Enzymes in Food and Beverage Processing

changes in the outer pericarp of tomato fruit during development and ripening. ..... Rutgers tomatoes remained firm and did not develop polygalac- tur...
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10 Enzymes Involved in Fruit Softening RUSSELL PRESSEY

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R. B. Russell Agricultural Research Center, Agricultural Research Service, U.S. Department of Agriculture, Athens, Ga. 30604

Softening o f the f l e s h i s one of the most dramatic changes accompanying the r i p e n i n g o f many fruits. Although other parame t e r s o f q u a l i t y are important, the peak o f fruit ripeness i s u s u a l l y a s s o c i a t e d with a fairly narrow range of firmness. Furthermore, texture that i s considered optimal f o r fruit consumed f r e s h may not be best f o r fruit that i s processed. S o f t e n ing undoubtedly r e f l e c t s changes i n c e l l walls of fruit t i s s u e s as the fruit progresses through r i p e n i n g i n t o senescence. Once the process i s initiated i n mature fruit, the period o f acceptable texture may be s h o r t , even with r e f r i g e r a t i o n and c o n t r o l l e d atmosphere s t o r a g e . A p r e r e q u i s i t e to improved methods o f c o n t r o l l i n g s o f t e n i n g , whether to initiate and a c c e l e r a t e it or to prevent s o f t e n i n g beyond the optimum stage, i s an understanding o f c e l l wall s t r u c t u r e , i t s changes, and the enzymes involved in its degradation. I.

H i s t o l o g y of Fleshy

Fruits.

A p p r e c i a t i o n o f the biochemical aspects o f fruit softening r e q u i r e s some knowledge o f the development and h i s t o l o g y o f fruits. F r u i t growth may be considered to begin i n the f l o r a l primordium (1_). The ovary wall becomes the p e r i c a r p o f the fruit. During development, the ground t i s s u e o f the p e r i c a r p remains r e l a t i v e l y homogeneous and p a r e n c h y m a l c . Initial development occurs mainly through c e l l m u l t i p l i c a t i o n . The p o s t f e r t i l i z a t i o n period i s marked by c e l l enlargement, although cell division a l s o continues i n ovaries o f l a r g e - f r u i t e d plants (2j. The p e r i c a r p may become d i f f e r e n t i a t e d i n t o three d i s t i n c t parts: the exocarp, the mesocarp, and the endocarp. F r u i t s are h i g h l y d i v e r s i f i e d i n t h e i r morphology, and f r u i t development i s not r e s t r i c t e d to the ovary but often involves n o n c a r p e l l a r y parts of the flower ( 2 ) . I f the e n t i r e ground t i s s u e develops i n t o f l e s h y t i s s u e , the f r u i t i s a b e r r y . A l l the f l e s h y t i s s u e of a berry may o r i g i n a t e from the ovary w a l l , 172

In Enzymes in Food and Beverage Processing; Ory, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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as in the grape. In c o n t r a s t , a considerable part of the tomato f r u i t c o n s i s t s of p l a c e n t a . The body of the tomato i s p e r i c a r p developed from the ovary wall and c o n s i s t s of o u t e r , r a d i a l , and inner w a l l s . During development, the placenta shows much more a c t i v e c e l l d i v i s i o n than the ovary w a l l . The r e s u l t i s l o c u l a r c a v i t i e s i n the p e r i c a r p that contain the seeds imbedded i n a j e l l y - l i k e tissue ( 2 J . Mohr and S t e i n (3) have described the f i n e s t r u c t u r a l changes in the outer p e r i c a r p of tomato f r u i t during development and r i p e n i n g . S h o r t l y a f t e r f e r t i l i z a t i o n , the vacuoles of each c e l l of the p e r i c a r p enlarge to form one l a r g e vacuole, so that a l a y e r of protoplasm l i n e s the inner surface of the c e l l w a l l s . I n t e r c e l l u l a r spaces develop subsequently, and c e l l walls par­ t i a l l y separate along the middle lamella r e g i o n , s t a r t i n g at the i n t e r c e l l u l a r spaces. As the f r u i t nears m a t u r i t y , the c e l l s become very l a r g e ( 1 0 0 - 5 0 0 μ diameter) and separation of c e l l w a l l s i n c r e a s e s . Degeneration of protoplasm begins, with the disappearance of membraneous s t r u c t u r e s . In the r i p e , red f r u i t , separation of adjacent c e l l s i s common, and degeneration of protoplasmic components i s pronounced. The development of many f r u i t s involves maturation of the ovary wall i n t o a p e r i c a r p with a conspicuous stony endocarp surrounded by a f l e s h y mesocarp. Important stone f r u i t s include the o l i v e , peach, plum, a p r i c o t , and c h e r r y . The f l e s h y t i s s u e of the peach c o n s i s t s of l o o s e l y packed parenchyma c e l l s that increase in s i z e from the periphery toward the i n t e r i o r . The mesocarp grows by r a p i d c e l l enlargement. I n t e r c e l l u l a r spaces are common in peach t i s s u e . C e l l wall thickness increases during peach development from 0 . 5 μ , a f t e r c e s s a t i o n of c e l l d i v i s i o n , to about l y , during the p i t hardening phase ( 4 ) . Increases in c e l l wall thickness are more marked in the l a t e phases of c e l l growth, and reach a maximum of about 2y in hard r i p e f r u i t . During subsequent r i p e n i n g , the c e l l w a l l s decrease in thickness. There are two d i s t i n c t types of peaches, the soc a l l e d "freestone" and " c l i n g s t o n e " v a r i e t i e s . E a r l y reports (5) described d i f f e r e n c e s in c e l l wall thickness between the two t y p e s , but Reeve ( 4 ) could not confirm t h i s and observed that the c e l l w a l l s decrease in thickenss to the same degree in c l i n g s t o n e as in freestone f r u i t . C e l l walls do not appear to rupture during r i p e n i n g ( 4 ) . The apple i s an example of another v a r i a t i o n in f r u i t development. The f l e s h of the apple i s derived from the f l o r a l tube as well as from the c a r p e l l a r y t i s s u e . The ovary region (the core) c o n s i s t s of f i v e c a r p e l s imbedded in f l e s h y paren­ c h y m a l c exocarp. The f l o r a l tube region of the a p p l e , which forms the bulk of the e d i b l e p a r t , a l s o c o n s i s t s of parenchyma cells. During growth, c e l l d i v i s i o n ceases at about 3 weeks a f t e r f u l l bloom (6j. Subsequent increase in f r u i t s i z e i s due mainly to c e l l enlargement and increase in the volume of i n t e r ­ c e l l u l a r spaces, which are abundant i n apple t i s s u e ( 7 J . L

In Enzymes in Food and Beverage Processing; Ory, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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According to Nelmes and Preston (7J, r a p i d wall synthesis during c e l l extension i s followed by r e a s s i m i l a t i o n o f the p e c t i c mater i a l s j u s t before m a t u r i t y . The c e l l walls decrease i n thickness at t h i s stage. Despite the d i v e r s i t i e s i n f r u i t s i n s i z e , shape, s t r u c t u r e , and development, patterns o f r i p e n i n g tend to be q u i t e s i m i l a r . Chlorophyll i n the c h l o r o p l a s t s o f the outermost c e l l s decreases and e v e n t u a l l y disappears. Carotenoids and anthocyanins develop and give f r u i t s t h e i r c h a r a c t e r i s t i c c o l o r s . Other changes are the development o f s p e c i f i c f l a v o r s , increases i n sweetness, and decreases i n a c i d content. The l o s s o f firmness associated with r i p e n i n g can be a t t r i b u t e d to changes i n the s t r u c t u r e o f c e l l w a l l s which w i l l be discussed next. II.

C e l l Wall S t r u c t u r e .

