Enzymic Lysis of Pectic Substances in Cell Walls - American Chemical

18

E n z y m i c Lysis o f Pectic Substances i n C e l l Walls: S o m e Implications for F r u i t Juice Technology 1

A. G. J. Voragen, H. A. Schols, H. A. I. Siliha , and W. Pilnik

Downloaded by MICHIGAN STATE UNIV on November 26, 2013 | http://pubs.acs.org Publication Date: June 5, 1986 | doi: 10.1021/bk-1986-0310.ch018

Department of Food Science, Agricultural University, De Dreyen 12, 6703 BC Wageningen, The Netherlands

Detailed knowledge of the role of various polysaccharide degrading enzymes in cell wall degradation and of the chemical nature of the solubilized fragments enable improvement of enzyme applications in fruit and vegetable processing and development of new applications with balanced enzyme formulations. This is demonstrated for apple juice production and cloud stabilization in apricot nectar. The enzyme systems effective in the solubilization of apple and apricot cell wall pectin are identified and their role in technological applications are discussed. Possible implications of solubilized fragments on product quality (color, haze formation) are indicated. Fruit juices are obtained from fruit pulps by mechanical separation (pressing, sieving, centrifuging) of cell liquid from cell wall fragments. For clear juices they are further clarified, for cloudy or pulpy juices only coarse and unedible particles are removed (1,2). The tissues of the edible parts of fruits and vegetables consist of parenchymatic cells which have a middle lamella consisting mainly of pectins, a primary wall which is a firm gel of pectin, cellulose, hemi-cellulose and some protein and sometimes a secondary wall in which cellulose and hemi-cellulose prevail (3,4). Technologically the pectic substances play the greatest role. They account for 0.5 to 4% of the weight of fresh material. When tissue is crushed this high molecular substance partly becomes water soluble and gives a high viscosity to the liquid phase and can form a protective colloid for cell fragments. Partly they remain in the pulp particles bound to cellulose fibrils by side chains of hemi1

Current address: Food Science Department, Zagazig University, Zagazig, Egypt. 0097-6156/86/0310-0230$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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VORAGEN ET AL.

Enzymic Lysis of Pectic Substances

231

c e l l u l o s e where they help to bind water. Pectins are complex heteropolysaccharides with a backbone of a-1,4 bound galacturonic acids. Some of the uronic acid groups are e s t e r i f i e d with methanol. In another c o n t r i b u t i o n to t h i s symposium s t r u c t u r a l features of pectins are discussed i n more d e t a i l (5)• Pectin esterases (E.C. 3.1.1.11), endo-polygalacturonases (E.C. 3.2.1.15) and endo-pectin lyases (E.C. 4.2.2.10) are by f a r the most relevant pectic enzymes i n f r u i t processing. P e c t i n esterases de-esterify pectins producing methanol and pectic acid. Endopolygalacturonase and endo-pectin lyase are both depolymerases which s p l i t the g l y c o s i d i c linkages i n t h e i r preferred substrates e i t h e r by hydrolysis (polygalacturonase) or by trans-elimination (pectin lyase). Endo-polygalacturonases hydrolyse low e s t e r i f i e d pectins i n a more or less random fashion. Endo-pectin lyases are the only depolymerases s p e c i f i c f o r highly e s t e r i f i e d pectins which they degrade more or less at random. The combined action of pectin-esterase and polygalacturonase can also depolymerize high methoxyl pectins. Pectic enzymes are reviewed i n more d e t a i l by Rombouts and P i l n i k (6). P i l n i k et a l (7) have noted that the combined a c t i o n of p e c t i nases and C-l (1,4-p-D-glucan cellobiohydrolase, E.C. 3.2.1.91) enriched c e l l u l a s e s are able to almost completely l i q u e f y pulped f r u i t s and vegetables. For the actual d i s s o l u t i o n of the c r i s t a l l i n e c e l l u l o s e f i b r i l s the C-l enzyme i s necessary which s p l i t s o f f cellobiose from the non-reducing end of the 1,4-p-D-glucan and which needs some C-x (1,4-p-D-glucan-glucanohydrolase, E.C. 3.2.1.4) to create such points of attack. Preparations r i c h i n C-l a c t i v i t y are u s u a l l y obtained from Trichoderma spp. (8). The extent and mode of bioconversions of pectic substances i n s i t u , t h e i r s o l u b i l i z a t i o n and depolymerization e f f e c t consistency, cloud behaviour, pressing c h a r a c t e r i s t i c s , j u i c e release, soluble s o l i d s , c l a r i f i c a t i o n , haze formation and browning p o t e n t i a l . We have been studying plant c e l l w a l l degradation with pure and w e l l characterized enzymes and from the results of these studies we are now able to better define the various stages i n c e l l w a l l degradat i o n and to understand implications for f r u i t processing. This w i l l be demonstrated i n apple j u i c e production and s t a b i l i z a t i o n of a p r i cot nectar. Apple j u i c e In processing of apples exogenous mould enzymes, mainly pectinases are frequently used. They are added to extracted j u i c e , to f a c i l i tate f i l t r a t i o n and prevent g e l l i n g i n concentrated j u i c e or they are added to the pulp to improve press y i e l d , or added together with c e l l u l a s e preparations to l i q u e f y the pulp. (2). Information about the c o n t r i b u t i o n of the various polysaccharide degrading enzymes i n the degradation of apple c e l l walls has been obtained by detailed studies of degradation of apple c e l l w a l l preparations with pure enzymes (9,10,11) and by c l o s e l y monitoring the chemical changes taking place i n apple pulp and apple j u i c e during processing (12). Our increased knowledge of the polysaccharide composit i o n and structure of f r u i t c e l l walls (13-17) and t h e i r changes during ripening and storage (18-20) contributes also to the understanding of the processes. Table I shows the sugar composition of


