Uses of Endogenous Enzymes in Fruit and Vegetable Processing

11 Uses of Endogenous Enzymes in Fruit and Vegetable Processing

Downloaded by UCSF LIB CKM RSCS MGMT on August 25, 2014 | http://pubs.acs.org Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0047.ch011

ROBERT C. WILEY Department of Horticulture, Food Science Program, University of Maryland, College Park, Md. 20742

The quality of fresh and processed fruits and vegetables may be improved by u t i l i z i n g naturally occurring enzymes which can alter appearance, flavor, nutrition, texture and functional properties. Research with endogenous enzymes as well as immobilized enzymes is finding renewed emphasis as consumers become more concerned about food additives. Management of endogenous enzyme systems to develop quality attributes of desirable characteristics is an extremely complex operation. Generally, it is d i f f i c u l t to modify a single enzyme system at a predetermined site and on a specific substrate without disturbing or activating other natural enzyme systems. This secondary activation may have an undesirable effect on the food product in question. Also Schwimmer (1) has indicated that some cell decomposition or disruption is continually taking place during maturation, ripening, handling, pre-processing and processing. These occurrances break natural compartmentalization and increase the d i f f i c u l t y of proper enzyme u t i l i z a t i o n . To a great extent, enzyme u t i l i z a t i o n rather than end-point control requires precise knowledge of enzyme systems, their substrates, cofactors, and inhibitors. These biocatalysts can probably best be managed in the pre-processing sector of the food chain. For example, Wiley and Winn (2) found polyphenol oxidase activity and subsequent browning in green beans and grapes could be controlled with f i e l d treatment by using a modified mechanical harvester. The products were treated at the moment of harvest with SO2 gas in the presence of CO2 and N2 to reduce oxygen levels. Because o f the obvious complexities and tremendous number o f plant products, the endogenous enzymes s e l e c t e d f o r study must be ranked f o r importance according to dominance o f the plant s p e c i e s , importance o f plant s t r u c t u r e , i.e. type o f p l a n t organ, and p o s s i b l e economic impact o f the enzyme utilization on r e s u l tant improvement o f q u a l i t y and f u n c t i o n a l p r o p e r t i e s . There seems to be little question that emphasis on enzyme management i n fruits and vegetables has centered on texture c o n t r o l , although

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

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some researchers have studied enzyme u t i l i z a t i o n i n r e l a t i o n to appearance f a c t o r s such as c o l o r - h u e , value and chroma, gloss and d e f e c t s , f l a v o r enhancement and n u t r i t i o n a l q u a l i t y . This paper emphasizes the i n f l u e n c e of endogenous enzymes on t e x t u r a l f a c t o r s and w i l l b r i e f l y review natural enzyme systems used to modify appearance, f l a v o r and n u t r i t i o n a l q u a l i t y .


APPEARANCE, FLAVOR AND NUTRITIONAL QUALITY The enzymes i n f r u i t s and vegetables which a f f e c t t h e i r appearance are g e n e r a l l y a s s o c i a t e d with negative and/or undes i r a b l e r e a c t i o n s such as browning, l o s s o f c h l o r o p h y l , l o s s of i n t e n s i t y of the anthocyanin pigments and the l i k e . Most enzymic browning reactions i n plant products are r e l a t e d to the polyphenol oxidases. Polyphenol oxidases which c a t a l y z e a number of r e a c t i o n s i n v o l v i n g phenolic compounds have engendered a great amount o f research i n t e r e s t ( 3 ) . Other enzymes such as peroxidases and c a t a l a s e s a l s o c o n t r i b u t e to the u n d e r s i r a b l e brown c o l o r r e a c t i o n s although t h e i r presence has t r a d i t i o n a l l y been used as an i n d i c a t o r o f inadequacy of blanch in frozen vegetables. Dehydrated prunes, r a i s i n s , dates and r e l a t e d f r u i t s are about the only plant products which are considered b e n e f i c i a r i e s of the browning r e a c t i o n s . This browning r e q u i r e s l i t t l e or no r e g u l a t i o n and occurs g e n e r a l l y during normal processing o p e r a t i o n s . C h l o r o p h y l l a s e which i s s p e c i f i c f o r c a t a l y z i n g the hydrolys i s of the phytyl e s t e r linkage of c h l o r o p h y l l i s involved in green hue s h i f t s i n many green vegetables and c h l o r o p h y l l continuing plant material. Braverman (4) i n d i c a t e s information i s unclear concerning the use o f endogenous c h l o r o p h y l l a s e to preserve the green c o l o r in t h i s type o f t i s s u e . Perhaps the best perspective r e l a t i n g to green c o l o r and c h l o r o p h y l l a s e i s the work of Van Buren et a l . (5) who suggested t h a t the conversion o f c h l o r o p h y l l to pheophytin in blanched green beans at 60-80°C was p r i m a r i l y due to pH l e v e l s in the pods which were s h i f t e d from 6.5 to 5.5 by endogenous p e c t i n e s t e r a s e (PE) a c t i v i t y . Anthocyanase enters plant products through bruises and i n s e c t damage and i s thought to be mainly from fungal o r i g i n s . The use o f endogenous anthocyanase a c t i v i t y has been l i m i t e d although Van Buren et a l . (6) have suggested that an anthocyanin degrading enzyme i s present in bruised sour c h e r r i e s . Natural c o l o r can be reduced in berry j u i c e s and wines by d e l i b e r a t e or inadvertent a d d i t i o n o f anthocyanase. Color and gloss have been improved i n r e c o n s t i t u t e d dehydrated sweet potato f l a k e s , according to Hoover ( 7 ) , by a c t i v a t i n g the natural a m y l o l y t i c enzyme systems (mainly 3 amylase). The pureed t i s s u e was held at 70-85°C f o r 2-60 minutes to get t h i s desirable conversion. In the area o f f l a v o r , i t i s c l e a r that few o f the t a s t e sensations such as sweet, sour, b i t t e r , s a l t y , or even p a i n , heat or c o l d are developed by u t i l i z a t i o n of endogenous enzyme

