Enzymes Affecting Flavor and Appearance of Citrus Products - ACS

1 Enzymes Affecting Flavor and Appearance of Citrus Products J. H . BRUEMMER, R. A. BAKER, and B. ROE

Downloaded by 117.255.251.27 on April 16, 2016 | http://pubs.acs.org Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0047.ch001

U. S. Citrus and Subtropical Products Laboratory, Winter Haven, Fla.

Enzymes that affect the quality of citrus products can be classified as: 1) enzymes in the intact f r u i t , stimulated by handling conditions, 2) enzymes in juice, stimulated by substrates, and 3) commercial enzymes used in processing. Intact Fruit Before citrus fruit are processed for juice they are subjected to handling conditions that adversely affect product quality. Although some are hand picked and bagged, others are harvested after they are shaken from the tree. As the fruit f a l l they may bounce off limbs and branches. They may also bounce when dumped into tractor t r a i l e r s for transporting to the processing plant. In t r a i l e r s the fruit at each layer are subject to pressure from weight of fruit above. Physical stresses increase fruit respiration. Vines et a l . (1) reported that oranges dropped 48 i n . onto a smooth surface or compressed with 29 lb pressure for 30 sec respired 100 and 70% more CO than controls. The dropped or squeezed fruit did not return to normal respiration within 7 days. Flavor of juice prepared from oranges dropped 36 i n . to a hard surface and stored overnight at 4°C was readily distinguished by a trained taste panel as different from juice flavor of fruit not dropped (2). Fruit respiration is also stimulated by abscission chemicals applied to the tree to loosen fruit for harvest. The chemicals promote ethylene formation in citrus fruit and ethylene stimulates respiration. Flavor of juice prepared from oranges sprayed with various abscission agents was readily distinguished by a trained taste panel as different from juice flavor of unsprayed fruit (3). The heat and CO generated during respiration are not readily dissipated from the fruit in tractor t r a i l e r s , which often rest for several days i n processing plant yards before they are unloaded. One consequence of these handling conditions is accummulation of fermentation products in the fruit from anaerobic metabolism. 2

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1 Ory and St. Angelo; Enzymes in Food and Beverage Processing ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

ENZYMES IN FOOD AND BEVERAGE PROCESSING

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Alcohol:NAD Oxidoreductase E.C.I.1.1.1. During anaerobic metabolism i n c i t r u s , the alcohol:NAD oxidoreductase r e a c t i o n (AOR) r e p l a c e s the ( ^ - r e s p i r a t o r y chain that r e o x i d i z e s reduced NAD, which f u n c t i o n s i n o x i d a t i o n r e a c t i o n s of carbohydrate, f a t , and p r o t e i n metabolism. The major s u b s t r a t e f o r the enzyme i s acetaldehyde formed from the r e d u c t i v e d e c a r b o x y l a t i o n of p y r u v i c acid. J u i c e from g r a p e f r u i t h e l d a n a e r o b i c a l l y f o r 40 hr at 40°C was r e j e c t e d by a t r a i n e d panel of 11 t a s t e r s and described as "fermented" and " o v e r r i p e " (4). Biochemical changes accompanied the f l a v o r change. The a c i d i t y was 20% lower and ethanol content was 15 times higher than i n j u i c e from c o n t r o l f r u i t . In another study, the ethanol content i n j u i c e from f r u i t h e l d f o r 16 hr at 40°C i n C0 was 10 times that of j u i c e from f r u i t h e l d i n a i r (Table I ) . Downloaded by 117.255.251.27 on April 16, 2016 | http://pubs.acs.org Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0047.ch001

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Table I Biochemical Changes i n G r a p e f r u i t During Anaerobic Metabolism (5).