232

CHEMISTRY AND FUNCTION OF PECTINS


the alcohol insoluble s o l i d s (AIS) prepared from apple c o r t i c a l tissue and canned apricots and the composition of t h e i r pectin and hemi-cellulose f r a c t i o n s . These data i l l u s t r a t e the differences i n c e l l w a l l composition of these f r u i t s . Apricot c e l l walls were found to contain more anhydrogalacturonic acid (AGA) but less glucose than apple c e l l walls. The pectin f r a c t i o n extracted from a p r i cot c e l l walls was almost twice as large as the apple p e c t i n f r a c t i o n . For the c e l l u l o s e content (data not shown) the reverse was the case. The hemi-cellulose fractions showed large differences i n sugar composition, p a r t i c u l a r l y i n arabinose, mannose and galactose content. Table I Sugar composition of AIS prepared from apples and canned apricots and of the p e c t i n and hemicellulose f r a c t i o n prepared from AIS (sugar composition expressed as mole % ) . Preparations

Composite sugars % AIS

AIS: apple apricot Pectin: apple apricot Hemicellulose: apple apricot

Rha/fuc

Arab

X y l Man

Gal Glc AGA

2 2

13 16

6 5

2 3

7 6

42 32

28 35

27. 2 52

1 2

23 20

2 1

-

4 4

1 1

68 73

16..9 14..7

4 5

34 8

19 21

12 1

17 9

30 21

9 11

Ref: Voragen et a l , (15) S i l i h a , (11). Some results of the studies on the enzymatic degradation of apple c e l l w a l l preparations are summarized i n Table I I and I I I . They show the amounts of sugars released from apple AIS by pure pect i c , c e l l u l o l y t i c and h e m i - c e l l u l o l y t i c enzymes. From the p e c t i c enzymes the combination of pectinesterase and endo-polygalacturonase (PE + PG) were found to release the largest amounts of neutral sugars and galacturonides followed by PL. PG alone released substant i a l l y less sugars. From these results i t can be derived that degradation of highly e s t e r i f i e d pectin (PE + PG, PL) coincides with an increased release of pectin associated sugars l i k e arabinose, galactose and rhamnose. C - l , an 1-4-p-D-glucan c e l l o b i o l y d r o l a s e enriched c e l l u l a s e preparat i o n released i n p a r t i c u l a r glucose and xylose (as cellobiose and xyloglucan fragments) and only minor amounts of galacturonides. A synergistic action was observed f o r the combination C-l and PE + PG, o v e r a l l 80% of a l l composite sugars of the c e l l walls were released r e s u l t i n g i n the almost complete l i q u e f a c t i o n of pulp p a r t i c l e s . Endo-1,5-a-L-arabanase and endo-1,4-p-D-galactanase were found to release mainly galactose and arabinose containing fragments. Galactanase however was also able to release 33% of the pectic material. The combination of arabanase and galactanase showed a cummulative


18.