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


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systems. This probably r e l a t e s to the f a c t that most t a s t e i n g r e d i e n t s can be added economically to food products and consequently there i s no p r a c t i c a l reason to a c t i v a t e s p e c i f i c enzyme systems. Although a great amount of research has been conducted on odor development by the use o f enzymes, r e c e n t l y a t t e n t i o n has been on the C r u c i f e r a e family and the A l l i u m genus. Odors appear to be a normal p h y s i o l o g i c a l process in plant m a t e r i a l s with development during the e n t i r e l i f e o f the p l a n t through prep r e c u r s o r s , precursors and t h e i r odor components. A large percentage o f these odors are developed by both chemical and/or enzymes means from amino a c i d s , isoprene groups, a c e t a t e s , and break-down products o f many more complex m o i t i é s . Konigsbacher (8) has v i s u a l i z e d that odor formation r e s u l t s from a s e r i e s o f chemical compounds i n a chain of r e a c t i o n s which probably begin with water, s u n l i g h t , and carbon d i o x i d e . I t i s obvious that each step i s chemical and enzymatic, with each enzyme system probably r e q u i r i n g s p e c i a l substrates and c o f a c t o r s to make the r e a c t i o n move forward. The problem i n t r i g g e r i n g an aroma r e a c t i o n during a processing operation i s the p o s s i b l e disappearance o f the aroma before i t reaches the consumer. The most s a t i s f a c t o r y way to protect aroma f a c t o r s in p l a n t t i s s u e s f o r l a t e r a c t i v a t i o n i s to preserve preprecursors and p r e c u r s o r s , maintain compartmentalization, and reduce as much as p o s s i b l e heat shock to the t i s s u e s which may destroy c e l l i n t e g r i t y or u t i l i z e precursor substances. Some ideas along t h i s l i n e were suggested by Puangnak, Wiley, and K a h l i l (9) studying heat a c t i v a t e d dimethyl s u l f i d e (DMS) in sweet c o r n . They found t h i s compound evolves mainly from the p e r i c a r p complex. Microwave blanching may p r o t e c t t h i s d e s i r a b l e c o r n - l i k e aroma in cooked frozen samples by reducing heat t r e a t ment to f l a v o r precursors in the p e r i c a r p a r e a . In the n u t r i t i o n a l q u a l i t y a r e a , i t appears that enzymes have been i n f r e q u e n t l y used i n f r u i t and vegetable t i s s u e to make food products more n u t r i t i v e . The p e c t i n a s e s , h e m i c e l l u l a s e s , and c e l l u l a s e s a l l have the p o t e n t i a l to break down polymers i n t o more s o l u b l e and more d i g e s t i b l e food substances, but these techniques have not been used e x t e n s i v e l y to provide more n u t r i t i o u s f o o d . Amylases have been used, however, to increase s o l u b l e s o l i d s in sweet potatoes f o r b e t t e r f u n c t i o n a l p r o p e r t i e s r a t h e r than improved n u t r i t i o n (7). A s c o r b i c a c i d o x i d a s e , enzymes involved i n carotene product i o n , and the a c t i o n of lipoxgenase f o r d e s t r u c t i o n of l i p i d s , are o f concern in the handling and processing o f f r u i t and vegetable products, but probably cannot be involved in worthwhile u t i l i z a t i o n programs. L i p a s e , which converts l i p i d s to f a t t y acids and g l y c e r o l , and polyphenol o x i d a s e s , which can destroy v i t a m i n s , must be c a r e f u l l y c o n t r o l l e d i n a l l f r u i t and vegetable products.


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TEXTURAL FACTORS The t e x t u r a l f a c t o r s emphasized in t h i s s e c t i o n w i l l r e l a t e mostly to softness and firmness of f r e s h apple f r u i t s and processed apple s l i c e s . The s t u d i e s of c e l l - w a l l p o l y s a c c a h r i d e s , p e c t i c enzymes, and calcium f i r m i n g conducted by several c o l eagues in t h i s department w i l l be presented. There w i l l be no attempt to i n c l u d e other t e x t u r a l and t a c t i l e sensations such as v i s c o s i t y , c o n s i s t e n c y , chewiness, s t r i n g i n e s s , b r i t t l e n e s s , cohesiveness, o i l n e s s , w a t e r i n e s s , e t c .