The f l e s h o f succulent f r u i t s c o n s i s t s l a r g e l y or e n t i r e l y o f parenchyma c e l l s (2J. These c e l l s are r e l a t i v e l y u n d i f f e r e n t i a t e d , but are h i g h l y complex p h y s i o l o g i c a l l y because they possess l i v i n g p r o t o p l a s t s . They are g e n e r a l l y polyhedral or elongated with t h i n primary w a l l s . The walls o f two contiguous c e l l s are separated by the i n t e r c e l l u l a r substance or middle l a m e l l a which i s r i c h i n p e c t i c substances (8). The d i s t i n c t i o n often i s not obvious between the middle lamella and the c e l l wall which appear as a u n i t . Mature parenchyma t i s s u e has abundant i n t e r c e l l u l a r spaces ( 9 ) . C e l l wall composition o f plants i s u s u a l l y determined on the e t h a n o l - i n s o l u b l e f r a c t i o n , which c o n s i s t s p r i m a r i l y of the c e l l wall and middle l a m e l l a . Jermyn and Isherwood (1_0) have found t h a t t h i s f r a c t i o n o f r i p e pears contains 21.4% glucosan, 3.5% g a l a c t a n , 1.1% mannan, 21% x y l a n , 10% araban, 11.5% p o l y g a l a c t u r o n i c a c i d , and 16.1% l i g n i n . Tomato c e l l w a l l s contain 17% c e l l u l o s e , 22% p e c t i n , 17% p r o t e i n , 21% araban-galactan, and 1323% xylose plus glucose (1J_). Knee (1_2) has reported the comp o s i t i o n o f a c e t o n e - i n s o l u b l e residues o f apples i n mg/g t i s s u e are: a r a b i n o s e , 2.58; x y l o s e , 0.52; g a l a c t o s e , 3.81; glucose 32.8; and g a l a c t u r o n i c a c i d , 3.53. The composition o f dates (percent, dry weight basis o f the whole f r u i t ) i s : 0.8% c e l l u l o s e , 1.5% h e m i c e l l u l o s e a , 0.8% hemicellulose b, 3.7% p e c t i n , and 0.3% l i g n i n ( 1 3 ) . C e l l u l o s e i s the s k e l e t a l substance o f the plant c e l l wall and, as the above data show, i t i s a major wall component i n most fruits. It i s a l i n e a r 3-1,4-glucosan. There have been occas i o n a l suggestions that other monosaccharides may be present (14), but c e l l u l o s e i s by f a r the most homogeneous polysaccharide i n the c e l l w a l l . The lengths o f i n d i v i d u a l c e l l u l o s e chains have been estimated a t 6000-8000 monomers (1_5). These chains are combined i n t o bundles, c a l l e d m i c r o f i b r i l s , that are up to 250°A wide and contain 2000 molecules i n a t r a n s e c t i o n (1_6). The

In Enzymes in Food and Beverage Processing; Ory, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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arrangement of molecules apparently i s o r d e r l y because X-ray studies have demonstrated regions of c r y s t a l l i n i t y in the microfibrils. The f r i b r i l l a r system i s i n t e r p e n e t r a t e d by c a p i l l a r i e s of various s i z e s , which are occupied by water, p a r a c r y s t a l l i n e c e l l u l o s e , and n o n c e ! l u l o s i c components of the c e l l w a l l . In the newly formed primary w a l l , the o r i e n t a t i o n of m i c r o f i b r i l s i s predominately t r a n s v e r s e , but becomes more d i s p e r s e during c e l l enlargement. The bulk of the primary wall i s n o n c e l l u l o s i c polysacchar i d e s , forming a dense, amorphous gel in which the c e l l u l o s e m i c r o f i b r i l s are imbedded. Meristematic and parenchymous t i s s u e s are p a r t i c u l a r l y r i c h in polysaccharides c a l l e d the p e c t i c substances ( p e c t i n ) . Pectin i s the main component of the middle l a m e l l a , as was i n d i c a t e d e a r l i e r , but i t i s a l s o present in the primary c e l l w a l l . The s t r u c t u r e of pectin may vary with the source. For example, a pure homogalacturonan has been i s o l a t e d from sunflower heads (17) and J a c k f r u i t (18). But the basic s t r u c t u r e of p e c t i n from most sources c o n s i s t s of long blocks of l i n e a r «-l ,4-galacturonan i n t e r s p e r s e d by rhamnose through CI and C2 (19., 20, 21_, 22). Talmadge et a l . (23) suggested that the p e c t i n from suspension c u l t u r e d sycamore c e l l s c o n s i s t s of blocks of 8 u n i t s of g a l a c t u r o n i c a c i d i n t e r r u p t e d by u n i t s of rhamnose-galacturonic acid-rhamnose. Neutral sugars other than rhamnose may be present i n f r u i t pectins. For example, p u r i f i e d pectin from apples contained, in a d d i t i o n to 1.2% rhamnose, 9.3% arabinose, 1.4% g a l a c t o s e , 0.80% x y l o s e , and t r a c e s of f u c o s e , 2-0-methylxylose and 2-0-methylfucose (21). Galacturonosylgalactose and g a l a c t u r o n o s y l x y l o s e have been t e n t a t i v e l y i d e n t i f i e d in the hydrolyzates of apple p e c t i n (21_), but the linkages remain unknown. It i s not c l e a r whether the neutral sugars occur in the galacturonan chain or as branches on the c h a i n . Rees and Wight (24) estimated that about 60% of the p e c t i n s from mustard cotyledons c a r r y side c h a i n s . Branching of g a l a c t u r o n i c a c i d i s thought to occur mainly through C3. Pectins from f r u i t t i s s u e s are considered to be much l e s s branched, however (20., 21_, 22). The only g a l a c t u r o n o s y l - g a l a c t u r o n i c a c i d linkage i d e n t i f i e d in p e c t i n i s «-1,4. On t h i s b a s i s , i t i s u n l i k e l y that branches of galacturonan, i f they e x i s t , are attached d i r e c t l y to the galacturonan backbone. It i s p o s s i b l e , however, that such branches are attached through neutral sugars on the c h a i n . The carboxyl group in g a l a c t u r o n i c a c i d introduces another v a r i a b l e i n the s t r u c t u r e of p e c t i n . These groups are p a r t l y e s t e r i f i e d with methanol, but the d i s t r i b u t i o n of e s t e r groups i s not known. It has been suggested that the f r e e carboxyl groups may be involved i n i n t e r m o l e c u l a r linkages (25). Calcium i n t e r a c t s with f r e e carboxyl groups to form i n s o l u b l e s a l t s . By forming i n t e r m o l e c u l a r b r i d g e s , calcium could c o n t r i b u t e to the s t r u c t u r e and binding p r o p e r t i e s of p e c t i n in the c e l l w a l l .

In Enzymes in Food and Beverage Processing; Ory, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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Doesburg (26) has proposed that movement of calcium i n c e l l walls may cause s o l u b i l i z a t i o n of p e c t i n during f r u i t r i p e n i n g . Because arabinan and galactan often accompany the g a l a c ­ turonan i n preparation of f r u i t p e c t i n s , many e a r l y workers considered the p e c t i n substances i n terms of the three p o l y s a c ­ charides. A p o s s i b l e reason f o r t h i s apparent a s s o c i a t i o n may be simply that t h e i r s o l u b i l i t i e s are s i m i l a r , although recent studies on p e c t i n from suspension-cultured sycamore c e l l s sug­ gested covalent i n t e r a c t i o n s (23). H i r s t and Jones (27) sep­ arated the arabinans from apple and c i t r u s pectins and found that they c o n s i s t mainly of arabinose i n a h i g h l y branched structure. Later workers (28) questioned the existence of a homoarabinan. B a r r e t t and Northcote (21) i s o l a t e d , from apple p e c t i n , an arabinan-galactan complex containing nearly equal q u a n t i t i e s o f the monomers, but could not separate the two components. In c o n t r a s t , Talmadge et a l . (23) showed that a branched arabinan and a l i n e a r galactan are released from syca­ more c e l l p e c t i n by endopolygalacturonase treatment. The galactan i n f r u i t pectins has not been c h a r a c t e r i z e d , but by analogy with Lupinus a!bus galactan (29), i s assumed to be a l i n e a r 3-1,4-polymer. In a d d i t i o n to the e s s e n t i a l l y homogeneous arabinans and g a l a c t a n s , h i g h l y branched polysaccharides c o n t a i n ­ ing both arabinose and galactose are common i n plants (21_). The remaining n o n c e l l u l o s i c polysaccharides i n the c e l l wall can be s o l u b i l i z e d with i n c r e a s i n g concentrations of KOH. The hemicelluloses are c h a r a c t e r i z e d by d i v e r s i t y i n composition, l i n k a g e s , and branching. Mannose, g a l a c t o s e , arabinose, x y l o s e , g l u c o s e , and other monomers may be present. These a l k a l i - s o l u b l e polysaccharides are complex mixtures that vary with the source and method o f e x t r a c t i o n . The stumbling block i n c h a r a c t e r i z i n g them has been d i f f i c u l t y in separating the components. There have been a few reports of homogeneous polysaccharides obtained by d i f f e r e n t i a l e x t r a c t i o n and p r e c i p i t a t i o n . A 3-1,4-xylan with g l u c u r o n i c a c i d s i d e chains has been i s o l a t e d from pear c e l l walls (30). A complex glucan has been extracted from mango f r u i t ( 3 1 ) . C a l l o s e , a 3 - 1 , 3 - g l u c a n , may be present i n f r u i t tissues"T32). Bauer et a l . (33) obtained from sycamore c e l l s , a neutral f r a c t i o n c o n s i s t i n g almost e x c l u s i v e l y of x y l o g l u c a n . The s t r u c t u r e of t h i s polymer i s a repeating u n i t containing 4 residues of β-1,4-1 inked glucose and 3 residues of x y l o s e , with s i n g l e xylose branches on 3 of the glucosyl r e s i d u e s . Plant c e l l w a l l s contain a small amount of p r o t e i n that has r e l a t i v e l y high proportions of seryl and hydroxyprolyl residues (34, 3 5 ) . I t has been proposed that t h i s p r o t e i n serves a s t r u c t u r a l r o l e on the basis that c e l l wall polysaccharides form g l y c o s i d i c linkages with the hydroxy-acid residues (36). Knee (37) confirmed that c e l l walls from apples contain a low l e v e l of g l y c o p r o t e i n r i c h i n hydroxyproline. This g l y c o p r o t e i n i s r e l a t i v e l y s o l u b l e and can be separated from the galacturonan by ion exchange chromatography. Knee concluded that the g l y c o p r o t e i n