Atmosphere* Air C0 2

°Brix % sugar 7.18 6.67

Titratable acidity % c i t r i c acid 1.46 1.34

Malate ion mM 6.0 3.0

Ethanol mM 2.2 23.6

*15 g r a p e f r u i t i n each atmosphere. D e c l i n e s i n sugar and malate concentrations under C0 indicate that g l y c o l y s i s and malate d e c a r b o x y l a t i o n produced the pyruvate. G r a p e f r u i t has a very a c t i v e "malic enzyme" (E.C. 1.1.1.40) that c a t a l y z e s t h i s d e c a r b o x y l a t i o n (5). C i t r u s f r u i t c o n t a i n other aldehydes that are r e a c t i v e w i t h AOR. However, they do not compete on the b a s i s of c o n c e n t r a t i o n and a f f i n i t y f o r the enzyme (8)· Two of these, o c t a n a l and decanal, are important f l a v o r components of orange j u i c e and are present at 12 and 7 times t h e i r f l a v o r thresholds (6) . Even so, orange j u i c e t i s s u e contains about 50 times as much acetaldehyde as e i t h e r o c t a n a l or decanal (Table I I ) . The r e a c t i v i t i e s with AOR of v a r i o u s aldehydes prepared from orange j u i c e c e l l s r e l a t i v e to the r e a c t i v i t y of acetaldehyde are shown i n Table I I I . 2

Ory and St. Angelo; Enzymes in Food and Beverage Processing ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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Table I I F l a v o r Thresholds o f Aldehydes and T h e i r Concentrations i n Orange J u i c e . Flavor Threshold (6)

Cone. i n OJ

PPb

PPb

5 7

3000 60 50

Aldehyde


Acetaldehyde Octanal Decanal

(1)

Table I I I R e l a t i v e R e a c t i v i t i e s o f Aldehydes with C i t r u s AOR (8).

Substrate Acetaldehyde Butanal Hexanal Octanal Decanal

Cone. mM 74 37 28 21 18

Rel. Activity* 100 80 80 33 6

Rel. Affinity

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100 8 6 5 4

* S p e c i f i c a c t i v i t y o f AOR with acetaldehyde was 15 ymoles NADH oxidized/min/mg p r o t e i n . **Substrate a f f i n i t i e s c a l c u l a t e d from M i c h a e l i s constants f o r the r e a c t i o n s (8). Butanal and hexanal were almost as r e a c t i v e as acetaldehyde; o c t a n a l and decanal were decidedly l e s s r e a c t i v e . Competition of the aldehydes f o r AOR can be estimated from t h e i r r e l a t i v e a f f i n i t i e s as s u b s t r a t e s f o r the r e a c t i o n (Table I I I ) . These r e l a t i o n s h i p s i n d i c a t e that acetaldehyde would dominate the AOR r e a c t i o n , and that r e l a t i v e l y l i t t l e of the f l a v o r compounds, o c t a n a l or decanal, would be l o s t i n the f r u i t through t h i s reaction. Malate:NAD Oxidoreductase (E.C. 1.1.1.37). I n h i b i t i o n o f malate:NAD oxidoreductase (MOR) probably caused the decrease shown i n Table I o f c i t r i c a c i d during anaerobic metabolism of c i t r u s fruit. By anaerobic treatment o f c i t r u s f r u i t , the r a t i o of reduced t o o x i d i z e d NAD increased by 100% from 0.21 to 0.43 (9). MOR a c t i v i t y i n e x t r a c t s of j u i c e c e l l s was completely i n h i b i t e d by reduced NAD a t 5% o f the t o t a l NAD content (Table IV) ( 9 ) .


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ENZYMES IN FOOD AND BEVERAGE PROCESSING Table IV E f f e c t o f NADH on Malate:NAD Oxidoreductase A c t i v i t y ( 9 ) . NADH μM 0 1 5 10 25