Polysaccahrides

Involved in Softening

Natural apple s o f t e n i n g during the post-harvest period has almost u n i v e r s a l l y been considered a breakdown of p e c t i n s , which by s t r i c t d e f i n i t i o n are galacturonans, b u i l t up of 1-4 l i n k e d D-galacturonic a c i d r e s i d u e s . Other i n v e s t i g a t o r s have f e l t that non-galacturonide material i s attached to the polyuronide chain and i s a l s o involved in f r u i t s o f t e n i n g . S p e i s e r et al (10) were among the f i r s t to i n d i c a t e that b a l l a s t m a t e r i a l s , or h e m i c e l l u l o s e s , are probably attached to the polyuronide chain by e s t e r linkages. L a t e r , Tavakoli and Wiley (11), in c o n s i d e r i n g the s t r u c t u r e o f apple c e l l w a l l s , found p e c t i c substances comprising u n i t s of D-galacturonic a c i d , hemicellui oses comprising units of D-xylose, L-arabinose, D-galactose, D-glucose, and c e l l u l o s e comprising r e s i d u a l material were 30%, 40% and 30% r e s p e c t i v e l y on a dry weight b a s i s . These c e l l wall components, except f o r c e l l u l o s e , were evaluated by gas l i q u i d chromtography (GLC) as trimethyl s i l y l (TMS) d e r i v a t i v e s of monomers formed a f t e r fungal pectinase and h e m i c e l l u l a s e h y d r o l y s i s o f ethanol p r e c i p i t a t e s . These p r e c i p i t a t e s were prepared from s e r i a l e x t r a c t i o n s of sugarf r e e alcohol i n s o l u b l e s o l i d s (AIS). Results showed the hexosans and the pentosans i n the hemicellui oses c o n s i s t e d o f 60% g l u c o s e , 27% galactose 6.5% xylose and 6% arabinose moieties i n about a 10:5:1:1 r a t i o . This GLC technique d i d not detect rhamnose which has been found i n the c e l l wall of a p p l e s , but d i d show subs t a n t i a l amounts o f the various glucose m o i e t i e s , even in the more mature and s t a r c h - f r e e f r u i t s . Tavakoli and Wiley (11) using apples o f a wide range of maturity and ripeness found, as shown in Table 1, through r e g r e s s ion a n a l y s i s o f o b j e c t i v e firmness measurements and c e l l wall components, that g a l a c t a n s , glucans, and water i n s o l u b l e p e c t i n i c acids were r e s p o n s i b l e f o r approximately 80% o f the v a r i a t i o n in firmness changes of the three major Appalachian apple v a r i e t i e s used in processing - Golden D e l i c i o u s , Stayman, and York I m p e r i a l . These workers a l s o r e p o r t e d , that according to t h e i r r e s u l t s the hemicelluloses and the p e c t i c substances which occur with c e l l u l o s e in apple parenchyma t i s s u e in a very c l o s e k n i t c e l l u l a r m a t r i x , should be regarded as the major chemical c o n s t i t u e n t s which g r a d u a l l y decompose as f r u i t undergoes r i p e n i n g . The


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Table 1. Multiple regression and correlation analysis of firmness (lbs. force) values with the cell wall constituents (per cent fresh weight basis) of Golden Delicious, Stayman, and York Imperial, η = 36.


Variable Galactose Glucose Water insoluble extractgalacturonic acid Residue Arabinose Xylose Water soluble cxtractgalacturonic acid Total galacturonic acid

Single correlation coefficients

Partial correlation coefficients

Cumulative regressions

0.867 0.836

0.294 0.298

0.741 0.772

0.867 0.879

0.857 -0.080 0.524 0.622

0.221 -0.175 0.113 0.049

0.788 0.797 0.802 0.803

0.888 0.893 0.896 0.896

-0.164 0.767

-0.047

0.804

0.896

R

R

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Table 2. Effect of variety on firmness and on galactose, water insoluble extract galacturonic acid, and the residue content of apple cell wall materials* (per cent fresh weight basis). Means Varietv

Shear-press (lb. force)

Golden Delicious Stayman York Imperial L . S . D . at 1% level a

Water insoluble extract galacturonic acid (Pectinic acids)

Galactose (Galactan)

Residue (Cellulose)

0.30 0.30 0.46 0.08

0.19 0.18 0.26 0.05

0.50 0.58 0.68 0.08

321 322 480 25

Combined data for maturation and storage treatments.

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p o r t i o n of apple f r u i t firmness which diminishes during storage l i f e should be r e f e r r e d to as "dynamic firmness" s i n c e i t i s g r e a t l y a f f e c t e d by changes in the i n t e r m i c e l l a r p o l y s a c c h a r i d e s . The starch content of f r u i t in a very immature stage and the osmotic p r o p e r t i e s of the c e l l s may a l s o produce some e f f e c t s on firmness. C e l l u l o s e , however, was not i n f l u e n c e d by m a t u r i t i e s and s t o r a g e s . T h e r e f o r e , c e l l u l o s e should be regarded as a s t a t i c c o n s t i t u e n t of the apple f r u i t c e l l wall which provides the c e l l s with r e l a t i v e l y f i x e d or b a s e - l i n e l e v e l s of s t r e n g t h . This strength may be r e f e r r e d to as " s t a t i c firmness" which p r e v a i l s throughout the storage l i f e of a p p l e s . The data in Table 2 by Tavakoli and Wiley (11), shows that York I m p e r i a l , a r e l a t i v e l y f i r m v a r i e t y , contained a s i g n i f i c a n t l y greater amount of w a t e r - i n s o l u b l e p e c t i n i c a c i d s , g a l a c t a n s , and c e l l u l o s e than e i t h e r Golden D e l i c i o u s or Stayman. The l e v e l s o f these substances should be used to c h a r a c t e r i z e the d i f f e r e n c e s in firmness among these v a r i e t i e s . It appears that a number of polysaccharides are involved i n apple f r u i t s o f t e n i n g . Rombouts (12) has r e c e n t l y reviewed p e c t i c enzyme research and presented several t h e o r i e s concerning the linkages of the various o l i g i s a c c h a r i d e s to the p e c t i c substances. F u r t h e r , Keegstra et al (13) have suggested that the various macromolecular components o f sycamore c e l l w a l l s are c o v a l e n t l y cross-linked. I t i s p o s s i b l e that the same linkages occur i n apple parenchma t i s s u e . Enzymes Systems Related_to