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Table I I Amounts of neutral sugars and galacturonides released from apple AIS by pure enzymes. Amounts expressed i n % of o r i g i n a l amounts present, corrected f o r control. Enzyme treatment

Neutral sugars released


Rha/ fuc

AGA re- Tot.sug. leased released

Arab

Gal Man X y l Glc Total

PE ( c i t r u s ) PG PL PE+PG

— 4 19 25

9 39 52

5 27 34

---

4 20 13

CI Cl+PE+PG

9 58

3 89

12 69

26 84

20 71

--

--

4

--

22 79

3 17 18

-21 57 75

0.1 8 28 33

16 78

5 82

13 80

Ref: (Voragen et a l , (9). Table I I I Amounts of neutral sugars and galacturonides released from apple AIS by pure enzymes. Amounts expressed i n % of o r i g i n a l amounts present, corrected f o r control. Enzyme treatment

PE+PG Endo-Arab. Endo-Gal Arab+Gal PE+PG+ Arab+Gal

Neutral sugars released

AGA re- Tot.sug. leased released

Rha/ fuc

Arab

Gal Man X y l Glc

Total

25 3 11 11

52 34 18 38

34 10 33 31

---

13 6 6

— —

18 10 11 15

36

33 8 18 22

59

56

89

--

24

11

36

73

48

—

75

-33

Ref: Voragen et a l . , (9,10) e f f e c t . The combination of PE + PG with both arabanase and galactanase showed an increased release of a l l neutral sugars, but not to the extent as observed f o r the combination with C - l . Chemical c h a r a c t e r i s t i c s of pulp residues and j u i c e s obtained by pressing of enzyme treated and non-enzyme treated pulp and by c e n t r i fugation of l i q u e f i e d pulp i n laboratory scale experiments are summar i z e d i n F i g . 1. Pulp residues were analysed f o r AIS content, pect i n content and c e l l u l o s e content, from the juices the anhydrogalacturonic acid content was estimated before and a f t e r d i a l y s i s . The differences between these values represent oligomeric pectin fragments consisting of less than ca. 6 sugar u n i t s . The data show that pulp enzyming r e s u l t s i n about 50% reduction of p e c t i n i n the press cake. A further reduction i n pectin content and i n addition a considerable reduction i n AIS and c e l l u l o s e content takes place during l i q u e f a c t i o n . The polymeric, oligomeric and monomeric sugars i n the In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

234



pressing

pulp enzyming

liquefaction

AIS = Alcohol Insoluble Solids C = Cellulose P = Pectin

AUA =Anhydro galacturonic acid % dialysable M = Mono galacturonic acid Di=Di # after enzymic clarification

\

cn E

[juice changes]

^

4000-

2000-

-

Im

Figure 1. Changes i n j u i c e and pulp as a r e s u l t of enzyme t r e a t ment of apple pulp. Ref: Kolkman (12)



18.

VORAGEN ET AL.

235


juices were further fractionated by g e l f i l t r a t i o n chromatography on Biogel P-2. F i g . 2 shows the e l u t i o n p r o f i l e for press j u i c e obtained from enzyme treated pulp. Mono - and o l i g a l a c t u r o n i c acid were i s o l a t e d from the large excess of glucose, fructose and saccharose by ion-exchange chromatography as outlined i n F i g . 2. I t can be seen that the s o l u b i l i z e d p e c t i c material occurs mainly as monomer i c and oligomeric galacturonides. From the absorbance at 232 nm measured for the oligogalacturonides i t was derived that they are p a r t i a l l y present i n the unsaturated form. In a l l j u i c e s about 5% of the dimers and trimers were present i n the unsaturated form, f o r tetramer t h i s was 20-25%. They are t y p i c a l reaction products of PL action. Preliminary studies have shown that oligogalacturonides and p a r t i c u l a r unsaturated oligogalacturonides are very reactive precursors for browning reactions (unpublished r e s u l t s ) . Table IV shows the o v e r a l l galacturonide content estimated for d i f f e r e n t l y processed j u i c e s , the release of soluble p e c t i c polymers and oligomers was found to increase strongly by enzym action. A standard t r e a t ment of j u i c e with bentonite and gelatine reduced the galacturonide content. Table IV Uronide content of apple j u i c e s (12°C to Kintner and van Buren (29)

