Sugar Beet Pectins: Chemical Structure and Gelation through

5 Sugar Beet Pectins: Chemical Structure and Gelation through Oxidative Coupling 1

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F. M . Rombouts and J. F. Thibault 1

Department of Food Science, Agricultural University, De Dreijen 12, 6703 BC Wageningen, The Netherlands Laboratoire de Biochimie et Technologie des Glucides, Institut National de la Recherche Agronomique, rue de la Géraudière, 44072 Nantes, France

Downloaded by UNIV OF ARIZONA on January 6, 2013 | http://pubs.acs.org Publication Date: June 5, 1986 | doi: 10.1021/bk-1986-0310.ch005

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Pectins were isolated from sugar beet pulp by sequential extraction with water, oxalate, acid, and alkali. They were found to be fairly low molecular mass products with a high degree of acetylation and a relatively high neutral sugar content. Neutral sugars were determined and their location in "hairy fragments" of the pectin molécules was studied by enzymic and chemical degradation experiments. Acetyl ester groups are bound to the anhydrogalacturonide backbone mainly. Feruloyl ester groups appear to be linked to the neutral sugar side chains. These can be used in the formation of cross-links with hydrogen peroxide and peroxidase or with ammonium persulfate. The products obtained are either pectin solutions with increased intrinsic viscosity or pectin gels. Cross-linked pectins isolated from these gels have an extremely high water-absorbing capacity.

Beet pulp i s the residue l e f t from ground sugar beet a f t e r sugar extraction. I n i t s dried form i t i s available a l l year round at r e l a t i v e l y low p r i c e s . Some 25% of i t s dry weight consists of anhydrogalacturonic acid and i t i s therefore a p o t e n t i a l l y r i c h source of pectin (1). However, several attempts i n the past to commercialise sugar-beet pectins have f a i l e d , due to the poor g e l l i n g properties of the pectins, compared to those from c i t r u s and apple. These poor g e l l i n g properties are a t t r i b u t a b l e to the presence of 0097-6156/ 86/0310-0049506.00/0 © 1986 American Chemical Society

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

CHEMISTRY AND FUNCTION OF PECTINS

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acetylester groups mainly, and further to the r e l a t i v e l y small s i z e of the beet p e c t i n molecules (2). Our recent studies on sugar-beet pectins have revealed some new features, which may increase t h e i r usefulness, and possibly lead to some new a p p l i c a t i o n s .


Experimental methods Materials. Pressed sugar-beet pulp, preserved i n four volumes of ethanol was obtained from a sugar factory at E p p e v i l l e , France. Endopolygalacturonase was p u r i f i e d from a preparation from A s p e r g i l lus niger, as described by Thibault and Mercier (3). Endopectate lyase was obtained from Pseudomonas fluorescens (4). Endopectin lyase (type 2) was i s o l a t e d from a preparation of A. niger (5). Pectinesterase was p u r i f i e d from an A. niger preparation according to Baron et a l (6). Exo a(1^5) arabinanase and endo β(1-4) galactanase were also p u r i f i e d from A. niger (unpublished r e s u l t s ) . Horse radish peroxidase (90 U/mg s o l i d ) was obtained from Sigma Chem. Comp., St. Louis, Mo. USA. E x t r a c t i o n and p u r i f i c a t i o n . An alcohol-insoluble residue was pre pared from sugar-beet pulp. Products successively extracted from t h i s material were: water-soluble p e c t i n (WSP), oxalate-soluble p e c t i n (OXP), acid-soluble p e c t i n (HP) and a l k a l i - s o l u b l e p e c t i n (OHP). Extractions were done according to the procedure described by Barbier and Thibault (7). The pectins were p u r i f i e d by chromato graphy at pH 4.8 on Whatman DEAE-cellulose DE 52 under the c o n d i t i ons described by Barbier and Thibault (7). As the a l k a l i - s o l u b l e p e c t i n binds i r r e v e r s i b l y to t h i s column, t h i s f r a c t i o n was p u r i f i e d by p r e c i p i t a t i o n with CuSO^ and extensive washing of the pre c i p i t a t e . Cupric ions were subsequently removed by d i a l y s i s against Na^EDTA at pH 4.8. A n a l y t i c a l methods. The anhydrogalacturonic acid content and neu t r a l sugar content (expressed as anhydro-arabinose) of pectins were determined automatically by the m-hydroxydiphenyl method (8) and the o r c i n o l method (9) respectively. In the l a t t e r method correc tions were made f o r interference from anhydrogalacturonic acid. Neutral sugar residues i n pectins were determined by g a s - l i q u i d chromatography of the a l d i t o l a c e t a t e s (10) prepared from the sugars a f t e r hydrolysis with 2 M t r i f l u o r o - a c e t i c acid during 1.5 h at 120°C. Methylester groups were determined according to Wood and Siddiqui (11). 0-acetyl groups were l i b e r a t e d from pectins by hy d r o l y s i s with 0.1 M sodium hydroxide f o r 1 h at room temperature. A f t e r n e u t r a l i s a t i o n , a c e t i c acid was determined by g a s - l i q u i d chromatography with formic acid-saturated nitrogen as c a r r i e r gas (12). Feruloylester groups were estimated spectrophotometrically at 375 nm, using f r e s h l y prepared p e c t i n solutions i n 0.1 M glycinesodium hydroxide b u f f e r , pH 10. The f e r u l o y l e s t e r content was c a l culated using a molar e x t i n c t i o n c o e f f i c i e n t of 31600 (13). Poly phenols were estimated with the F o l i n - C i o c a l t e u reagent without copper treatment (14), using f e r u l i c acid as standard. I n t r i n s i c v i s c o s i t i e s and viscosity-average molecular weights of pectins were determined with the method of Owens et a l (15).