Softening

The enzymes systems involved in apple s o f t e n i n g and o v e r a l l t e x t u r a l changes are s t i l l under q u e s t i o n . There have been few reports of e i t h e r endogenous polygalacturonases (PG) or p e c t i n lyases(PL) in disease f r e e apple parenchma t i s s u e . The presumed absence o f these important enzymes in t h i s t i s s u e places i n f u r t h e r jeopardy the theory that apple f r u i t s o f t e n i n g i s due to galacturonan 1-4 linkage breaks and/or t r a n s i i m i n a t i o n i n p o l y galacturonic acids. Mannheim and S i v (14) have a l s o reported that PG was absent in oranges. PE, which removes methyl e s t e r s from methylated p e c t i c subs t a n c e s , i s the remaining endogenous p e c t i c enzyme that i s thought to be involved in p h y s i o l o g i c a l s o f t e n i n g changes of apple f r u i t . Rombouts (12) pointed o u t , Schultz et al (15) were the f i r s t to suggest that the mode o f a c t i o n of PE resembles that o f a z i p p e r . Zipper a c t i o n may account f o r the major softening i n apple f r u i t up to and i n c l u d i n g the f i r s t 60-90 days o f c o l d storage, by separating the p e c t i n and h e m i c e l l u l o s e complexes from the c e l l u lose m a t r i x , or by causing i n t e r n a l separation of the "dynamic" c o n s t i t u e n t s such as the galacturonans, pentosans, and hexosans. Some c o l d storage work (1°C) conducted by Lee (16) on Golden D e l i c i o u s and York Imperial v a r i e t i e s showed PE a c t i v i t y increased in storage up to 60 days and then l e v e l e d o f f a t a high p l a t e a u . This plateau may have been due to accumulation of



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products or l i m i t a t i o n s i n substrate l e v e l , which can have an i n h i b i t i n g e f f e c t on enzyme a c t i v i t y . A negative c o r r e l a t i o n c o e f f i c i e n t of -0,85 (n=40) was shown between PE a c t i v i t y in acetone powder from raw s l i c e s and shear press readings on raw slices. Results i n d i c a t e d that as PE a c t i v i t y increases in raw f r u i t , presumably in a Ca2 ion d e f i c i t , the f r u i t became s o f t e r . He a l s o showed that PE a c t i v i t y was approximately 2 times higher in the s o f t v a r i e t y Golden D e l i c i o u s as compared with the f i r m v a r i e t y York I m p e r i a l . In c o n s i d e r i n g the problems a s s o c i a t e d with processing apple s l i c e s , i t i s c l e a r that a s u b s t a n t i a l amount of PE a c t i v i t y has taken place in the apple c e l l wall during maturation and r i p e n ing of the f r u i t and the pH i n t r e g i t y of the c e l l wall has been maintained by the slow demethylation of the p e c t i c substances. Lee (16) suggested that PE a c t i v i t y i s very low in apple t i s s u e which normally has a pH of about 3.4. He p r e d i c t e d PE s p e c i f i c a c t i v i t y at t h i s pH could be 5-10 yg CH3OH per mg p r o t e i n in a 30 min period at 35°C. Cold storage f u r t h e r reduces t h i s PE a c t i v i t y and might account f o r the very slow softening changes which take place in c o n t r o l l e d atmosphere (CA) and r e f r i g e r a t e d storage. Apple f r u i t received by processors then would be q u i t e v a r i a b l e in terms of PE a c t i v i t y which had taken place during maturation and r i p e n i n g and subsequent storage.


+

Calcium Firming Plant T i s s u e Locenti and Kertesz (17) were among the f i r s t to i n d i c a t e treatment o f f r u i t t i s s u e with Ca2+ s a l t s would f i r m t i s s u e s and that the t i s s u e f i r m i n g compound was calcium pectate. Archer (18) and C o l l i n s and Wiley (19) reported C a s a l t s were not used to any great extent to f i r m apple s l i c e s because of problems in g e t t i n g a uniform f i r m i n g response. They found that calcium l a c t a t e was a good f i r m i n g agent and gave a more d e s i r a b l e f l a v o r response than e i t h e r calcium c h l o r i d e or calcium gluconate Van Buren et al (20),working with calcium f i r m i n g of green beans, found PE a c t i v i t y r e s u l t e d i n firmness o f t i s s u e and a decreased s o l u b i l i t y of the p e c t i c substances, p a r t i c u l a r l y in the presence of Ca^+ s a l t s . They suggested use o f the endogenous PE system, under proper processing c o n d i t i o n s , to allow the enzyme to a c t on the p e c t i n s . Penetration of Ca2 s a l t s i n t o heat processed apple t i s s u e s using C a C l 2 as shown in Figure 1 was studied by C o l l i n s and Wiley (21). The dark areas in the autoradiograms of the apple pieces show 45caCl2 d i s t r i b u t i o n . Section A shows only very s l i g h t amount of Ca2+ penetration occurs i f the f r u i t i s submerged 15 min i n a 45caCl2 s o l u t i o n at 20°C. Section Β presents sub merged f r u i t which was then canned and heat processed f o r 8 min at 100°C These samples do not show complete penetration of 45ca to the center of the p i e c e . S e c t i o n C shows a more complete penetration of the p i e c e , when p r e v i o u s l y submerged s l i c e s were 2 +