In Enzymes in Food and Beverage Processing; Ory, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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and galacturonan are not l i n k e d c o v a l e n t l y , but he suggested that the two components may be p h y s i c a l l y a s s o c i a t e d in the c e l l wall. The current view on c e l l wall s t r u c t u r e i s that the p o l y saccharides and p r o t e i n form a polymeric network, i n v o l v i n g covalent linkages and hydrogen bonding (38). The model proposed f o r the s t r u c t u r e of suspension-cultured sycamore c e l l walls i s based on the premise that xyloglucan i s hydrogen-bonded to cellulose. The reducing ends of the xyloglucan side chains are connected to the rhamnogalacturonan through the l i n e a r g a l a c t a n . The rhamnogalacturonan, in t u r n , i s connected to the hydroxyp r o l i n e - r i c h p r o t e i n through a branched a r a b i n o g a l a c t a n . A second chain of rhamnogalacturonan chain i s a t t a c h e d , through a r a b i n o g a l a c t a n , to the same protein molecule and the order of components i s reversed to another c e l l u l o s e c h a i n . The s t r u c t u r a l component of the c e l l wall can thus be considered as a macromolecule. III.

Enzymes Involved in C e l l Wall Changes of Ripening

Fruits.

1. Cellulase. Because of i t s abundance in f r u i t t i s s u e and prominent r o l e in c e l l wall s t r u c t u r e , c e l l u l o s e should be an important f a c t o r in f r u i t t e x t u r e . Working with many v a r i e t i e s o f a p p l e s , Kertesz et a l . (39) found that the l e v e l of c e l l u l o s e c o r r e l a t e s with i n i t i a l firmness of f r e s h l y harvested f r u i t , but they concluded that subsequent softening of apples i s not due to changes in c e l l u l o s e . B a r t l e y (40) confirmed that the c e l l u l o s i c glucose content of apple c e l l w a l l s d i d not change in the r i p e n i n g f r u i t . In peaches, Nightingale et a l . (41) observed a decrease in c e l l u l o s e content during r i p e n i n g , but they used a method that i s now considered u n r e l i a b l e . Jermyn and Isherwood (10) found a s i m i l a r small decrease in c e l l u l o s e in r i p e n i n g pears. In a highly organized component l i k e c e l l u l o s e , changes in molecular o r i e n t a t i o n i n the microf i b r i l s could be more important than changes in c e l l u l o s e l e v e l s . Using t h i s approach, S t e r l i n g (42) examined the physical s t a t e of c e l l u l o s e in r i p e n i n g peaches with X-ray techniques. He found that the c r y s t a l l i n e m i c e l l e s enlarge in diameter during peach r i p e n i n g . S t e r l i n g a t t r i b u t e d the mi c e l l a r enlargement to c e l l u l o s e degradation r a t h e r than to formation of new m i c r o f i brils. He conceded, however, that the l i m i t e d degradation of c e l l u l o s e cannot c o n t r i b u t e g r e a t l y to peach s o f t e n i n g . The i n s o l u b i l i t y of c e l l u l o s e has hindered d e t e c t i o n of c e l l u l o l y t i c enzymes in f r u i t s . Many workers have circumvented the problem by using carboxymethylcellulose (CMC), a s o l u b l e d e r i v a t i v e of c e l l u l o s e , as the s u b s t r a t e . The high v i s c o s i t y of aqueous s o l u t i o n s of CMC allows the use of the very s e n s i t i v e v i s c o m e t r i c method f o r measurement of c e l l u l a s e a c t i v i t y which i s extremely low i n many plant t i s s u e s . The f i r s t report of c e l l u l a s e in higher plants was by Tracy (43) who found a c t i v i t y

In Enzymes in Food and Beverage Processing; Ory, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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i n tobacco and other p l a n t s . Dickinson and McCollum (44J and Hall (45) detected s i m i l a r a c t i v i t y in e x t r a c t s of r i p e tomatoes. The long incubation periods (20 hr) required to measurably reduce the v i s c o s i t y of CMC i n d i c a t e the low a c t i v i t y in tomatoes. The c e l l u l a s e in the 1 o c u l a r material of tomatoes i s s o l u ble in water, whereas a high concentration of NaCl i s required to s o l u b i l i z e the enzyme from p e r i c a r p t i s s u e (45). Hall (46) found that c e l l u l a s e i s present in the outer p e r i c a r p of young, green tomatoes. The a c t i v i t y decreases during tomato maturation, and then increases at the onset of r i p e n i n g (46.). In c o n t r a s t , the c e l l u l a s e in the inner p e r i c a r p and the placental t i s s u e i s low i n young f r u i t and remains low u n t i l the turning stage, when the a c t i v i t y increases s h a r p l y , e s p e c i a l l y in the placental portion. Hall concluded that c e l l u l a s e may be involved not only i n tomato softening during r i p e n i n g but a l s o in c e l l enlargement during f r u i t development. Sobotka and Watada (47) confirmed that c e l l u l a s e i s present in mature green tomatoes and that i t increases sharply with ripening. They measured f r u i t firmness and c e l l u l a s e v i s c o m e t r i c a l l y in two commercial v a r i e t i e s and several breeding lines. The v a r i e t i e s with f i r m f r u i t e x h i b i t e d lower c e l l u l a s e a c t i v i t y than those with s o f t f r u i t . The r a t e of softening of each v a r i e t y was c l o s e l y a s s o c i a t e d with an increase in c e l l u l a s e , but a sharp decrease in tomato firmness e a r l y in the r i p e n i n g process was not accompanied by an increase in c e l l u l a s e . Hobson (48), who conducted a d e t a i l e d study on the r e l a t i o n ship between tomato softening and c e l l u l a s e , used acetone prec i p i t a t e s of tomato e x t r a c t s and a reductometric rather than a v i s c o m e t r i c assay. In agreement with other workers, he found high c e l l u l a s e in young tomatoes. The a c t i v i t y decreased s t e a d i l y , as f r u i t s i z e i n c r e a s e d , u n t i l the mature green stage and then began to i n c r e a s e . C e l l u l a s e continued to increase up to the red s t a g e , but not i n t o the o v e r r i p e stage. In c o n t r a s t to other workers, Hobson found higher c e l l u l a s e in the f i r m e r of two v a r i e t i e s , and firmness was not c o r r e l a t e d with c e l l u l a s e in f r u i t in the sub-genera E r i o p e r s i c o n and E u l y c o p e r s i c o n . He concluded that the c o n t r i b u t i o n of c e l l u l a s e to softening i s of minor importance in tomatoes. An explanation f o r the d i f f e r e n c e in opinion concerning the r o l e of c e l l u l a s e in tomato softening may l i e in the r e c e n t l y revealed complexity of tomato c e l l u l a s e . Pharr and Dickinson (49) i d e n t i f i e d two enzymes i n 1 o c u l a r contents of r i p e n i n g tomatoes. One enzyme reduced the v i s c o s i t y of CMC s o l u t i o n s and generated reducing groups, but i t d i d not attack i n s o l u b l e cellulose. The second enzyme hydrolyzed c e l l o b i o s e r a p i d l y , and the r a t e of cleavage decreased as the chain length of the substrate increased. Sobotka and S t e l z i g (50) provided evidence f o r four c e l l u l o y t i c enzymes in tomatoes. Two of the enzymes were e n d o c e l l u l a s e s that degraded both CMC and i n s o l u b l e c e l l u -