NADH χ 100 NAD 0 0.2 1 2 5

MOR a c t i v i t y % max 100 73 65 10 0


+

Thus, during anaerobic metabolism the i n c r e a s e i n reduced NAD r e l a t i v e t o the o x i d i z e d form probably decreased the r a t e o f o x i d a t i o n o f malate t o o x a l o a c e t i c a c i d i n the o x i d a t i v e pathway f o r the s y n t h e s i s o f c i t r i c a c i d . I f c i t r i c a c i d was metabolized f a s t e r than i t was s y n t h e s i z e d , i t s net c o n c e n t r a t i o n would have been reduced. Malate would not have accumulated under these c o n d i t i o n s because the very a c t i v e "malic enzyme" decarboxylates malate t o pyruvate. Enzymes i n J u i c e C i t r u s j u i c e i s a t i s s u e homogenate and must be heated f o r the i n a c t i v a t i o n o f i t s enzymes which a r e r e l e a s e d from t h e i r normal r e s t r a i n t i n the t i s s u e . C i t r u s j u i c e t i s s u e contains peroxidase (E.C. 1.11.1.7), diphenol oxidase (E.C. 1.10.3.1), p y r u v i c decarboxylase (E.C. 4.1.1.1), carboxyesterase (E.C. 3.1.1.1), and p e c t i n e s t e r a s e (E.C. 3.1.1.11). When r e l e a s e d , these enzymes c a t a l y z e r e a c t i o n s , i n v o l v i n g j u i c e c o n s t i t u e n t s , that could a f f e c t f l a v o r and appearance. Peroxidase. When orange j u i c e was h e l d at 30°C, peroxidase a c t i v i t y d e c l i n e d r a p i d l y but s t a b i l i z e d a f t e r 1 h r t o about onet h i r d o f the o r i g i n a l a c t i v i t y (10). The amount o f a c t i v e s o l u b l e peroxidase i n commercial j u i c e s before p a s t e u r i z a t i o n depends upon the e x t r a c t o r f i n i s h e r f o r c e . Hard e x t r a c t i o n t o o b t a i n h i g h j u i c e y i e l d s i n c r e a s e d peroxidase a c t i v i t y of the j u i c e ; l i g h t e x t r a c t i o n pressure reduced the amount o f s o l u b l e peroxidase (10). J u i c e q u a l i t y was i n v e r s e l y c o r r e l a t e d with j u i c e y i e l d and peroxidase a c t i v i t y . A s c o r b i c a c i d and the p h e n o l i c a c i d s , c a f f e i c and p-coumaric a c i d s , were e f f e c t i v e donors i n the p e r o x i d a s e - ^ 0 r e a c t i o n with enzyme p r e p a r a t i o n s from orange j u i c e t i s s u e (10;. However, a s c o r b i c and c a f f e i c a c i d s were u n r e a c t i v e when added t o orange j u i c e . These compounds d i d not o x i d i z e t o a measurable extent when the j u i c e was h e l d a t 30° f o r 4 hr. C i t r u s peroxidase i s not d e t r i m e n t a l t o j u i c e f l a v o r i f j u i c e i s processed w i t h i n 4 h r o f extraction. 2



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Diphenol Oxidase. E x t r a c t s o f orange j u i c e c e l l s o x i d i z e d oand £-diphenols and £-methoxyphenolics (11). Table V l i s t s a number o f simple d i p h e n o l i c s and f l a v o n o i d compounds that supported 0^ uptake with the enzyme preparation. A l l of the f l a v o n o i d s and many o f the simple d i p h e n o l i c s have been reported present i n c i t r u s (12). Rates f o r the o- and p - d i p h e n o l i c s were about equal, and were higher than f o r the o-methoxyphenolics (11). Rates f o r the o-methoxyphenolic compounds decreased i n order as the s u b s t i t u t e d group changed from a c i d t o amine t o α-β-unsaturated a c i d . Quercetin and h e s p e r i d i n supported the same r a t e of 0^ uptake as t h e i r aglycones d i d . Diphenol oxidase was unstable at a c i d pH (11). Only 25% o f the a c t i v i t y was recovered a f t e r treatment at pH 4 f o r 5 min. I t s poor s t a b i l i t y might e x p l a i n why enzymic browning i s not a problem i n c i t r u s processing and why a s c o r b i c a c i d , an e f f e c t i v e donor f o r the £-dihydroxyphenols, i s s t a b l e i n the presence o f the many phenolic compounds i n c i t r u s j u i c e s . Carboxyesterase. Carboxyesterase i s r e l a t i v e l y s t a b l e i n orange j u i c e . A f t e r 2 h r a t 25°C, about one-half of the a c t i v i t y of the f r e s h l y reamed j u i c e remained (13). T r i a c e t i n was the p r e f e r r e d substrate but other acetates, i n c l u d i n g e t h y l , l i n a l y l , t e r p i n y l and o c t y l acetates and e t h y l butyrate were r e a c t i v e . At the pH of j u i c e (3.5), the a c t i v i t y of a p a r t i a l l y p u r i f i e d enzyme was about 15% of i t s a c t i v i t y , which was optimum, a t pH 7; but even a t 15% e f f i c i e n c y , the enzyme hydrolyzed about 15 ppm of e t h y l b u t y r a t e i n 1 h r a t s a t u r a t i n g l e v e l of substrate. The r a t e of e s t e r h y d r o l y s i s i n j u i c e has not been measured, but i t i s probably much l e s s than the c a l c u l a t e d p o t e n t i a l r a t e . The change i n aroma o f orange j u i c e at 25°C was determined by d i f f e r e n c e t a s t e t e s t (13). A panel of t a s t e r s detected a d i f f e r e n c e i n aroma between f r e s h l y reamed j u i c e , and j u i c e h e l d f o r 2 and 5 h r a t 25°C a f t e r reaming. When f r e s h l y reamed j u i c e was t a s t e d immediately a f t e r a d d i t i o n of p u r i f i e d orange flavedo esterase or commercial esterase, no d i f f e r e n c e was detected by the t a s t e panel. But 1 and 2 h r a f t e r a d d i t i o n o f the esterase, the t a s t e panel determined that the j u i c e was d i f f e r e n t , at the 0.1% l e v e l of s i g n i f i c a n c e , from the untreated j u i c e . These t e s t s suggest that e s t e r a s e - c a t a l y z e d hydrolyses, which occur p r i m a r i l y during the f i r s t few hours i n freshly-prepared j u i c e , c o n t r i b u t e to change i n aroma. Pyruvic Decarboxylase. Acetaldehyde accumulates i n orange j u i c e from the r e d u c t i v e decarboxylation o f p y r u v i c a c i d . At 30°C, 1.4 ppm acetaldehyde was formed i n orange j u i c e i n 4 h r by the r e a c t i o n c a t a l y z e d by p y r u v i c decarboxylase (14). Pyruvic acid-dependent accumulation o f acetaldehyde i n f r e s h orange j u i c e was demonstrated by the a d d i t i o n of sodium pyruvate (Table V I ) .