ROMBOUTS AND THIBAULT

5.

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Sugar Beet Pectins


Chromatography. P u r i f i e d pectins and t h e i r degradation products were studied by gel-permeation chromatography on Sepharose CL-2B or Sepharose CL-6B (Pharmacia), under conditions as described by Barb i e r and Thibault (7). High-performance s i z e exclusion chromato graphy was done with a series of Biogel TSK columns types 6000, 5000, 4000 and 3000 PW (Bio-Rad Labs., Richmond, Ca., USA). The solvent was 0.1 M sodium s u l f a t e i n sodium acetate, pH 3.7, i o n i c strength 0.34 (16). Degradation studies. Degradation l i m i t s by endopolygalacturonase and by β-elimination were determined as described by Thibault (17) and by endopectate lyase as described by Rombouts et a l (4). For p e c t i n lyase the reaction conditions were 0.25 (w/v) p e c t i n , 0.01 M sodium phosphate buffer, pH 5.2 and 0.52 U/ml of pectin lyase, 30°C, 24 h. The p e c t i n lyase reaction was monitored by measuring 235 ^ l i < l s , d i l u t e d t h i r t y f o l d with 0.1 Ν hydrochloric acia and the degradation l i m i t was calculated, using a molar e x t i n c t i o n c o e f f i c i e n t of 5500 (18). A l k a l i n e d e e s t e r i f i c a t i o n of pectins was done by d i a l y s i s of pectin solutions (4 mg/ml) against 0.05 Ν sodium hydroxide at 2°C for 6 h. Conditions for enzymatic demethylation were 0.4% (w/v) p e c t i n , 0.1 M sodium acetate buffer, pH 4.5 and 5.7 U/ml of pectinesterase, 30°C, 24 h. I n a c t i v a t i o n of enzymes was done by heating of the reaction mixtures for 5 min i n a b o i l i n g water bath. The reaction conditions f o r exoarabanase and endogalactanase were 0.75% (w/v) p e c t i n , 0.05 M sodium phosphate buffer pH 6 and 7 U/ml of exoara banase and 0.66 U/ml of endogalactanase, 30°C, 24 h. The reactions were monitored by measuring increase i n reducing groups with the method of Nelson-Somogyi (19). A