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lose. The e n d o c e l l u l a s e s d i f f e r e d in pH optimum, degradation of short chain c e l l o d e x t r i n s , and the s i z e of product released from i n s o l u b l e c e l l u l o s e . One of the other enzymes was c h a r a c t e r i z e d as a n o n s p e c i f i c 3-glucosidase. It hydrolyzed c e l l o b i o s e r a p i d l y ; the r a t e of cleavage decreased as the chain length i n c r e a s e d . The f o u r t h enzyme a l s o hydrolyzed c e l l o b i o s e , but the rate of r e a c t i o n decreased very slowly with i n c r e a s i n g chain l e n g t h . This enzyme produced glucose as the main product, and t h e r e f o r e can be c l a s s i f i e d as an e x o c e l l u l a s e . C e l l u l a s e , as measured v i s c o m e t r i c a l l y with CMC, has a l s o been studied in r e l a t i o n to the softening of dates and peaches. A c t i v i t y was absent in dates at the green stage but began to develop between the green and e a r l y red stages (51_). Activity increased sharply at the e a r l y red stage and reached a maximum at the l a t e red stage. The development of c e l l u l a s e c l o s e l y p a r a l l e l e d the l o s s of f i r m n e s s , suggesting a r o l e f o r t h i s enzyme in date s o f t e n i n g . S i m i l a r l y , c e l l u l a s e was not detected in green peaches, but a c t i v i t y developed and increased during r i p e n i n g (52). The increase in c e l l u l a s e was g r e a t e s t before peach firmness decreased s i g n i f i c a n t l y , suggesting that c e l l u l a s e may not only be involved in peach s o f t e n i n g , but a l s o in i n i t i a t i o n of the process. 2. P e c t i c Enzymes. The p e c t i c substances have received more a t t e n t i o n i n r e l a t i o n to f r u i t softening than any other c e l l wall component. The reasons f o r the concentration on p e c t i n are i t s predominance in the middle l a m e l l a , the r e l a t i v e l y high l e v e l of p e c t i n in most f r u i t s , and above a l l , the appearance of w a t e r - s o l u b l e p e c t i n that accompanies the softening of many f r u i t s . Pectin s o l u b i l i z a t i o n occurs in apples (40), pears (10), peaches (53, 54, 55), tomatoes (56), and other fruits. The s o l u b l e p e c t i n content of apples increased more than 3 - f o l d during a change in firmness of 4.8 to 3.4 kg (40). The p e c t i c changes in r i p e n i n g pears are q u i t e s i m i l a r to those in a p p l e s . The proportion of s o l u b l e p e c t i n i s very low in unripe pears, but increases markedly with r i p e n i n g (10). Some e a r l i e r workers (56) found a d i r e c t r e l a t i o n between r i p e n i n g and s o l u b l e p e c t i n i n pears and proposed the use of s o l u b l e p e c t i n as an index of m a t u r i t y . There i s controversy over whether p e c t i n i s s o l u b i l i z e d in tomatoes, however. Woodmansee et a l . (57) were not able to confirm e a r l i e r claims of p e c t i n s o l u b i l i z a t i o n in tomatoes. Changes in p e c t i n s o l u b i l i t y are most pronounced in f r e e stone peaches. Postlmayr et a l . (54) found that 5% of the dry matter of unripe Fay E l b e r t a peaches was p e c t i n , a f o u r t h of which was w a t e r - s o l u b l e . On r i p e n i n g , the p e c t i n content decreased to 4.1%, but the proportion of water-soluble p e c t i n increased to 71% o f the t o t a l . In c o n t r a s t to the changes in the freestone v a r i e t y , r i p e n i n g of H a l f o r d c l i n g s t o n e peaches was accompanied by a decrease in t o t a l p e c t i n from 4.4 to 3.8%,

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but the s o l u b l e p e c t i n content remained r e l a t i v e l y unchanged. Shewfelt (55) studied three pectin f r a c t i o n s ( w a t e r - s o l u b l e , V e r s e n e - s o l u b l e , and V e r s e n e - i n s o l u b l e ) in f i v e freestone and one c l i n g s t o n e v a r i e t i e s . In the c l i n g s t o n e peaches, the prop o r t i o n of the three p e c t i n f r a c t i o n s remained e s s e n t i a l l y constant. But in freestone peaches, the water-soluble p e c t i n increased r a p i d l y at the expense of the other two f r a c t i o n s . Only 20% of the p e c t i n in unripe peaches was w a t e r - s o l u b l e , whereas the l e v e l increased to 80% in r i p e peaches. Shewfelt et a l . (58) subsequently i s o l a t e d and c h a r a c t e r i z e d the three pectin f r a c t i o n s . They demonstrated that peach r i p e n i n g i s accompanied not only by p e c t i n s o l u b i l i z a t i o n but a l s o by reduct i o n i n molecular weights, as determined v i s c o m e t r i c a l l y . In y e t another study on peaches, Pressey et a l . (59) found that the t o t a l p e c t i n content of E l b e r t a and Red Haven peaches d i d not change s i g n i f i c a n t l y during f r u i t softening while the s o l u b l e p e c t i n increased s h a r p l y . They analyzed the s o l u b l e p e c t i n by gel f i l t r a t i o n and found that the molecular weights of the p e c t i n decreased p r o g r e s s i v e l y during r i p e n i n g . The complexity of p e c t i n seems to o f f e r a number of p o s s i b i l i t i e s f o r enzyme a c t i o n leading to s o l u b i l i z a t i o n . At one extreme, r e l e a s e of p e c t i n molecules may represent cleavage of i t s linkages to other c e l l wall components. Cleavage could i n v o l v e terminal residues of the galacturonan chains or hydrol y s i s of the polymers attached to the c h a i n s . Evidence i s accumulating to show that t h i s may be the case in apples because the pectin does not appear to be degraded during s o l u b i l i z a t i o n . On the other hand, p e c t i n may be s o l u b i l i z e d by h y d r o l y s i s of l o n g , i n s o l u b l e molecules. In t h i s c a s e , h y d r o l y s i s could occur in the galacturonan chains or at the linkages with rhamnose. Two enzymes that could be involved in the mechanism are p e c t i n esterase and polygalacturonase. a. Pectinesterase. P e c t i n e s t e r a s e c a t a l y z e s the h y d r o l y s i s of methyl e s t e r s in p e c t i n . It i s h i g h l y s p e c i f i c f o r the methyl e s t e r and the e s t e r as i t occurs in p e c t i n , and does not hydrolyze other e s t e r s and the methyl e s t e r in short chain galacturonans. The enzyme i s a c t i v a t e d by d i v a l e n t cations or monovalent c a t i o n s at high c o n c e n t r a t i o n s . The optimum pH range i s 5 to 8 and i s a f f e c t e d by c a t i o n s . The a c t i o n of p e c t i n esterase on p e c t i n produces blocks of f r e e carboxyl groups (60), i n d i c a t i n g that d e e s t e r i f i c a t i o n occurs in a l i n e a r manner. Solms and Deuel (61 ) suggested t h a t . e s t e r h y d r o l y s i s occurs only adjacent to f r e e carboxyl groups, in d e t a i l e d studies on the mode o f a c t i o n of tomato p e c t i n e s t e r a s e , Lee and Macmillan (62) demonstrated that i t acts a t both the reducing ends and i n t e r i o r l o c i on h i g h l y e s t e r i f i e d p e c t i n c h a i n s . P e c t i n e s t e r a s e occurs commonly in various parts of higher p l a n t s , i n c l u d i n g the f r u i t . Kertesz (63) found that tomatoes are a p a r t i c u l a r l y r i c h source of the enzyme. The a c t i v i t y i s