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subjected to 2 min vacuum treatment f o r d e a e r a t i o n , steam blanched f o r 30 s e c . at 10 p s i , canned and then processed f o r 8 min a t 100°C. Figure 2 shows that canning and then processing of apple s l i c e s in b o i l i n g water f o r 8 min using 45caCl? as a packing medium, did not r e s u l t in f u l l penetration of ' C a C l 2 to the middle o f the p i e c e s . These s l i c e s received the usual vacuum and steam treatments p r i o r to heat processing Results i n d i c a t e Ca^ * ions are f a i r l y e f f e c t i v e in f i r m i n g apple f r u i t i f handled p r o p e r l y , however, very mature f r u i t and c e r t a i n s o f t v a r i e t i e s , such as overmature Golden D e l i c i o u s , Red D e l i c i o u s , and Romes continue to present texture control problems. Van Buren (22), working with green beans, a l s o found blanching treatments in the presence o f C a d i d not always e f f e c t i v e l y f i r m the pods. The continuing problems of f i r m i n g plant t i s s u e s during processing has stimulated the idea that endogenous PE could be used to a c t i v a t e s i t e s f o r Ca - bonding. However, considerable research information i s required so that the PE can be measured e a s i l y under simulated processing c o n d i t i o n s . These c o n d i t i o n s should include wide ranges in temperature, pH, s o l u b l e s o l i d s and the l i k e . 5


4

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PE Measurement and P a r t i a l

Characterization

Lee and Wiley (23) developed a d i a l y s i s procedure to c h a r a c t e r i z e apple PE a c t i v i t y . The d i a l y s i s and GLC procedure to measure methanol produced by PE demethylation involved using a u n i t i z e d d i a l y s i s c e l l under a g i t a t e d c o n d i t i o n s . Each d i a l y z e r comprised 2 polymethyl-methacrylate h a l f c e l l s separated by a V i s k i n g membrane with 4.8 my pore diameters. Each h a l f c e l l has a c i r c u l a r c a v i t y (4.0 cm diameter, volume 7 ml) and a f i l l i n g hole. One side o f the compartment, designated the f r o n t was charged with 5 ml of the r e a c t i o n mixture. The charge contained 0.5% o f apple acetone powder and 0.15M NaCl in 1% phosphate buffered p e c t i n s o l u t i o n s adjusted to various pH l e v e l s . The other s i d e of the compartment, designated the back, was charged with an equal volume o f d i s t i l l e d water. The f i l l i n g holes were sealed with tape and the c e l l s were shaken in a temperature c o n t r o l l e d oven. These phosphate b u f f e r s ranging from pH 4 . 5 9.0 allowed s a t i s f a c t o r y measurement of PE a c t i v i t y over t h i s pH range. The methanol produced was taken as PE a c t i v i t y and the d i a l y s i s procedure allowed simultaneous production and separation of methanol from the p e c t i n substrate i n t o the d i s t i l l e d water. A f t e r d i a l y s i s and depending on the methanol concentration in the p e c t i n f r e e , d i s t i l l e d water side o f the d i a l y s i s c e l l , a 5 μ£ sample of the methanol and d i s t i l l e d water mixture was i n j e c t e d d i r e c t l y i n t o the gas chromatograph.




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Figure 1. Autoradiograms of transverse sections of mature Stayman apple slices submerged in a solution of Ca Cl for 15 min at 68°F. A , submerged only; B , submerged and processed; and C , submerged, subjected to vacuum and steam, and processed. 45

2

Figure 2. Autoradiograms of longitudinal and transverse sections of mature Stayman apple slices subjected to vacuum and steam. Slices were processed in cans containing Ca Cl solution. 45

2


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The concentration e q u i l i b r i u m f o r the closed system d i a l y ser with compartments charged with equal volumes of s o l u t i o n were as f o l l o w s : I n i t i a l concentration of methanol, in f r o n t : Cf = C-j; Ci i s designated as concentration of methanol as a r e s u l t of PE a c t i v i t y . At e q u i l i b r i u m c o n c e n t r a t i o n , i f a l l methanol i s d i f f u s i b l e , Cf = C = 0.5 C i . R equals the r a t i o of 0 / 0 . 5 · . At e q u i l i b r i u m , R = 1 i f a l l methanol i s d i f f u s i b l e ; i f some of the methanol i s not d i f f u s i b l e , R w i l l be l e s s than 1. D


ο

η

Table 3 shows a substrate of 1% p e c t i n and r e a c t i o n times of about 3-4 hrs (at 35°C) gave s a t i s f a c t o r y r e s u l t s using t h i s method Apple PE e x h i b i t s the c h a r a c t e r i s t i c b e l l - s h a p e d curve showing optimum a c t i v i t y between pH 6.5 and 7.5. Figure 3 shows the PE curves f o r Golden D e l i c i o u s apple f r u i t of 4 d i f f e r e n t maturities. The GO code i n d i c a t e s Golden D e l i c i o u s apple samples were harvested and tested immediately without f u r t h e r storage. GOA, GOB, GOC, and GOD d e p i c t maturity l e v e l s . The GOA f r u i t was harvested Sept. 11, the GOD f r u i t Oct. 3 i n d i c a t i n g a spread o f 23 days between f r u i t o f the f i r s t and l a s t harvest. I t seems c l e a r that PE a c t i v i t y i s s i g n i f i c a n t l y higher in the more mature f r u i t or f r u i t from l a t e r h a r v e s t s . Lee and Wiley (23) found the optimal r e a c t i o n temperature f o r apple PE i s 55°C and the a c t i v a t i o n energy (E) c a l c u l a t e d by the graphic procedure i s 5800 c a l o r i e s . A c t i v a t i o n energy should be regarded as the amount of energy needed to place the substrate molecules in a r e a c t i v e s t a t e . I f Ε i s l a r g e , the r a t e of r e a c t i o n w i l l increase r a p i d l y f o r a f i x e d increase i n temper ature. The substrate molecules must f i r s t absorb that amount of E. They then can r e a c t and be converted to products. This means that PE a c t i v i t y can be expected to be 2.5 times higher a t 25°C as compared with 0°C. This compares f a v o r a b l y to data from storage research where firmness of apples held at 0°C f o r 2 months was about twice as f i r m as those held at 20°C f o r the same p e r i o d . Apple PE shown in Table 4 i s completely i n a c t i v a t e d at 80°C f o r 10 min or 90°C f o r 5 min. According to published f i g u r e s , the temperature of i n a c t i vation of plant PE shows a wide range. Heating f o r 5 min at 70°C gave almost complete i n a c t i v a t i o n o f p u r i f i e d PE, whereas a s i m i l a r preparation from orange was i n a c t i v a t e d by 30 min at 5 6 ° C Other l e s s p u r i f i e d e x t r a c t s have been found more r e s i s t a n t to heat; f o r example, McColloch and Kertesz (24) found 50% a c t i v i t y l e f t in tomato e x t r a c t s a f t e r one hour at 70°C, J o s l y n and Sedky (25) found a marked d i f f e r e n c e in the thermal s t a b i l i t y of PE i n c i t r u s j u i c e s according to species and v a r i e t y .