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ENZYMES IN FOOD AND BEVERAGE PROCESSING Table V Substrate S p e c i f i c i t y o f Orange Diphenol Oxidase (11). yliters0 / min/ o

o-Dihydroxyl DOPA Pyrogallol* Catechol**

3-(3,4-Dihydroxyphenyl)alanine 1,2,3-Trihydroxybenzene 1,2-Dihydroxybenzene

7.0 4.6 4.0

1,4-Dihydroxybenzene

4.5

4-Hydroxy-3-Methoxyphenylacetic A c i d 4-Hydroxy-3-Methoxyphenylethylamine 4-Hydroxy-3-Methoxycinnamic A c i d N-(4-Aminobutyl)-4-hydroxy-3methoxycinnamide 3 ,5,7-Trihydroxy-4-methoxyflavonone Hesperetin-7-rutinoside

1.4 0.34 0.14


p-Dihydroxyl ρ-Hydroquinone o-Hydroxyl-methoxy Homovanillic A c i d 3-Methoxytyramine Ferulic Acid*** Feruloylputrescine*** Hesperetin Hesperidin

1

0.21 0.56 0.56

o-Dihydroxyl Catechin Chlorogenic A c i d Eriodictyol Rutin Quercetin Quercetrin

f

1

3,5,7,3 ,4 -Pentahydroxyflavan 3-(3,4-Dihydroxycinnamoyl)quinic a c i d 3 ,4 ,5,7-Tetrahydroxyflavonone Quercetin-3-rutinoside 5,7,3 ,4'-Tetrahydroxyflavonol Quercetin-3-rhamnoside f

f

1

2.7 0.81 1.1 0.56 1.3 1.3

*Assayed at pH 5^6 i n s t e a d o f pH 7. . **Catechol 1 χ 10" M w i t h ascorbate 1 χ 10" M and EDTA 1 χ 10 * M. * * * F e r u l i c a c i d or f e r u l o y l p u t r e s c i n e 3 χ 10" M, ascorbate 1 χ 10 M and EDTA 1 χ 10 M.


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P y r u v i c a c i d i s the most r e a c t i v e s u b s t r a t e f o r the enzyme, 2k e t o b u t y r i c a c i d i s only o n e - t h i r d as r e a c t i v e , and higher homologues o f 2-keto a c i d s (5-7 carbons) are l e s s than 20% as r e a c t i v e . The enzyme i s slowly d e a c t i v a t e d i n orange j u i c e a f t e r a r a p i d 80% d e a c t i v a t i o n during the f i r s t hour a f t e r reaming o f the j u i c e . Acetaldehyde i s a precursor o f a c e t o i n and d i a c e t y l so that i t s accumulation could i n c r e a s e the formation of more d i a c e t y l during storage of j u i c e (14). D i a c e t y l produces a "cardboard" o f f - f l a v o r i n c i t r u s products a t 0.25 ppm (15). Table V I Pyruvate Dependent Accumulation


Sodium pyruvate ymoles/ml 0 1 5 10 15

of Acetaldehyde

(14).