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high in green tomatoes and, according to Kertesz, increases about 4 - f o l d during r i p e n i n g . This suggests that softening may be r e l a t e d to high p e c t i n e s t e r a s e a c t i v i t y . However, Hamson (64) reported that p e c t i n e s t e r a s e i s higher in f i r m than in s o f t varieties. In a study on s i x breeding l i n e s and one v a r i e t y , Hall and Dennison (65) d i d not f i n d a s i g n i f i c a n t c o r r e l a t i o n between firmness and p e c t i n e s t e r a s e . Furthermore, Hobson (66) observed only a 40% increase in p e c t i n e s t e r a s e during r i p e n i n g of tomatoes. In c o n t r a s t to the f i n d i n g s of Hamson, the f i r m e r of the two v a r i e t i e s contained lower p e c t i n e s t e r a s e . Part of the confusion about p e c t i n e s t e r a s e in tomatoes may be due to the complexity of the enzyme. The a c t i v i t y can be resolved i n t o a number of components (67). The number of p e c t i n esterases and t h e i r r e l a t i v e l e v e l s vary with both tomato v a r i e t y and stage of r i p e n e s s . For example, green Marion tomatoes contained two p e c t i n e s t e r a s e s . On r i p e n i n g , one enzyme decreased while the other nearly t r i p l e d , and a t h i r d form of p e c t i n esterase appeared. One of the enzymes appears to be the major component in most v a r i e t i e s . The same three enzymes were present in Homestead tomatoes in the same p r o p o r t i o n s . The dwarf v a r i e t y P i x i e contained two of the above enzymes and a d i f f e r e n t enzyme. The four tomato p e c t i n e s t e r a s e s d i f f e r e d i n molecular weight, s t a b i l i t y to heat, pH optimum, and a c t i v a t i o n by c a t i o n s . An even greater heterogeneity of tomato p e c t i n e s t e r a s e has been demonstrated by t h i n - l a y e r i s o e l e c t r i c focusing (68). At l e a s t e i g h t enzymes were separated, although one component predominated in a l l samples. The pectinesterases appear to have s i m i l a r molecular weights, and to d i f f e r mainly with respect to charge p r o p e r t i e s . P e c t i n e s t e r a s e has been detected in many other f r u i t s , although the a c t i v i t i e s are u s u a l l y much lower than i n tomatoes. There have been c o n f l i c t i n g reports on the presence of the enzyme in a p p l e s . Apparently i t i s present in low amounts and does not change during apple r i p e n i n g (69). Nagel and Patterson (70) reported that p e c t i n e s t e r a s e in pears decreases during maturation. The l e v e l of p e c t i n e s t e r a s e in peaches v a r i e s with the v a r i e t y , and the changes in a c t i v i t y show no d i s t i n c t trend with advancing ripeness (55). The degree of e s t e r i f i c a t i o n of p e c t i n in avocados g r a d u a l l y decreases during r i p e n i n g (71). Pectinesterase i s high in young avocados (72). A pronounced decrease in a c t i v i t y was observed during the i n t e n s i v e growth stage, and s t i l l f u r t h e r reduction during f r u i t maturation. S i m i l a r l y , p e c t i n e s t e r a s e decreased sharply in stored avocados through the softening p e r i o d . The more mature the f r u i t at h a r v e s t , the more pronounced was the decrease in p e c t i n e s t e r a s e during storage (72). H u l t i n and Levine {73) separated the p e c t i n e s t e r a s e a c t i v i t y in bananas i n t o three f r a c t i o n s by d i f f e r e n t i a l e x t r a c t i o n . The enzymes d i f f e r e d in pH optimum, thermal s t a b i l i t y , and s e n s i t i v i t y to SDS, as well as in s o l u b i l i t y . In c o n t r a s t to the

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decrease in p e c t i n e s t e r a s e in avocados, the a c t i v i t y in bananas increased about 1 0 - f o l d during r i p e n i n g . One of the p e c t i n esterases increased c o n t i n u a l l y during r i p e n i n g , whereas the other two enzymes were maximal a f t e r about 2 days of r i p e n i n g . The g r e a t e s t increase in a l l three enzymes occurred as the bananas changed from green to y e l l o w . Thus, the t o t a l p e c t i n e s t e r a s e a c t i v i t y increases in bananas and decreases in avocados during r i p e n i n g , but in most f r u i t s the enzyme i s present at the immature stage and appears to remain r e l a t i v e l y constant during r i p e n i n g . A r o l e f o r t h i s enzyme in f r u i t s o f t e n i n g , t h e r e f o r e , i s not apparent. Multiple forms of p e c t i n e s t e r a s e are present in bananas and tomatoes, and a s p e c i f i c form of enzyme might be involved in s o f t e n i n g . A l s o , the d i r e c t r o l e of pectinesterase in p e c t i n s o l u b i l i z a t i o n i s not obvious because a decrease in e s t e r i f i c a t i o n alone would not be expected to increase s o l u b i l i t y . To the c o n t r a r y , an increase in f r e e carboxyl groups would lead to greater i n t e r a c t i o n with C a and to decreased s o l u b i l i t y . But p e c t i n e s t e r a s e a c t i o n must precede degradation of p e c t i n by polygalacturonase, and in t h i s way p e c t i n e s t e r a s e could exert r e g u l a t i o n on the process of f r u i t s o f t e n i n g . Judging from the lack of a c o n s i s tent pattern of f l u c t u a t i o n of the enzyme, r e g u l a t i o n of p e c t i n esterase probably involves a mechanism other than changes in enzyme c o n c e n t r a t i o n . The f a c t that pectin i s never completely d e e s t e r i f i e d in tomatoes, f o r example, even though p e c t i n e s t e r a s e i s always p r e s e n t , suggests that other r e g u l a t o r y mechanisms are active. 2 +

b. Polygalacturonase. P e c t o l y t i c enzymes are c l a s s i f i e d on the basis of mode of a c t i o n as e i t h e r hydrolases or l y a s e s . The l a t t e r degrade galacturonans by a t r a n s e l i m i n a t i o n mechanism i n a random or terminal manner with preference f o r low or high ester substrates. The hydrolases a l s o can f u n c t i o n in a random or uniform manner, but a l l known enzymes in t h i s group act only on d e e s t e r i f i e d substrates and t h e r e f o r e are c a l l e d p o l y g a l a c turonases. They are the only p e c t o l y t i c enzymes known to occur in f r u i t tissues. Polygalacturonase was f i r s t detected in r i p e tomatoes which remain the r i c h e s t plant source. The l i s t of f r u i t s c o n t a i n i n g the enzyme i s now f a i r l y l o n g , and the f o l l o w ing d i s c u s s i o n w i l l deal with each commodity s e p a r a t e l y . i. Tomatoes. In c o n t r a s t to p e c t i n e s t e r a s e and c e l l u l a s e , polygalacturonase i s not present in green, immature tomatoes (63, 66). It appears i n f r u i t near the onset of r i p e n i n g and increases as the f r u i t s o f t e n s . It i s t h i s apparent coincidence of polygalacturonase with softening that has suggested a key r o l e f o r t h i s enzyme in the process. Numerous studies have been d i r e c t e d at e s t a b l i s h i n g such a r e l a t i o n s h i p . One approach i s to study the enzyme l e v e l in r e l a t i o n to tomato firmness which d i f f e r s considerably with a v a r i e t y .

In Enzymes in Food and Beverage Processing; Ory, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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Tomato l i n e s with high pigment c h a r a c t e r i s t i c s tend to be f i r m e r than other l i n e s (74). Foda (75) found that high pigment l i n e s contained more protopectin and l e s s polygalacturonase than a commençai v a r i e t y . Sobotka and Watada (76) a l s o observed that two l i n e s c o n t a i n i n g the high pigment c h a r a c t e r i s t i c had cons i d e r a b l y lower polygalacturonase than l i n e s without the chara c t e r i s t i c . The a c t i v i t y was not only lower, but i t increased at a slower rate than in other v a r i e t i e s . Hobson (77_) reported that r i p e f r u i t o f the v a r i e t y Potentate were both f i r m e r and lower in polygalacturonase than those of the v a r i e t y Immuna. A f u r t h e r study by Hobson (78) revealed a highly s i g n i f i c a n t p o s i t i v e c o r r e l a t i o n between polygalacturonase and the compression o f 6 d i f f e r e n t v a r i e t i e s at the commerical p i c k i n g stage. A f r u i t r i p e n i n g i n h i b i t o r ( r i n ) mutant of tomato can be produced by backcrossing (79). Tomatoes containing the r e c e s s i v e gene soften much more slowly than normal v a r i e t i e s . Hobson (80) observed that Potentate tomatoes backcrossed in t h i s manner were a s s o c i a t e d with markedly reduced s o l u b i l i z a t i o n of p e c t i n and c o n s i d e r a b l y lower l e v e l s of polygalacturonase. This was confirmed by Buescher and T i g c h e l a a r (81.) who found that backcrossed Rutgers tomatoes remained f i r m and did not develop p o l y g a l a c turonase. The p h y s i o l o g i c a l d i s o r d e r "blotchy" r i p e n i n g has a l s o been evaluated in terms of polygalacturonase a c t i v i t y . The red areas of blotchy f r u i t contained l e s s than two-thirds the a c t i v i t y in evenly red f r u i t , and the a c t i v i t y was even lower in non-red areas (77). Polygalacturonase a c t i v i t y i s highest in the outer l o c u l e wall of the p e r i c a r p t i s s u e followed by the inner l o c u l e walls and the placental t i s s u e (77). The enzyme i s not present in the l o c u l a r contents. The a c t i v i t y f i r s t appears i n the placenta which u s u a l l y shows i n c i p i e n t yellowness. It then develops in both the inner and outer l o c u l e walls as the change in c o l o r spreads to the p e r i c a r p . Hobson suggested that the p a r a l l e l appearance of polygalacturonase and c o l o r a t i o n i s f u r t h e r e v i dence f o r a r e l a t i o n s h i p between the enzyme and tomato r i p e n i n g . McColloch et a l . (82) a l s o noted that deep red c o l o r i n tomatoes was a s s o c i a t e d with high polygalacturonase. Tomato polygalacturonase 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 (83, 84). It cleaves pectate randomly f i r s t to o l i g o g a l a c t u r o n a t e s and u l t i m a t e l y to g a l a c t u r o n i c a c i d , but the r a t e of h y d r o l y s i s decreases r a p i d l y with decreasing chain length. I f the i n i t i a l r a t e o f pectate h y d r o l y s i s i s 100, the rates of h y d r o l y s i s of t e t r a - , t r i - , and d i - g a l a c t u r o n a t e are approximatley 7, 1.6, and 1, r e s p e c t i v e l y (84). Some of the p r o p e r t i e s of the a c t i v i t y in crude e x t r a c t s suggest that the a c t i v i t y i s produced by more than one polygalacturonase. McColloch and Kertesz (85) observed t h a t most of the a c t i v i t y was destroyed by heating at r e l a t i v e l y low temperatures, but part of i t survived heating to 90°. Patel and Phaff (84) examined the h e a t - s t a b l e component and concluded that i t was