B r i x ) estimated

according

Method of preparation

Uronide content mg/ml

Pressing Water extraction Pulp enzyming and pressing Enzymic l i q u e f a c t i o n

0.58 1.31 2.51 4.32

(1.6)*

* A f t e r c l a r i f i c a t i o n with bentonite and gelatine Ref: Kolkman, (12). The polysaccharides i n the j u i c e were i s o l a t e d by u l t r a f i l t r a t i o n i n a tubelar system (PCI) equiped with a BX3 polysulfonmembrane (Mw cutoff 60,000 Dalton). The retentate and permeate f r a c t i o n were then dialysed, centrifuged and freeze-dried. F i g . 3 shows a flow sheet of t h i s process for l i q u e f a c t i o n j u i c e . From t h i s j u i c e ca. 0.3% of fresh apple weight was obtained as retentate f r a c t i o n and ca. 0.1% as permeate f r a c t i o n . Table V shows the sugar and glycosidic-linkage composition of the retentate f r a c t i o n . The presence of an a-1,5-arabinan with single unit or short arabinose containing side chains at C-2 and C-3 i s evident (16,21). These arabinans are probably linked to C-4 of rhamnose which i n turn i s 1,2 l i n k e d i n the rhamnogalacturonan backbone. This backbone further c a r r i e s single unit and larger xylose containing side chains. Also the presence of 1,4 and 1,3/1,6 linked-galactan i s indicated. The galacturonan was methylated for 42% and s u r p r i s i n g l y a degree of acetylat i o n of 60% was estimated (based on anhydrogalacturonic acid content) . The retentate f r a c t i o n was r e s i s t e n t to further degradation by p e c t i c enzymes. With an arabinofuranosidase 80% of the arabinose could be removed, endo-p-l,4-D galactanase was able to release some oligomeric galactose. The retentate f r a c t i o n obviously represents hairy regions from pectin molecules (21). In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.


236

neutral sugars (ug /ml)


A.U.A. (ug/ml) neutral sugar AU.A.

•1200

bio-gel P2 (100x2.6cm) sample: 1 ml apple juice

100-

(1'. 1 concentrate) eluent: 0.1 M sodium acetate pH 3.6 800

50400

lit

/

180

sacch-glc.fruct. di-mono G.U.A.

N

— — I

1

300 elution volume (ml)

DOWEX 1 X 8 . (25x2cm,200/400 mesh) eluents 250 ml water 150 ml 0.8M.NaAc pH 6

dituted (1:10)

Figure 2. Analysis of oligomeric galacturonides by g e l f i l t r a t i o n chromatography and ion-exchange chromatography. Sample: apple j u i c e from enzyme treated pulp. Ref: Kolkman (12).


18.

VORAGEN E T A L .

237


Table V Sugar and g l y c o s i d i c linkage composition of u l t r a f i l t r a t i o n retentate.


Sugar composition (as mole %)

Glycosidic linkage composition (as mole %)

Rhamnose

5.6

Arabinose

55.1

1,2,4-Rhap

100

T-Araf 1,2-Araf 1,3-Araf 1,5-Araf 1,3,5-Araf 1,2,5-Araf 1,2,3,5-Araf

25 1 8 47 11 3 5

Xylose

8.4

T-Xylp 1,4-Xylp 1,2-Xylp

65 32 3

Galactose

9.9

T-Galp 1,3-Galp 1,4-Galp 1,6-Galp 1,2,4-Galp 1,3,6-Galp

45 19 14 4 4 14

1,4-GalpA * 1,3,4-GalpA*

80 20

Galacturonic acid

20.9

* Determined a f t e r carboxyl reduction. G e l f i l t r a t i o n chromatography of the retentate f r a c t i o n of l i q u e f a c t i o n j u i c e gave 2 overlapping peaks with s i m i l a r uronide content and neutral sugar composition when buffer was used as eluent ( F i g . 4b). However with water as eluent the negatively charged p e c t i c polymers appeared i n the void volume because they are excluded from the p a r t i c l e matrix due to t h e i r charge (22). A galacturonide free, almost pure arabinan representing 7% of the retentate eluted i n the included volume ( F i g . 4a). Table VI shows the glycosyl linkage comp o s i t i o n of t h i s arabinan f r a c t i o n and of haze i s o l a t e d from apple j u i c e concentrate. In the retentate arabinan 15.7% of the arabinosyl units are branched, i n haze arabinan t h i s i s only 4%. Haze arabinan has been i d e n t i f i e d as an almost l i n e a i r a-1,5-L-arabinan (10,23). Native arabinans are described as highly branched (13,14,16). Table VII summarizes the degree of branching estimated f o r arabinans i n apple c e l l w a l l fractions and i n fractions i s o l a t e d from apple j u i ces. From these data i t i s obvious that during enzyme treatment arabinofuranosidases commonly present i n commercial pectinase preparations reduce the degree of branching and increase the chance f o r chain association r e s u l t i n g i n haze formation. Several p o s s i b i l i t i e s can be suggested f o r avoiding haze problems. Enzyme manufacturers can t r y to avoid the presence of arabinofuranosidase i n t h e i r pectinase preparations or they can add an arabanase enzyme complex which degrades the branched arabinans completely. We are presently