Acetaldehyde nmoles/ml 95 120 330 485 485

Orange j u i c e was incubated with sodium pyruvate f o r 2 h r a t 30°C. P e c t i n e s t e r a s e . C i t r u s p e c t i n i s a polymer o f 1,4 l i n k e d , α-D-galactopyranosyluronic a c i d u n i t s with about 65% o f the a v a i l a b l e c a r b o x y l groups methylated (degree o f e s t e r i f i c a t i o n , DE). P e c t i n i s p a r t o f the s t a b l e c o l l o i d a l system that gives c i t r u s j u i c e s the c h a r a c t e r i s t i c t u r b i d appearance. The c o l l o i d i s "broken", o r the j u i c e i s c l a r i f i e d , by p e c t i n e s t e r a s e (PE), a hydrolase that demethylates p e c t i n to p e c t i c a c i d s and methanol. Demethylation proceeds block-wise, s t a r t i n g a t a methoxyl group adjacent to a f r e e carboxyl group. When the average DE o f the p e c t i n reaches a c r i t i c a l stage, the j u i c e c l a r i f i e s . Baker (16) found the c r i t i c a l DE f o r orange j u i c e to be 28% and recorded p a r t i a l c l a r i f i c a t i o n a t 35% DE. The d e s t a b i l i z a t i o n of c i t r u s j u i c e s by PE i s prevented commercially by p a s t e u r i z a t i o n a t 92°C to i n a c t i v a t e the enzyme. Use of Enzymes i n C i t r u s Processing. Polygalacturonase (E.C. 3.2.1.15), n a r i n g i n a s e , and limonoate dehydrogenase have been shown u s e f u l i n improving the q u a l i t y and q u a n t i t y of c i t r u s products by s t a b i l i z i n g c o l l o i d , decreasing v i s c o s i t y , and reducing b i t t e r n e s s .


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ENZYMES IN FOOD AND BEVERAGE PROCESSING

Polygalacturonase. Heat i n a c t i v a t i o n o f PE i s not the only method o f s t a b i l i z i n g orange j u i c e against c l a r i f i c a t i o n . Orange j u i c e was s t a b i l i z e d by treatment with polygalacturonase (PG) (Table V I I ) . Table V I I E f f e c t of PG on T u r b i d i t y o f OJ (17).


PG Treatment ppm Klerzyme* 0 50 200 400

T u r b i d i t y (g/1 bentonite) a f t e r 4° storage 0 12d 20d 28d 40d 1.6 1.6 1.6 1.6

0.4 0.9 1.1 1.4

0.3 1.0 1.3 1.6

0.3 1.1 1.5 1.6

0.3 1.1 1.5 1.6

*Commercial pectinase. Both 200 and 400 ppm of a commercial pectinase sustained t u r b i d i t y f o r more than 40 days a t 4°C. The e f f e c t s of PG on the d i s t r i b u t i o n o f s o l u b l e and i n s o l u b l e p e c t i n s , i n s o l u b l e pectates and o l i g o g a l a c t u r o n a t e s show that 61% o f the p e c t i n i n orange j u i c e was hydrolyzed to s o l u b l e o l i g o g a l a c t u r o n a t e s i n 8 days at 4°C (Table V I I I ) . Table V I I I E f f e c t o f PG on P e c t i c Substances of Orange J u i c e (17).

P e c t i c substances Insoluble pectins Soluble p e c t i n s I n s o l u b l e pectates Total Oligogalacturonates*

Control mg/1 AGA** 556 118 185 859

PG-Treated mg/1 % of AGA** c o n t r o l 141 103 95 339 520

25 87 51 39 61

*By d i f f e r e n c e ( c o n t r o l t o t a l minus t r e a t e d total; % of control total. **AGA » anhydrogalacturonic a c i d . These data were used to e x p l a i n s t a b i l i z a t i o n o f orange j u i c e by PG. The intermediate s t a t e s o f demethylated p e c t i n are s u s c e p t i b l e t o depolymerization. Before the c r i t i c a l s t a t e of 28% DE i s reached, PG hydrolyzes the a-l,4-D-galacturonide l i n k s o f the intermediates t o short chain p e c t i n s , which are demethylated by PE t o c o l l o i d s t a b l e o l i g o g a l a c t u r o n a t e s .