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E N Z Y M E S IN FOOD A N D B E V E R A G E PROCESSING Table 3. Diffusibility of methanol' through a cellulose membrane under agitated conditions. R values Concentration of methanol 100 200 300 400

b

0

Reaction times (hours) 1

ppm ppm ppm ppm

2

0.65 0.66 0.54 0.38

0.95 0.98 0.96 1.00

3

4

0.96 0.95 0.95 0.99

0.95 0.99 0.98 1.02

Methanol was mixed in 1 % pectin solution at 3 5 ° C . A Visking membrane obtained from Arthur H . Thomas Co., Phila., Pa., 4465-A2 dialyzer tubing. R value represents the ratio of methanol concentration between front and back sides of membrane in a dialyzer. a

b


C

Journal of the American Society for Horticulture Science

Table 4. Temperatures for apple pectinesterase" inactivation. Relative velocity ^ g C H 3 O H in 5 ml dialysate)

Inactivation temperatures (°C)

Control

ambient

Inhibition (%)

655

0

100 90 80 70 60

5 min. heating 0 0 50 80 325

100 100 92 88 50

100 90 80 70 60

10 min. heating 0 0 0 40 195

100 100 100 94 70

Dialysis under agitation for 4 hr at 3 5 ° C in a p H 6.5 phosphate buffer. a

Journal of the American Society for Horticulture Science

Table 5. Effects of buffer treatments on Ca in alcohol insoluble solids (AIS) of canned Stayman apple slices. 1

Treatments Buffer (1.0M) lactic acid— sodium lactate plus 1% CaCl

2

Control Unbuffered plus Ca pH 3.0 pH 3.5 pH 4.0 pH 4.5 pH 5.0 pH 5.5

Tissue PH 3.48 3.27 3.20 3.57 3.94 4.27 4.52 4.88

Ca μ.%1 g/AIS' 3.70 a 12.79 b 16.29 c 17.51 d 18.49 ef 18.95 f 17.94 de 32.59 g

1 All fruit stored (34 F) 50 days. 2 Means (average 10 observations) not fol lowed by the same letter are significantly dif ferent at the 5% level. e

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According to Lee and Wiley (23) PE can a l s o be i n h i b i t e d by sucrose and the percent of apple PE i n h i b i t i o n p r o g r e s s i v e l y increased in s o l u t i o n s c o n t a i n i n g from 5 to 15% sucrose. Approximately 5, 25, and 40% PE i n h i b i t i o n were found in the presence of 5, 10, and 15% sucrose c o n c e n t r a t i o n s , r e s p e c t i v e l y . These r e s u l t s are s i m i l a r to those of Chang et al (26) who reported that a 13% sucrose concentration i n h i b i t e d papya PE. The actual mechanism by which sucrose i n h i b i t s PE a c t i v i t y i s now known. However, an environment o f lower water a c t i v i t y due to sucrose may be the most important f a c t o r i n v o l v e d , since PE requires water f o r i t s r e a c t i o n . Sugar s o l i d s , added to f r u i t , should be c a r e f u l l y managed i f i t becomes d e s i r a b l e to a c t i v a t e PE during pre-processing o p e r a t i o n s . Endogenous

PE_Utilization

Once a method i s developed to determine enzymatic a c t i v i t y under simulated pre-processing and processing c o n d i t i o n s , the u t i l i z a t i o n of that endogenous enzyme system may commence. Wiley and Lee (27) have pointed out that texture m o d i f i c a t i o n of apple s l i c e s during processing i s necessary to accomodate customer's specifications. S o f t texture i s d e s i r a b l e in no-cook p i e s , whereas some products such as apple-turnovers and the l i k e may need f i r m e r f r u i t to withstand baking. As i n d i c a t e d e a r l i e r , there i s great v a r i a t i o n in firmness o f raw apple products and even though heat processing tends to make f o r more t e x t u r a l u n i f o r m i t y , the Ca-firming r e a c t i o n has been d i f f i c u l t to control because of unknown or undesirable handling p r a c t i c e s used on f r u i t p r i o r to p r o c e s s i n g . Modifying apple texture by a c t i v a t i n g the PE enzyme system during the r e l a t i v e l y short pre-processing periods of s l i c i n g , trimming, vacuumizing, steaming and/or blanching and during the e a r l y heating phases o f thermal processing appears possible. Wiley and Lee (27) have i n v e s t i g a t e d PE a c t i v a t i o n and have vacuumized apple s l i c e s in a wide range o f b u f f e r s o l u t i o n s prepared from 0.1 M c i t r i c acid-disodium phosphate mixtures c o n t a i n ing 0.5% calcium c h l o r i d e . This procedure was based on e a r l i e r research o f C o l l i n s (28) who showed an improved up-take o f calcium in apple AIS as the pH of the t i s s u e increased from about 3.5 to 5.0 (Table 5 ) . These r e s u l t s were probably due to increased PE a c t i v i t y a t the higher pH l e v e l s . Figure 4 shows the r e l a t i o n s h i p between PE a c t i v i t y in the acetone powder of raw Golden D e l i c i o u s t i s s u e and the firmness of a s i m i l a r sample o f Golden D e l i c i o u s s l i c e s which were vacuumi z e d over a wide pH range i n the presence o f 0.5% CaCl2. The s l i c e s were then blanched i n steam at 10 psi f o r 2 minutes, cooled and measured in the Kramer shear p r e s s . I t appears that maximum firmness i n the C a t r e a t e d blanched s l i c e s , held i n a concentration o f 0.5% CaCl2 over the wide pH range, occurred a t 2 +