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s i m i l a r to the heat-unstable enzyme. But they observed double pH optima f o r the enzyme a c t i n g on t r i - a n d t e t r a - g a l a c t u r o n a t e s and a t t r i b u t e d t h i s to the presence of two enzymes. The observat i o n that pectate i n h i b i t s the h y d r o l y s i s of small substrates at low pH, but only to the extent of 70%, i s f u r t h e r evidence f o r a complex system o f polygalacturonase in tomatoes (86). McColloch (87) separated two polygalacturonases from tomatoes by moving boundary e l e c t r o p h o r e s i s . One of the peaks was more a c t i v e than the o t h e r , but the enzymes were not c h a r a c t e r ized. Pressey and Avants (88) separated the a c t i v i t y i n tomato e x t r a c t s i n t o two peaks (PG I and PG II) by chromatography on DEAE-Sephadex A-50. The enzymes d i f f e r e d in molecular weight and s t a b i l i t y to temperature and pH, although the more s t a b l e component d i d not s u r v i v e heating to 90°. The pH optima f o r both enzymes were near 4 . 5 . The a c t i v i t i e s of both enzymes s h i f t e d to the a c i d side with decreasing substrate s i z e and i n c r e a s i n g NaCl c o n c e n t r a t i o n , but PG I was l e s s dependent on these f a c t o r s . At enzyme l e v e l s producing equal numbers of reducing groups, PG II was much more e f f e c t i v e than PG I in reducing the v i s c o s i t y of pectate. However, the e f f e c t of PG I on the v i s c o s i t y o f pectate i s too r a p i d f o r a mechanism of end group cleavage. ii. Peaches. The changes in f l e s h firmness and p e c t i n s o l u b i l i t y are p a r t i c u l a r l y pronounced in r i p e n i n g freestone peaches. Shewfelt et a l . (58) demonstrated that p e c t i n i s not only s o l u b i l i z e d , but that the molecular weights of the s o l u b l e f r a c t i o n g r a d u a l l y decrease, suggesting that peach r i p e n i n g i s accompanied by p e c t i n degradation. Pressey et a l . (59) confirmed t h a t reduction in p e c t i n molecular weights occurs during both t r e e and postharvest r i p e n i n g . Although e a r l i e r attempts to detect polygalacturonase in peaches were u n s u c c e s s f u l , the presence of t h i s enzyme was revealed by incubation of waterwashed residues of peach t i s s u e with polygalacturonate (59). Polygalacturonase a c t i v i t y was absent during the f r u i t e n l a r g e ment stage; i t appeared as the peaches began to ripen and then increased s h a r p l y . The appearance of the enzyme p a r a l l e l e d the formation o f s o l u b l e p e c t i n . Both processes were preceded by some f r u i t s o f t e n i n g , but they coincided with the most r a p i d l o s s of firmness. Pressey and Avants (89) subsequently s o l u b i l i z e d and chara c t e r i z e d the peach polygalacturonase. The procedure involved e x t r a c t i o n o f water-washed residues of r i p e freestone peaches with 0.2 M NaCl followed by u l t r a f i l t r a t i o n of the macromolecules. Attempts to p u r i f y the a c t i v i t y by chromatography on Sephadex G-100 revealed two enzymes (PG I and PG I I ) . The enzymes d i f f e r e d in a number o f ways, i n c l u d i n g pH optimum, c a t i o n a c t i v a t i o n , and e f f e c t of substrate s i z e . PG I functioned o p t i m a l l y at pH 5.5 and required Ca2+ f o r a c t i v i t y (89). It cleaved d i g a l a c t u r o n a t e very s l o w l y , but the r a t e of cleavage

In Enzymes in Food and Beverage Processing; Ory, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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increased with substrate chain length to a maximum f o r substrates with a degree of polymerization of about 20. This enzyme exh i b i t e d the highest a f f i n i t y f o r the l a r g e s t substrates (90). PG II was most r e a c t i v e with l a r g e s u b s t r a t e s ; i t s pH optimum was near 4.5 f o r the l a r g e s t substrate ( p e c t a t e ) , but i t s h i f t e d to the a c i d s i d e as the substrate s i z e decreased (89). The two peach polygalacturonases d i f f e r e d markedly in mode o f a c t i o n . PG II was a t y p i c a l endopolygalacturonase; i t r a p i d l y reduced the v i s c o s i t y of pectate r e l a t i v e to a slow r e l e a s e of reducing groups. T h i s enzyme was very e f f e c t i v e in s o l u b i l i z i n g p e c t i n from washed peach c e l l w a l l s . In c o n t r a s t , PG I had a very small e f f e c t on the v i s c o s i t y of pectate. The product of i t s r e a c t i o n was i d e n t i f i e d as g a l a c t u r o n i c a c i d . The evidence presented showed that cleavage occurs at the nonreducing ends of the substrate c h a i n s , and that a l l chains are p r o g r e s s i v e l y shortened. The polygalacturonase in peaches t h e r e f o r e , c o n s i s t s of endo- and exo-cleaving enzymes. T h i s was the f i r s t report of an exopolygalacturonase in f r u i t t i s s u e , but we now know that t h i s enzyme i s widespread in higher p l a n t s . i i i . Dates. Hasegawa et a l . (91) found a t r a c e of p o l y galacturonase, as measured by the r e l e a s e of reducing groups from p e c t a t e , in green dates. The a c t i v i t y remained low during r i p e n i n g o f dates u n t i l the l a t e red stage at which time the a c t i v i t y rose sharply to a maximum at the 50% s o f t stage. The greatest part of the increase in polygalacturonase, t h e r e f o r e , occurred immediately before date s o f t e n i n g . The development of a c t i v i t y a l s o followed softening w i t h i n s i n g l e d a t e s , which commences at the a p i c a l end and progresses toward the stem end. They concluded from the c l o s e r e l a t i o n between polygalacturonase and s o f t e n i n g t h a t t h i s enzyme may be involved in c o n t r o l l i n g the texture of d a t e s . P r o p e r t i e s of the date enzyme have not been determined. iv. Avocado. McCready and McComb (92) f i r s t reported that polygalacturonase i s absent in green avocados, but considerable a c t i v i t y develops during r i p e n i n g . Reymond and Phaff (93) found t h a t the enzyme f i r s t appears at the blossom end and spreads toward the stem end. In a more d e t a i l e d study, Zauberman and Schiffmann-Nadel (72) d i d not detect polygalacturonase in f r u i t at various stages of development immediately a f t e r harvest. In harvested f r u i t , the enzyme developed e a r l i e r in more mature f r u i t s , although the maximum l e v e l s of enzymes appeared to be independent of f r u i t maturity. Avocado polygalacturonase e x h i b i t s optimal a c t i v i t y at pH 5.5 and in the presence of 0.14 M Na (93). Phosphate and Calgon were i n h i b i t o r y , but Ca2+ at 0.1 M was without e f f e c t . The enzyme appears to be an endopolygalacturonase; i t hydrolyzes pectate to intermediate o l i g o g a l a c t u r o n a t e s which are then slowly hydrolyzed to g a l a c t u r o n i c a c i d (93, 9 4 ) . Reymond and +