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65% DE P e c t i n Short Chain Pectins


28% DE P e c t i n

pç Oligogalactp • uronates; stability E

Insoluble pectates; clarification

Depolymerization of p e c t i n by PG r e s u l t s i n r e d u c t i o n of v i s c o s i t y and pulp v o l u m e — p h y s i c a l changes that a i d p r o c e s s i n g of c i t r u s j u i c e s (18). Reduced v i s c o s i t y permits more e f f i c i e n t evaporation to h i g h s o l i d s concentrates; reduced pulp volume increases the y i e l d of f i n i s h e d j u i c e . Commercially, PG i s used to i n c r e a s e y i e l d of pulp-wash l i q u i d s from orange and g r a p e f r u i t j u i c e pulps and to f a c i l i t a t e the evaporation of these l i q u i d s to high s o l i d s concentrates; but i t i s p r e s e n t l y not used i n j u i c e p r o c e s s i n g (19). A l s o PG can be used to reduce the v i s c o s i t y of c l a r i f i e d j u i c e s f o r evaporation to syrups. C i t r u s j u i c e s are r a p i d l y c l a r i f i e d by added p o l y g a l a c t u r o n i c a c i d or p e c t i n s with low DE (20). Orange j u i c e c l a r i f i e d with PG was concentrated to a c l e a r , 90°Brix syrup. T h i s syrup i s p r e s e n t l y being t e s t e d as a p r e s e r v a t i v e f o r the formulation of a low pulp, orange j u i c e concentrate that can be s t o r e d at room temperature (21). Naringinase. N a r i n g i n , the 7-rhamnoside-g-glucoside of 4 ,5,7-trihydroxyflavonone i s a b i t t e r component of g r a p e f r u i t . Most of the compound i s found i n the albedo and core of the f r u i t (2 to 4% by weight of wet t i s s u e ) ; but at times, the content i n j u i c e i s as high as 0.1%. Naringinase hydrolyzes n a r i n g i n to prunin (naringenin-7,β-glucoside) and rhamnose, reducing the bitterness. The enzyme was used to d e b i t t e r g r a p e f r u i t j u i c e (22), grape f r u i t concentrate (23), and g r a p e f r u i t pulp (24). At present, a l i m i t e d amount of g r a p e f r u i t concentrate i s d e b i t t e r i z e d commercially. Use of n a r i n g i n a s e to d e b i t t e r i z e albedo and core of f r e s h i n t a c t g r a p e f r u i t i s under development at the C i t r u s and S u b t r o p i c a l Products Laboratory i n Winter Haven, F l o r i d a (25). A n a r i n g i n a s e s o l u t i o n , f r e e from PG and c e l l u l a s e , i s vacuum i n f u s e d i n t o flavedo-shaved i n t a c t g r a p e f r u i t . A f t e r 1 hr at 50°C, the albedo i s l e s s b i t t e r . Solutions containing n a r i n g i n a s e , p r o t e i n concentrates, vitamins, m i n e r a l s , f l a v o r , and c o l o r i n g compounds, sweeteners, g e l l i n g compounds, and b a c t e r i o s t a t s have been i n f u s e d i n t o seedless g r a p e f r u i t i n t h i s manner. The product i s f l a v o r f u l with a pleasant c o l o r f u l appearance, and, when eaten e n t i r e l y , (albedo and core as w e l l as the j u i c e s e c t i o n s ) would supply food f i b e r as w e l l as n u t r i e n t s . 1