o f

a

u o t

Crosslinking and gelation. To sugar-beet p e c t i n solutions of va rying concentrations between 0.25 and 3% (w/v) i n 0.1 M sodium phosphate buffer pH 6.0 were added (per ml of p e c t i n s o l u t i o n ) : 10 m i c r o l i t r e s of peroxidase s o l u t i o n of a concentration of 1 mg of enzyme per ml and, a f t e r mixing, 0.1 ml of 0.1 M hydrogen peroxide s o l u t i o n . At higher pectin concentrations (£1.0% w/v) gelation oc curred immediately upon mixing while at lower p e c t i n concentrations v i s c o s i t y increased. Pectin gels or solutions with increased v i s c o s i t y were also obtained by treatment with 0.01 M ammonium persulf a t e , at 25°C for up to 15 h (measured by reduced s p e c i f i c v i s c o sity). Results and discussion The y i e l d s of crude pectins, as w e l l as t h e i r anhydrogalacturonic acid content are given i n Table I. This Table shows that about one t h i r d of the alcohol-insoluble residue can be s o l u b i l i s e d as crude pectins, but the anhydrogalacturonide content of these pectins i s very low. Most of the pectins are extracted with acid and subse quently with a l k a l i . Even a f t e r these extractions not a l l of the pectin i s s o l u b i l i s e d from the alcohol-insoluble residue: i t s an hydrogalacturonic acid content i s s t i l l some 5%. P u r i f i c a t i o n and composition. The p u r i f i c a t i o n of these pectins, carried out as described i n "Experimental Methods r e s u l t s i n an 11


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Table I. Pectins extracted from sugar-beet pulp Pectin f r a c t i o n

Yield (%


Water-soluble Oxalate-soluble Acid-soluble Alkali-soluble Pectin extracted

(WSP) (OXP) (HP) (OHP)

w/w)

2.2 0.5 20 11 33.7

Anhydrogal. acid content (% w/w) 31 50 36 41

appreciable increase i n anhydrogalacturonic acid content (Table I I ) . Neutral sugar residues present i n a l l four pectins are arabinose and galactose mainly and further rhamnose, fucose, xylose, mannose and glucose. Total neutral sugar content varies from 5.7 to 24.3%, i n d i f f e r e n t pectins which i s r e l a t i v e l y high when compared to pectins from apple pomace and c i t r u s wastes (20). Degrees of methylation are comparable to those of c e r t a i n types of commercial pectins (from apples and c i t r u s ) which, unlike these pectins, cont a i n few or no acetylester groups. The weight percentage values i n Table I I do not add up to 100% as the p u r i f i e d pectins s t i l l cont a i n some p r o t e i n , unbound polyphenols and sodium counterions. V i s cosity-average molecular masses are low, as compared to those of pectins from apple, c i t r u s f r u i t s or cherries, which are i n the order of 70000 to 90000 (20,7). Gel-permeation chromatograms (Figure 1) of p u r i f i e d pectins on Sepharose CL-2B (WSP, HP, OHP) and CL-6B (OXP) show a f a i r l y c o n t i nuous v a r i a t i o n of the d i s t r i b u t i o n of neutral sugars over the pect i n molecules of varying molecular mass, except f o r OHP, which appar e n t l y consists of two d i f f e r e n t populations of molecules: high mol e c u l a r mass p e c t i n r i c h i n neutral sugars and low molecular mass pectin with a low neutral sugar content. As w i l l be clear from degradation studies, t h i s p a r t i t i o n points towards breakdown of the p e c t i n molecules during a l k a l i n e extraction. Table I I . Composition and properties of p u r i f i e d pectins from sugar-beet pulp. Anhydrogalacturonic acid Neutral sugars Rhamnose + fructose Arabinose Xylose Mannose Galactose Glucose Feruloylester groups Methylester groups Acetylester groups Viscosity-average M.W.

WSP

OXP

54..4 16..5 0..89 8..44 0..14 0..18 6..46 0..39 0..10 7..24 5..71 47700

77.9 5.7 0.86 1.85 0.16 0.14 2.43 0.21 0.04 8.19 4.04 15400

Numbers are given as % (w/w), except

HP 65..1 18..9 2..25 9..97 0..17 0..12 5..93 0..44 0..48 7..09 7..53 42800

OHP 54..9 24..3 3..17 12..49 0..23 8..09 0..31 0..57 0..72 0.,54 36400

M.W.


5.

ROMBOUTS AND THIBAULT

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Sugar Beet Pectins

Table I I I . Degradation l i m i t s of sugar-beet pectins before and after alkaline deesterification. Pectins

Pretreatment and mode of degradation WSP


No-pretreatment β-elimination p e c t i n lyase polygalacturonase pectate lyase Alkaline deesterification polygalacturonase pectate lyase

7.2 8.6 1.2 1.0 37.5 33.4

OXP a)