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Phaff [93_) concluded that avocados contain an i n h i b i t o r o f polygalacturonase because recovery of the enzyme during p u r i f i c a t i o n often exceeded 100%. Furthermore, the a d d i t i o n o f ext r a c t s o f immature avocados to p u r i f i e d polygalacturonase r e s u l t ed i n 48% i n h i b i t i o n , confirming the existence o f an i n h i b i t o r . P r e c i p i t a b i l i t y o f the i n h i b i t o r by 50% ammonium s u l f a t e suggests that i t i s a p r o t e i n . v. Pears. McCready and McComb (92J presented evidence showing that r i p e n i n g o f pears i s accompanied by degradation o f p e c t i c substances. During the r i p e n i n g o f pears, the s o l u b i l i t y of p e c t i n increased and p r e c i p i t a b i l i t y with alcohol decreased. They d i d not detect polygalacturonase i n unripe pears, but they found considerable a c t i v i t y i n r i p e pears. Pressey and Avants (95) have demonstrated that the a c t i v i t y c o n s i s t s o f endo- and exo-polygalacturonases, s i m i l a r to the system o f p o l y g a l a c turonases found i n peaches. The exopolygalacturonase was o p t i m a l l y a c t i v e at pH 5.5 and was a c t i v a t e d by Ca2+ and Sr2+. It was most r e a c t i v e with polygalacturonate possessing a chain length o f about 12 u n i t s . The endopolygalacturonase had a pH optimum a t 4.5; i t was most r e a c t i v e with a substrate possessing a chain length o f about 80. vi. Cucumbers. B e l l (96) detected p e c t o l y t i c a c t i v i t y i n cucumbers by using p e c t i n at pH 4 as the substrate i n the v i s c o metric assay. The choice o f p e c t i n over pectate f o r the subs t r a t e may have been the reason f o r the low l e v e l o f a c t i v i t y found. When Pressey and Avants (97) reexamined the p o l y g a l a c turonase i n cucumbers, they found that the a c t i v i t y c o n s i s t s s o l e l y o f an e x o s p l i t t i n g enzyme. This enzyme i s s i m i l a r to peach exopolygalacturonase i n pH optimum (5.5), molecular weight (59,000), and mode o f a c t i o n . In c o n t r a s t to the exopolygalacturonases, the cucumber enzyme was r e a d i l y extracted by water. It was a c t i v a t e d by C a , but not by S r . Substrates with chain lengths from about 6 to 12 were cleaved most r a p i d l y . 2 +

2 +

v i i . Cranberries. Patterson et a l . (98) found p o l y g a l a c turonase i n c r a n b e r r i e s that were breaking down i n storage and softened by b r u i s i n g , but not i n sound b e r r i e s . It i s p o s s i b l e that the a c t i v i t y detected was of microbial o r i g i n . However, A r a k j i and Yang (99) i s o l a t e d and c h a r a c t e r i z e d a p o l y g a l a c turonase from McFarlin c r a n b e r r i e s . They found that phenol binding agents increased the y i e l d s of a c t i v i t y . The enzyme was endo-cleaving with a pH optimum near 5. c. Other Enzymes. Enzymatic cleavage o f any linkage involved i n maintaining the s t r u c t u r e and r i g i d i t y o f the middle lamella and c e l l wall could a f f e c t f r u i t firmness. Even the phenomenon o f p e c t i n s o l u b i l i z a t i o n may be due to enzymatic cleavage of linkages between pectin and other c e l l wall components

In Enzymes in Food and Beverage Processing; Ory, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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r a t h e r than to degradation of the p e c t i n c h a i n . T h i s may be the case in a p p l e s . Attempts to f i n d polygalacturonase in r i p e apples have been unsuccessful (100). Furthermore, s o l u b l e p e c t i n i s formed during apple r i p e n i n g , but the p e c t i n chains are not shortened (100). The s o f t e n i n g of apples i s accompanied by not only p e c t i n s o l u b i l i z a t i o n but also by the loss of galactose residues from the c e l l wall (101). A small decrease i n arabinose a l s o o c c u r s , but the x y l a n , c e l l u l o s e , and n o n c e l l u l o s i c glucans are not hydrolyzed. The l o s s of galactose i s s u b s t a n t i a l , decreasing to about a t h i r d of the i n i t i a l l e v e l , and precedes the increase in s o l u b l e p e c t i n and the decrease in apple firmness. This has prompted searches f o r galactan-degrading enzymes in apples. B a r t l e y (102) has found that both s o l u b l e and c e l l wall preparat i o n s from apple cortex hydrolyze 3-galactan and p-nitrophenyl d e r i v a t i v e s of «- and 3 - g a l a c t o s i d e . The 3-galactosidase a c t i v i t y was present at a high l e v e l in f i r m apples and increased l e s s than 2 - f o l d during r i p e n i n g . B a r t l e y concluded that 3g a l a c t o s i d a s e may control p e c t i n s o l u b i l i z a t i o n , but the f a c t that the l o s s of galactose precedes the r e l e a s e of p e c t i n led him to suggest that the h y d r o l y s i s of other bonds may be i n volved (102). Wallner and Walker (103) measured the enzymatic a c t i v i t y a g a i n s t a number of g l y c o s i d e s and polysaccharides to determine whether the cleavage of c e r t a i n linkages v a r i e s with the stage of ripeness of tomatoes. E x t r a c t s of tomatoes hydrolyzed glucomannan, galactomannan, l a m i n a r i n , a r a b i n o g a l a c t a n , x y l a n , and p-nitrophenyl d e r i v a t i v e s of «-D and 3 - D - g a l a c t o s i d e , «-Dg l u c o s i d e , «-D-and 3 - D - x y l o s i d e , as well as c e l l u l o s e and p o l y galacturonate. The major enzymes, in terms of reducing group f o r m a t i o n , were l a m i n a r i n a s e , polygalacturonase, and 3 - g a l actosidase. With the exception of glucomannanase, xylanase, and p o l y g a l a c t u r o n a s e , the enzymes were present at a l l stages of ripeness. During r i p e n i n g , the 3-galactosidase increased 4 - f o l d in the p l a c e n t a , but l e s s in other parts of the f r u i t . The other enzymes did not change s i g n i f i c a n t l y . However, on the basis of s u s c e p t i b i l i t y of c e l l walls to degradation by the mixture of tomato enzymes, Wallner and Walker concluded that changes in the c e l l wall occur before the appearance of p o l y galacturonase. An enzyme other than polygalacturonase may t h e r e f o r e be i n v o l v e d , but has not been i d e n t i f i e d . Conclusions F r u i t s o f t e n i n g probably i s due to breakdown of the middle lamellae and c e l l w a l l s . T h i s d é s i n t é g r a t i o n may i n v o l v e movement of calcium in the c e l l walls (26) or a l t e r a t i o n of hydrogen bonding between c e l l u l o s e and xyloglucan (38) in response to changes in pH. But evidence of covalent bond cleavage in c e l l wall components points to r o l e s f o r several of the h y d r o l y t i c

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enzymes found in f r u i t t i s s u e s . There has been a tendency to emphasize the p e c t i c enzymes because of the apparent r e l a t i o n s h i p between p e c t i n s o l u b i l i z a t i o n and f r u i t s o f t e n i n g . The impor­ tance of polygalacturonase i s supported by i t s widespread occur­ rence i n f r u i t s and i t s appearance during r i p e n i n g . The d i s ­ covery of exopolygalacturonase in f r u i t s suggests that cleavage of terminal linkages in c e l l wall macromolecules may be involved in s o f t e n i n g . But the process i s complex and undoubtedly r e ­ q u i r e s numerous enzymes, some of which remain to be i d e n t i f i e d .

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Literature 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.

Cited

N i t s c h , J. P. Ann. Rev. Plant P h y s i o l . (1953), 4, 199. Esau, K. "Plant Anatomy", Wiley and Sons, New York. 1965. Mohr, W. P . , and S t a i n , M. Can. J. Plant S c i . (1969), 49, 549. Reeve, R. M. Amer. J o u r . Bot. (1959), 46, 241. Addoms, R. Μ., N i g h t i n g a l e , G. T., and Blake, M. A. New Jersey A g r i c . Exp. S t a . B u l l . 507, (1930). Smock, R. W., and Neubert, A. M. "Apple and Apple Products". I n t e r s c i e n c e P u b l i s h e r s , New York. 1950. Nelmes, B. J., and Preston, R. D. J. Exp. Bot. (1968), 19, 496. P o r t e r , K. R., and Machado, R. D. J. Biophys. Biochem. C y t o l . (1959), 7, 167. S i f t o n , H. B. Bot. Rev. (1957), 23, 303. Jermyn, Μ. Α., and Isherwood, F. A. Biochem. J. (1956), 64, 123. W i l l i a m s , Κ. Τ., and Bevenue, A. J. A g r i c . Food Chem. (1954), 2, 472. Knee, M. Phytochemistry (1973), 13, 2207. Coggins, C. W., Knapp, J. C. F., and R i c h e r , A. L. Date Growers Report. (1968), 45, 3. Dennis, D. T., and Preston, R. D. Nature (1961), 191, 667. Ranby, B. G. In "Encyclopedia of Plant Physiology" (W. Ruhland, ed.) 6, p. 268, S p r i n g e r , B e r l i n (1958). F r e y - W y s s l i n g , A. "Die p f l a n z l i c h e Zellwand". Springer, B e r l i n (1959). Bishop, C. T . Can. J. Chem. (1955), 33, 1521. Sen-Gupta, U. Κ., and Rao, C. V. N. B u l l . Chem. Soc. Japan (1963), 36, 1683. A s p i n a l l , G. O., and Canas-Rodrigruez, A. J. Chem. Soc. (1958), 4020. A s p i n a l l , G. O., and Fanshawe, R. S. J. Chem. Soc. (1961), 4215. B a r r e t t , Α., and Northcote, D. Biochem. J. (1965), 94, 617. A s p i n a l l , G. O., C r a i g , J. W. T., and Whyte, J. Z. Carbo­ hydrate Res. (1968), 7, 442.