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210 :

4.5

5.5 PHOSPHATE

65 7.5 BUFFERED PH VALUES

8.5

9.0

Journal of the American Society for Horticultural Science Figure 3.

Effects of pH on PE specific activity



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3.0

3.5

4Ό

4.5

5.0

5.5

PHOSPHATE

6.0

6.5

7.0

205

7.5

8.0

85

9.0

BUFFERED pH VALUES

Food Technology

Figure 4.

Effects of pH on PE activity andfirmnessof calciumtreated blanched Golden Delicious slices

approximately the same pH l e v e l which i s optimum f o r p e c t i n esterase a c t i v i t y . Apple s l i c e s can a l s o be t r e a t e d with Ca(0H)2 to f i r m the t i s s u e and modify pH. Golden D e l i c i o u s s l i c e s impregnated with a 1% Ca(0H)2 s o l u t i o n gave a firmness value of 158 l b / f o r c e . This was much f i r m e r than control which was 78 l b / f o r c e . The samples firmed with the Ca(0H)2 treatment were then pH reversed with a 1% c i t r i c a c i d s o l u t i o n to about 3.95. T h i s reduction in pH d i d not s i g n i f i c a n t l y reduce the firmness of the t i s s u e which had been p r e v i o u s l y induced a t the higher pH l e v e l i n the presence o f Ca(0H)2The Ca(0H)2 t r e a t e d s l i c e s showed a d u l l brownish c o l o r a t i o n and had to be a c i d i f i e d to return the c o l o r of s l i c e s to normal. Case hardening was noticed i n the 1% Ca(0H)2 treated s l i c e s and concentrations below t h i s l e v e l be used in commercial o p e r a t i o n s .


should

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E N Z Y M E S IN FOOD AND B E V E R A G E PROCESSING


P o s s i b l e Processing A p p l i c a t i o n s Apple processors should consider u t i l i z i n g endogenous PE systems to standardize the t e x t u r a l q u a l i t y of t h e i r products. Techniques f o r enzyme management have already been approved in p r i n c i p l e by the Food and Drug A d m i n i s t r a t i o n . Recent research by Wagner et al (29) and r e s u l t a n t r e g u l a t i o n s (30) f o r tomato puree and paste allow a c i d i f i c a t i o n by HC1 and n e u t r a l i z a t i o n with NaOH p r i o r to s t r a i n i n g . This technique, to improve the consistency of tomato products, c o n t r o l s and u t i l i z e s c h a r a c t e r i s t i c s o f endogenous PE and PG enzyme systems. Apple s l i c e manufacture presents a somewhat more complex set o f o p e r a t i o n s . The f r u i t may be u t i l i z e d in a thermally processed system or as a frozen item r e q u i r i n g d i f f e r e n t enzyme i n h i b i t i n g operations than f o r canned s l i c e s . For example, many apples f o r f r e e z i n g are s u l f i t e d without the use of high blanching temperatures. Most g e n e r a l l y problems occur with s l i g h t l y s o f t f r u i t and/or f r u i t almost too s o f t and mushy to be u t i l i z e d . U t i l i z a t i o n o f endogenous PE i s p r i m a r i l y f o r s o f t f r u i t . Although i t i s recognized t h a t apple s l i c e s become s o f t and mushy during thawing a f t e r f r e e z i n g , the f o l l o w i n g examples deal with canned s l i c e s . A general schematic flow sheet used by apple s l i c e processors i s as f o l l o w s : c l a s s i f y f r u i t according to firmness and v a r i e t y , s i z e grade, a u t o m a t i c a l l y p e e l , c o r e , s l i c e , t r i m , dry or wet flume, vacuum blanch, convey, f i l l and heat p r o c e s s : The PE u t i l i z a t i o n procedure should be used a f t e r the fluming step and would incorporate a v a r i a b l e speed steam or hot water treatment and a 2nd vacuumizing u n i t with s u i t a b l e c o n t r o l s to moniter temperature, time of h o l d , pH, s o l u b l e s o l i d s , and the l i k e . For the various catagories of s o f t f r u i t the v a r i a b l e speed treatment chamber would be used to a c t i v a t e PE at about 55°C The 1st vacuum u n i t would be used to impregnate s l i c e s with C a s a l t s and/or Ca2+ bases. The 2nd vacuum u n i t would only be used where pH r e v i s i o n was necessary to r e t u r n firmed s l i c e s to t h e i r normal pH, c o l o r and f l a v o r . 2 +