238


apples

T mill enzyme treatment bentonite treatment

t

sieve


centrifuge | permeate [

-ultraf i Urate -

dialyse

dialyse centrifuge-^) pel let |

| pe!1et|«-centrifuge

L

purified retentate

purified permeate

~

retentate

T

r

freeze-dry

freeze-dry

Figure 3. Scheme f o r the preparation of u l t r a f i l t r a t i o n permeate and retentate fractions from apple l i q u e f a c t i o n j u i c e .

neutral sugar AiiA. sephacryl S 500 ( 60x1.2 cm) sample:+10 mg UF retentate eluent: a) water

elution volume (ml)

Figure 4. G e l f i l t r a t i o n chromatography of the u l t r a f i l t r a t i o n retentate from apple l i q u e f a c t i o n j u i c e and sugar composition of f r a c t i o n s i n mol %.


18.

VORAGEN ET AL.

239


Table VI Glycosidic linkage composition for arabinosyl residues i n arabinan f r a c t i o n of u l t r a f i l t r a t i o n retentate and i n haze. Arabinosyl residue

Occurrence g l y c o s y l linkage type i n % Haze


U.F. Retentate Arabinan T-Araf T-Arap 1,2-Araf 1,3-Araf 1.5-Araf 1,3,5-Araf 1,2,5-Araf 1,2,3,5-Araf

4.2 0.2

11.4 0.2 0.4 6.6 65.7 9.4 3.4 2.9

2.3 88.4 3 1

Table VII Degree of branching (branched arabinosyl units i n % of t o t a l arabinosyl u n i t s ) of arabinan fractions i s o l a t e d from apple c e l l walls and apple j u i c e . Apple c e l l w a l l

Degree of branching

Pectin fractions Hemi-cellulose f r a c t i o n

36.5-49* 30

Apple j u i c e U.F. retentate (liquefaction) Arabinan f r a c t i o n retentate Haze

19 15.7 4

* (de Vries et a l , 21). studying a powerful arabinan degrading enzyme preparation produced by a fungus and k i n d l y supplied by G i s t Brocades ( D e l f t , The Netherlands). Following l a r g e l y i s o l a t i o n procedures as described by K a j i (30) we are able to i s o l a t e 3 enzymes active on branched (ex sugar beets) and l i n e a i r (haze and u l t r a f i l t r a t i o n retentate) arabinan ( f i g . 5). Two of them are arabinofuranosidases because of t h e i r act i v i t y on p-nitrophenylarabinofuranoside; type I i s able to degrade oligomers and i s only s l i g h t l y active on branched arabinan. Type I I r e a d i l y debranches sugar beet arabinan and i n addition i t can also s p l i t arabinose from the a-1,5-arabinan backbone s t a r t i n g from the non-reducing end. So type I I can degrade branched arabinans complet e l y on i t s own. The t h i r d enzyme i s an endo-a-1,5-arabanase which i s only active on l i n e a i r arabinan, i t can degrade haze and u l t r a f i l t r a t i o n retentate. Only i n the presence of arabinofuranosidase a c t i v i t y i t can completely degrade branched arabinan. Emperical studies have shown that Pectinase preparations with a high a c t i v i t y on high e s t e r i f i e d p e c t i n were most e f f e c t i v e i n i n creasing j u i c e y i e l d s (24,25). We understand now that such enzyme




c*-L-arabi not uranose

a - arabinofuranosKJQse II

p

a (X-L-arabtnofuranosidase I

endo-arabanase cK-L-arabinofuranosidose II o

Enzymic Lysis of Pectic Substances in Cell Walls - American Chemical

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