In Enzymes in Food and Beverage Processing; Ory, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

10.

PRESSEY

23. 24. 25. 26. 27. 28.

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29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52.

Fruit Softening

189

Talmadge, K. W., Keegstra, K . , Bauer, W. D . , and Albersheim, P. Plant Physiol. (1973), 51, 158. Rees, D. Α., and Wight, N. J. Biochem. J. (1969), 115, 431. Albersheim, P. In "Plant Biochemistry" ( J . Bonner and J. E. Varner, eds.) Academic Press, New York, 1965, p. 313. Doesburg, J. J. J. S c i . Food Agric. (1957), 8, 206. Hirst, E. L., and Jones, J. K. N. J. Chem. Soc. (1939), 454. McCready, R. M . , and Gee, M. J. Agric. Food Chem. (1960), 8, 510. Hirst, E. L. J. Chem. Soc. (1942), 70. Chanda, S. Κ., Hirst, E. L., and Perceval, E. G. V. J. Chem. Soc. (1951), 1240. Das, Α., and Rao, C. V. N. Aust. J. Chem. (1965), 18, 845. Dekazos, E. D. J. Food S c i . (1972) 37, 562. Bauer, W. D . , Talmadge, K. W., Keegstra, K . , and Albersheim, P. Plant Physiol. (1973), 51, 174. Lamport, D. T. Α., and Northcote, D. H. Nature (1960), 188, 665. Lamport, D. T. Α., Katona, L., and Roerig, S. Biochem. J. (1973), 173, 125. Lamport, D. T. A. Nature (1967), 216, 1322. Knee, M. Phytochemistry (1975), 14, 2181. Keegstra, Κ., Talmadge, K. W., Bauer, W. D . , and Albersheim, P. Plant Physiol. (1973), 51, 188. Kertesz, Ζ. I., Eucare, M . , and Fox, G. Food Res. (1959), 24, 14. Bartley, I. M. Phytochemistry (1976), 15 625. Nightingale, G. T., Addoms, R. M . , and Blake, M. A. New Jersey Agr. Expt. Sta. Bull. 494 (1930). Sterling, C. J. Food S c i . (1961), 26, 95. Tracy, M. V. Biochem. J. (1960), 47, 431. Dickinson, D. B . , and McCollum, D. P. Nature (1964), 203, 525. H a l l , C. B. Nature (1963), 200, 1010. H a l l , C. B. Bot. Gaz. (1964), 125, 156. Sobotka, F. E., and Watada, A. E. J. Amer. Soc. Hort. S c i . (1971), 96, 705. Hobson, G. E. J. Food S c i . (1968), 33, 588. Pharr, D. M. and Dickinson, D. B. Plant Physiol. (1973), 51, 577. Sobotka, F. E., and Stelzig, D. A. Plant Physiol. (1974), 53, 759. Hasegawa, S . , and Smolensky, D. C. J. Food S c i . (1971), 36, 966. Hinton, D. M. and Pressey, R. J. Food S c i . (1974), 39, 783.

In Enzymes in Food and Beverage Processing; Ory, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

190

53. 54. 55. 56. 57. 58.

Downloaded by UNIV OF MINNESOTA on October 1, 2013 | http://pubs.acs.org Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0047.ch010

59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 80. 81. 82.

E N Z Y M E S I N FOOD A N D B E V E R A G E PROCESSING

Appleman, C. O., and Conrad, C. M. Maryland Univ. Expt. Sta. B u l l . 283 (1926). Postlmayr, H. L., Luh, B. S., and Leonard, S. J. Food Technol. (1956), 10, 618. Shewfelt, A. L . J. Food S c i . (1965), 30, 573. Kertesz, Ζ. I. "The Pectic Substances". Interscience, New York, 1951. Woodmansee, C. W., McClendon, J. H . , and Somers, G. F. Food Res. (1959), 24, 503. Shewfelt, A. L., Paynter, V. Α . , and Jen, J. J. J. Food Sci. (1971), 36, 573. Pressey, R . , and Avants, J. K. J. Food S c i . (1971), 36, 1070. Deuel, H . , and Stutz, E. Advan. Enzymol. (1958), 20, 341. Solms, J., and Deuel, H. Helv. Chim. Acata (1955), 38, 321. Lee, M. and Macmillan, J. D. Biochemistry (1970), 9, 1930. Kertesz, Ζ. I. Food Res. (1938), 3, 481. Hamson, A. R. Food Res. (1952), 17, 370. H a l l , C. B . , and Dennison, R. A. Proc. Amer. Soc. Hort. S c i . (1960), 75, 629. Hobson, G. E. Biochem. J. (1963), 86, 358. Pressey, R. and Avants, J. K. Phytochemistry (1972), 11, 3139. Delincee, H. Phytochemistry (1976), 15, 903. Pollard, Α . , and Kieser, M. E. J. S c i . Food Agric. (1951), 2, 30. Nagel, C. W., and Patterson, J. E. J. Food S c i . (1967), 32, 294. Dolendo, A. L., Luh, B. S., and Pratt, Η. K. J. Food S c i . (1966), 3 1 , 332. Zauberman, G . , and Schiffmann-Nadel, M. Plant Physiol. (1972), 49, 864. Hultin, H. O., and Levine, A. S. J. Food S c i . (1965), 30, 917. Thompson, A. E. Science (1955), 121, 986. Foda, Y. H. Ph.D. Dissertation, University of I l l i n o i s (1957). Sobotka, F. E., and Watada, A. E. Proc. W. Va. Acad. S c i . (1970), 141. Hobson, G. E. Biochem. J. (1964), 92, 324. Hobson, G. E. J. Hort. S c i . (1965), 40, 66. Rich, C. M . , and Butler, L. Advan. Genet. (1956), 8, 267. Hobson, G. E. Phytochemistry (1967), 6, 1337. Buescher, R. W., and Tigchelaar, E. C. Hortscience (1975), 10, 624. McColloch, R. J., Keller, G. J., and Beavens, E . A. Food Tech. (1952), 6, 197.

In Enzymes in Food and Beverage Processing; Ory, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

Downloaded by UNIV OF MINNESOTA on October 1, 2013 | http://pubs.acs.org Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0047.ch010

10.

PRESSEY

Fruit Softening

191

83. Patel, D. S., and Phaff, H. J. Food Res. (1960), 25 37. 84. Patel, D. S., and Phaff, H. J. Food Res. (1960), 25, 47. 85. McColloch, R. J., and Kertesz, Ζ. I. Arch. Biochem. (1948), 17, 197. 86. Pressey, R . , and Avants, J. K. J. Food S c i . (1971), 36, 486. 87. McColloch, R. J. Ph.D. thesis, Kansas State College, (1948). 88. Pressey, R . , and Avants, J. K. Biochem. Biophys. Acta. (1973), 309, 363. 89. Pressey, R . , and Avants, J. K. Plant Physiol. (1973), 52, 252. 90. Pressey, R . , and Avants, J. K. Phytochemistry (1975), 14, 857. 91. Hasegawa, S., Maier, V. P . , Kaszycki, H. P . , and Crawford, J. K. J. Food S c i . (1969), 34, 527. 92. McCready, R. M. and McComb, E. A. Food Res. (1954), 19, 530. 93. Reymond, D . , and Phaff, H. J. J. Food S c i . (1965), 30, 266. 94. McCready, R. M . , McComb, Ε. Α . , and Jansen, E. F. Food Res. (1955), 20, 186. 95. Pressey, R . , and Avants, J. K. Phytochemistry (1976), 15, 1349. 96. B e l l , T. A. Bot. Gaz. (1951), 113, 216. 97. Pressey, R. and Avants, J. K. J. Food S c i . (1975), 40, 937. 98. Patterson, M. E., Doughty, C. C., Graham, S. O., and Allan, G. Proc. Amer. Soc. Hort. S c i . (1967), 90, 498. 99. Arakji, O. Α . , and Yang, H. Y. J. Food S c i . (1969), 34 340. 100. Doesburg, J. J. I.B.V.T. Commun. No. 25 (1965). 101. Knee, M. Phytochemistry (1973), 12, 1549. 102. Bartley, I. M. Phytochemistry (1974), 13, 407. 103. Wallner, S. J. and Walker, J. E. Plant Physiol. (1975), 55, 94.

In Enzymes in Food and Beverage Processing; Ory, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.