v e r a l t y p e s o f l y a s e s o r t r a n s e l i m i n a s e s a r e known ( 6 8 ) . A l l c a t a l y z e d e p o l y m e r i z a t i o n by a b e t a - e l i m i n a t i o n r e a c t i o n l i k e that which occurs during base-catalyzed depolymerization. Pectin l y a s e s , a l l o f which a r e endo-enzyraes, c a t a l y z e b e t a - e l i m i n a t i o n s at e s t e r i f i e d D - g a l a c t u r o n i c a c i d u n i t s (See F i g u r e 1) ( 6 8 ) . Pectate l y a s e s c a t a l y z e beta-eliminations at n o n e s t e r i f i e d D - g a l a c t u r o n i c a c i d u n i t s . B o t h exo- and endo-pectate l y a s e s a r e lôiown (68). P o l y g a l a c t u r o n a s e s d e p o l y m e r i z e p e c t i n s by c a t a l y z i n g h y d r o l y s i s o f g l y c o s i d i c bonds ( 6 8 , 7 J . ) . The f a c t t h a t t h e r a t e o f d e p o l y m e r i z a t i o n i s i n v e r s e l y p r o p o r t i o n a l t o t h e DE s u g g e s t s a requirement f o r a n o n e s t e r i f i e d D-galacturonic a c i d u n i t . Exo-polygalacturonases r e l e a s e mono- o r d i s a c c h a r i d e s from n o n r e d u c i n g t e r m i n i ; e n d o - p o l y g a l a c t u r o n a s e s a t t a c k randomly ( 6 8 ) . -

Literature Cited 1. Kertesz, Ζ. I.; Baker, G. I.; Joseph, G. H.; Mottern, H. H.; Olsen, A. G. Chem. Eng. News 1944, 22, 105. 2. Pedersen, J. "Pectin", in "Industrial Gums"; Academic Press: Orlando, 3rd ed., 1986. 3. Stephen, A. M. "Other Plant Polysaccharides", in "The Polysaccharides"; Academic Press: New York, Vol. 2, 1983. 4. Fishman, M. L.; Pepper, L.; Damert, W. C.; Barford, R. A. This Volume. 5. Rombouts, F. M. This Volume. 6. Rees, D. Α.; Wight, A. W. J. Chem. Soc. Β 1971, 1366. 7. de Vries, J. Α.; Rombouts, F. M.; Voragen, A. G. J.; Pilnik, W. Carbohydr. Polym. 1982, 2, 25. 8. de Vries, J. Α.; Voragen, A. G. J.; Rombouts, F. M.; Pilnik, W. This Volume.


I. BeMILLER 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. 41.


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Chemistry and Function of Pectins - American Chemical Society

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