ENZYMES

10

I N FOOD A N D B E V E R A G E PROCESSING

Limonoate Dehydrogenase. Limonin b i t t e r n e s s i s recognized as a q u a l i t y problem i n g r a p e f r u i t and n a v e l orange j u i c e s . This t r i t e r p e n o i d d i l a c t o n e i s formed i n the j u i c e from the n o n - b i t t e r limonoate Α-ring l a c t o n e , which occurs n a t u r a l l y i n f r u i t t i s s u e s . Limonoate dehydrogenase (LD) o x i d i z e s the hydroxyl group on carbon 17 of limonoate Α-ring lactone to the 17-dehydro compound, which cannot l a c t o n i z e t o the b i t t e r D - r i n g lactone i n a c i d i c media + Tljie enzyme i s an NAD(P) oxidoreductase which i s a c t i v a t e d by NAD(P) . I t was i s o l a t e d from c u l t u r e s o f Pseudomonas grown on media c o n t a i n i n g limonoate as s o l e carbon source (27). Although a c t i v i t y was optimum at pH 8, LD decreased the limonin content o f n a v e l orange j u i c e (pH 3.5 t o 4) t o an acceptable l e v e l (28) i n 1 h r . Commercial use o f LD must await f u r t h e r i n v e s t i g a t i o n s on growth o f the organism and i t s production. Downloaded by 117.255.251.27 on April 16, 2016 | http://pubs.acs.org Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0047.ch001

(26).

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

Vines, Μ. Η . , Edwards, G. J. and Grierson, W. Proc. F l a . State Hortic. Soc. (1965) 78:198-202. Bryan, W. L., personal communication. Moshonas, M. G., Shaw, P. E . and Sims, D. A. J. Food Sci. (1976) 41:809-811. Bruemmer, J. Η . , and Roe, B. Proc. F l a . State Hortic. Soc. (1969) 82:212-215. Bruemmer, J. H. and Roe, B. Proc. F l a . State Hortic. Soc. (1970) 83:290-294. Lea, C. H. and Swoboda, P. A. Chem. & Ind. (1958) 1289-1290. Kirchner, J. G. and M i l l e r , J. M. J. Agric. Food Chem. (1957) 5:283-291. Bruemmer, J. H. and Roe, B. J. Agric. Food Chem. (1971) 19:266-268. Bruemmer, J. H. and Roe, B. Phytochem. (1971) 10:255-259. Bruemmer, J. Η . , Roe, B . , Bowen, E . R. and Buslig, B. J. Food S c i . (1976) 41:186-189. Bruemmer, J. H. and Roe, B. J. Food S c i . (1970) 35:116-119. Horowitz, R. M. in "The Orange" (334) Univ. of C a l i f . , Berkeley, 1968. Bruemmer, J. H. and Roe, B. Proc. F l a . State Hortic. Soc. (1975) 88:300-303. Roe, B. and Bruemmer, J. H. J. Agric. Food Chem. (1974) 22:285-288. Beisel, C. G., Dean, R. W., Kichel, R. L., Rowell, Κ. Μ., Nagel, C. W., Vaughn, R. H. J. Food Res. (1954) 19:633-643. Baker, R. A. Proc. F l a . State Hortic. Soc. (1976) 89:0-0. Baker, R. A. and Bruemmer, J. H. J. Agric. Food Chem. (1972) 20:1169-1173. Baker, R. A. and Bruemmer, J. H. Proc. F l a . State Hortic. Soc. (1971) 84:197-200.


1.

19. 20. 21.

22. 23. 24. 25.


26. 27. 28.

BRUEMMER

ET

AL.

Flavor and Appearance of Citrus Products

11

Braddock, R. J. and Kesterson, J. W. J. Food S c i . (1976) 41:82-85. Baker, R. Α . , J. Food S c i . (1976) 41:1198-1200. Bruemmer, J. H. Citrus Chem. & Techn. Conf. (1976) USDA Citrus & Subtropical Products Laboratory, Winter Haven, Florida. Ting, S. V. J. Agric. Food Chem. (1958) 6:546-549. Olsen, R. W. and Hill, E . C. Proc. F l a . State Hortic. Soc. (1964) 77:321-325. G r i f f i t h s , F. P. and Lime, B. J. Food Technol. (1959) 13: 430-433. Roe, B. and Bruemmer, J. H. Proc. F l a . State Hortic. Soc. (1976) (in press). Hasegawa, S., Bennett, R. D . , Maier, V. P. and King, A.D. J r . J. Agric. Food Chem. (1972) 20:1031. Hasegawa, S., Maier, V. P. and King, A. D. Jr., J. Agric Food Chem. (1974) 22:523. Brewster, L . C., Hasegawa, S. and Maier, V. P. J. Agric. Food Chem. (1976) 24:21-24.


Enzymes Affecting Flavor and Appearance of Citrus Products - ACS

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