CONCLUSIONS I t i s evident that only a few endogenous enzyme systems have been used to a l t e r q u a l i t y c h a r a c t e r i s t i c s and/or f u n c t i o n a l p r o p e r t i e s of processed f r u i t s and vegetables. The most important studies can be summarized as f o l l o w s : (1) Heat a c t i v a t e d 3 amylase i s used to improve f u n c t i o n a l p r o p e r t i e s of sweet potato f l a k e s . (2) Heat and pH a c t i v a t e d PE can be used to provide s i t e s f o r calcium f i r m i n g of thermally processed apple slices. A c t i v a t i o n of PE during maturation and r i p e n i n g considered in the l i g h t o f the f o l l o w i n g obervations:


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Endogenous Enzymes in Processing (a)

Apple s o f t e n i n g during maturation and r i p e n ing appears t o be due to the cumulative changes o f several macromolecular components. They are g a l a c t a n s , glucans and i n s o l u b l e p e c t i n i c acids. Therefore linkages between these components must be considered i n any Ca2 related-PE u t i l i z a t i o n f i r m i n g o p e r a t i o n . (b) C e l l u l o s e tends to lend b a s e - l i n e o r background strength to mature o r r i p e apple t i s s u e and i s not an important f a c t o r during enzyme u t i l i z a t i o n operations. (c) PE a c t i v i t y shows a highly s i g n i f i c a n t negative c o r r e l a t i o n (-O.85) with firmness changes i n apple f r u i t and thus appears to be a f a c t o r i n apple t i s s u e s t r u c t u r a l i n t e g r i t y . (d) A d i a l y s i s - GLC procedure t o measure methanol produced by PE a c t i v i t y provides an a n a l y t i c a l tool t o provide information f o r the management of the PE system during pre-processing and processing procedures. (e) PG i s presumed to be absent i n disease f r e e apple parenchma t i s s u e thus may be disregarded during PE a c t i v a t i o n o p e r a t i o n s . PG, i f present, would be a c t i v a t e d by Ca2+ ions and decrease e f f e c t i v e f i r m i n g . Further research i s needed to develop and u t i l i z e endogenous enzyme systems i n f r u i t and vegetable products.


+

Literature Cited 1. 2. 3. 4. 5. 6. 7. 8.

9. 10. 11.

Schwimmer, J. Food S c i . (1972) 37: 530. Wiley, R.C. and Winn, P . N . , N.Y. State Proc. Veg. Conf. (1970) Whitaker, J.R. "Principles of Enzymology for the Food Science" 636P Marcel Dekker Inc. N.Y. 1972. Braverman,J.B.S."Introduction ot the Biochemistry of Foods, 336p Elsevier Pub. Co. Amsterdam, 1963. Van Buren, J.P., Moyer, J.C. and Robinson W.B. Food Technol (1964) 18: 1204. Van Buren, J.P., Scheiner, D.M. and Wagenknecht, A . C . Nature (1960) 185: 165. Hoover, M.W. Food Technol. (1967) 21: 322. Kongisbacher, K.S. "Enzymes Use and Control in Foods" Institute of Food Technologist Short Course pG-1 Chicago, 1976. Puangnak, W., Wiley, R.C. and K a h l i l , T. 1976 Private Communication 12p. Speiser, R . , Eddy, C.R. and Hills, C.H. J.Phys, Chem. (1945) 49: 563. Tavakoli, M. and Wiley, R.C. Proc. Amer. Soc. Hort. S c i . 1968) 92: 780.


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13. 14. 15.


16.

17. 18. 19.

20. 21. 22. 23. 24. 25. 26. 27. 28.

29. 30.

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

PROCESSING

Rombouts, F.M. "Occurrence and Properties of Bacterial Pectate Lyases" Ph.D. Dissertation Wageningen Netherlands p. 132 1972. Keegstra, K., Talmadge, K.W., Bauer, W.D. and Albersheim, P. Plant Physiol (1973) 51: 188. Mannheim, C.H. and Siv, S. Fruchtsaft Industrie (1969) 14: 98. Schultz, T . H . , Lotzkar, H . , Owens, H.S. and Maclay, W.D. J. Phys. Chem. (1954) 49: 554. Lee, Y.S. "Measurement, Characterization and Evaluation of Pectinesterase in Apple Fruits" Ph.D. Dissertation University of Maryland, College Park 105p. 1969. Loconti, J.D. and Kertesz, Z . I . Food Res (1941) 6: 449. Archer, R.W. Canner and Packer (1962) 131: 28. C o l l i n s , J.S. and Wiley, R.C. Maryland Agric. Exp. State. Bul. (1963) A-130 Influence of added calcium salts on texture of thermal-processed apple slices 62p. Van Buren, J.P., Moyer, J.C. and Robinson, W.B. J. Food S c i . (1962) 27: 291. C o l l i n s , J.L. and Wiley, R.C. (1967) J. Food S c i . 32: 185. Van Buren, J.P. Food Technol. (1968) 22: 132. Lee, Y.S. and Wiley, R.C. J. Amer. Soc. Hort. S c i . (1970) 95: 465. McColloch, R . J . and Kertesz, Z . I . Food Technol (1949) 3: 94. Joslyn, M.A. and Sedky, A. Food Res (1940) 5: 223. Chang, L.W.S., Morita, L . L . and Yamamoto, H.Y. J. Food S c i . (1965) 30: 218. Wiley, R.C. and Lee, Y.S. Food Technol (1970) 24: 126. Collins, J.L. Effect of Calcium Ion-Structural Polysaccahride Interaction on Texture of Processed Apple Slices. Ph.D. Dissertation, University of Maryland, College Park 99p 1965. Wagner, J.R. and Miers, J.C. Food Technol (1967) 21: 920. Anon "Code of Federal Regulations" Food and Drugs Parts 10-129 (1974) Part 53:20 General Services Admin., Washington, D.C.


Uses of Endogenous Enzymes in Fruit and Vegetable Processing

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