4.9 4.3 3.7 4.8 36.7 34.5

HP

OHP

4.6 4. 1 1. 7 1. 3

26.9 23.7

30. 8 31. 0

29.3 26.4

a) Percentage of galacturonide bonds broken. Degradation studies. In order to study the d i s t r i b u t i o n of s u b s t i tuents, notably neutral sugar side chains along the rhamnogalacturonan backbone of the p e c t i n s , degradation studies were done by β-elimination (heating at 80°C and pH 6.8 for up to 6 h) and with various enzymes before and a f t e r a l k a l i n e demethylation and deacet y l a t i o n . Table I I I summarizes the degradation l i m i t s obtained with the various methods. β-Elimination and pectin lyase degradation are known to require methylesterified anhydrogalacturonide residues. The degrees of methylesterif i c a t i o n of WSP, OXP, HP and OHP were 76%, 60%, 62% and 7,5%, respectively. I t i s obvious that both break-down mechanisms were strongly promoted with pectins with higher degrees of m e t h y l e s t e r i f i c a t i o n . The inverse was true f o r degradation with endopolygalacturonase and pectate lyase. I t has been shown by Rexovâ-Benkova et a l . (21) that acetylester groups at C-2 and C-3 of anhydrogalacturonic acid residues decrease the extent of degradation by lowering the a f f i n i ty of endopolygalacturonase f o r i t s substrate through blocking of binding s i t e s . In t h i s case a further l i m i t a t i o n of the degree of degradation through a c e t y l a t i o n i s also observed. The degrees of a c e t y l e s t e r i f i c a t i o n (expressed as moles per 100 moles of anhydrogalacturonide residues) are 31%, 16%, 35% and 4%, f o r WSP, OXP, HP and OHP, respectively. Although the degrees of m e t h y l e s t e r i f i c a t i o n of OXP and HP are quite s i m i l a r , the degradation l i m i t s of these two pectins towards endopolygalacturonase and pectate lyase are quite d i f f e r e n t , and probably r e f l e c t the difference i n acetylest e r i f i c a t i o n . A l k a l i - s o l u b l e p e c t i n (OHP) i s l a r g e l y d e - e s t e r i f i e d during extraction which explains the high degrees of degradation with polygalacturonase and pectate lyase. Methylester groups and acetylester groups are both completely removed by a l k a l i n e d e e s t e r i f i c a t i o n . That i s why the degradation l i m i t s for polygalacturonase and pectate lyase are raised to the values shown i n Table I I I . Higher degradation l i m i t s f o r polygalacturonates are reported i n l i t e r a t u r e , both for endopolygalacturonase and endopectate lyase. Rexovâ-Benkova et a l . (21) found 38%, for pectate from c i t r u s p e c t i n degraded by an Aspergillus niger endopolygalacturonase. Rombouts et a l . (4) found pectate from apple




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p e c t i n to be degradated to a l i m i t of 35% by the endopectate lyase used i n t h i s study. I t i s l i k e l y that the differences i n degradat i o n l i m i t s of the alkali-demethoxylated and -deacetylated pectates r e f l e c t the number of neutral sugar side chains attached to the pectins. Although WSP has a much higher neutral sugar content than OXP, the a l k a l i - d e e s t e r i f i e d products are degraded to p r a c t i c a l l y the same extent. This can be explained i f indeed the r a t i o of side chains per unit of chain length i n both pectins were simular; those i n WSP would then on average be four times as long as those i n OXP (compare Tables I I and I I I ) . Gel-permeation chromatograms of the digests obtained i n various degradation experiments reveal a general trend towards separation of the products into several peaks, i n those cases where the degrees of degradation are s u f f i c i e n t l y high. The products of sodium-hydroxide d e e s t e r i f i e d , pectate-lyase degraded pectins are shown i n F i g . 2. With WSP, HP and OHP, the peaks near the void volume (K =0-0.5) represent r e l a t i v e l y large fragments, very r i c h i n neutraî sugar residues and r e l a t i v e l y poor i n anhydrogalacturonide residues. In contrast, the peaks i n a l l p e c t i n digests which elute towards the included volume (K =l),are small products ( o l i gogalacturonide fragments) with a very low neutral-sugar content. The oligogalacturonide fragments account f o r 80%, 87%, 84% and 66% of t o t a l anhydrogalacturonide present i n WSP, OXP, HP and OHP d i gests, respectively. Intermediary sized fragments, also r e l a t i v e l y r i c h i n neutral sugar residues are present i n a l l four pectin d i gests (0.7

Sugar Beet Pectins: Chemical Structure and Gelation through

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