Enzymes in Food and Beverage Processing

Enzymes in Food and Beverage Processing In Enzymes in Food and Beverage Processing; Ory, R., et al.; ACS Symposium Seri...

Author: Robert L. Ory | Allen J. St. Angelo

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Enzymes in Food and Beverage Processing

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

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

Enzymes in Food and Beverage Processing Robert L . Ory, EDITOR Southern Regional Research Center

Allen J . St. Angelo, EDITOR Southern Regional Research Center

A symposium sponsored by the Division of Agricultural and Food Chemistry at the 172nd Meeting of the American Chemical Society, San Francisco, Calif., Aug. 30-31, 1976

ACS SYMPOSIUM SERIES 47

AMERICAN

CHEMICAL

SOCIETY

WASHINGTON, D. C. 1977

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

Library of CongressCIPData Enzymes in food and beverage processing (ACS symposium series; 47 ISSN 0097-6156) Includes bibliographical references and index. 1. Food industry and trade—Congresses. 2. Enzymes— Industrial applications—Congresses. I. Ory, Robert L . , 1925II. St. Angelo, Allen J., 1932. III. American Chemical Society. Division of Agricultural and Food Chemistry. IV. Series: American Chemical Society. ACS symposium series; 47. TP368.E59 ISBN 0-8412-0375-X

664

77-6645

Copyright © 1977 American Chemical Society All Rights Reserved. N o part of this book may be reproduced or transmitted in any form or by any means—graphic, electronic, including photocopying, recording, taping, or information storage and retrieval systems—without written permission from the American Chemical Society. P R I N T E D I NTHEU N I T E D S T A T E S OF A M E R I C A

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

ACS Symposium Series Robert F. Gould, Editor

Advisory Board

Jeremiah P. Freeman E. Desmond Goddard Robert A. Hofstader John L. Margrave Nina I. McClelland John B. Pfeiffer Joseph V. Rodricks Alan C. Sartorelli Raymond B. Seymour Roy L. Whistler Aaron Wold

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

FOREWORD The ACS SYMPOSIU a medium for publishing symposia quickly in book form. The format of the SERIES parallels that of the continuing ADVANCES IN CHEMISTRY SERIES except that in order to save time the papers are not typeset but are reproduced as they are submitted by the authors in camera-ready form. As a further means of saving time, the papers are not edited or reviewed except by the symposium chairman, who becomes editor of the book. Papers published in the ACS SYMPOSIUM SERIES are original contributions not published elsewhere in whole or major part and include reports of research as well as reviews since symposia may embrace both types of presentation.

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

PREFACE TVTany desirable, or undesirable, changes inflavorsand properties of fruits, vegetables, oilseeds, cereals, and uncooked animal products are catalyzed by one or more enzymes. Whether activated intentionally or not, these enzymes affect the ultimate quality of the food or beverage in which they are present. From the dawn of history man has used enzyme systems (albeit unknowingly) for food preservation, for fermentation, and for breadmaking. However, great advances made in enzyme chemistry over the past fe specific enzymes to achieve a desired end product without the guesswork inherent in the older systems. Today winemaking, cheese processing, and breadmaking are no longer just an art perpetuated in unknown microbial cultures, but they are now under strict scientific control. In addition to these older applications of enzyme systems, we now use proteases for meat tenderization and for stabilization of chill haze in brewing, two processes so well established that they are no longer considered unusual applications of enzymes. Even the development of the unique flavor of milk chocolate is believed to have been caused by a lipase that was in the milk added during processing. Today it is hard to recall a time when we did not have milk chocolate. The number and types of foods and beverages we consume today are continually changing; foods are becoming more refined. They are being processed by heat or by freezing as frozen-raw or precooked convenience food items designed for long storage and easy preparation at home. The ultimate quality of these products will often depend upon the quality of the material prior to processing or on certain processing steps during their preparation. Food scientists are playing an important role in the expanding food industry by improving the flavor, texture, organoleptic, and nutritional quality of virtually all types of beverages, fruits, vegetables, animal and plant protein foods, and even animal feeds. Improvements can be achieved by the use of chemicals, by processing conditions, or by applications of selected enzymes that can directly or indirectly affect the quality of thefinalproduct. The title and subjects for this symposium were selected in early 1974 when we were asked to organize it under the sponsorship of the Division of Agricultural and Food Chemistry. The participants, experts in their respectivefields,were chosen to provide a well-balanced program ix In Enzymes in Food and Beverage Processing; Ory, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

covering the major beverages, plus fruits, vegetables, oilseeds, and both animal and plant protein foods. Noticeably absent are papers reporting enzyme applications in winemaking, meat tenderization, chill-haze re moval in brewing, and traditional cheese manufacturing. We felt that these areas have been thoroughly reviewed in other publications and symposia ("Food Related Enzymes," J . R. Whitaker, Ed., Advances in Chemistry Series (1974) 136; 170th ACS National Meeting, Chicago, symposium on "Microbial and Enzymatic Modification of Proteins"). Because of the growing number of enzymes being utilized in other areas of food and beverage processing, we tried to assemble a balanced pro gram to highlight some of these newer applications. The topics include applications of enzymes related to flavor and quality changes in products used in preparing beverages, enzymes that affect flavor and texture of fresh fruits and vegetables considered as sources of protein for supplementation of traditional foods), plus some reports of current research on potential future applications of enzymes in immobilized systems, as biological indicators, and in con version of abattoir wastes—a pollution problem—into food or feed-grade products. This book, therefore, is a development of the symposium presented at the August, 1976, ACS Meeting in San Francisco. It contains new information on the roles and utilization of endogenous and exogenous enzymes derived from plant, animal, and microbial sources for improving the quality of certain foods. We hope it is useful for food and beverage processors, food chemists, and others engaged in these or related areas of research. We have not reached the limits of our ability to use enzymes to produce new and better quality foods and beverages. Our objective in this book is to provide information that will stimulate others engaged in research on food enzymes to come closer to those limits. New Orleans, La. November 1976

ROBERT

L.

ORY

ALLEN J . ST. ANGELO

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

1 Enzymes Affecting Flavor and Appearance of Citrus Products J. H . BRUEMMER, R. A. BAKER, and B. ROE U. S. Citrus and Subtropical Products Laboratory, Winter Haven, Fla.

Enzymes that affec 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

2

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

ENZYMES IN FOOD AND BEVERAGE PROCESSING

2

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 ) . 9

Biochemical Change 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

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

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3

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 AO

Substrate Acetaldehyde Butanal Hexanal Octanal Decanal

Cone. mM 74 37 28 21 18

Rel. Activity* 100 80 80 33 6

Rel. Affinity

1

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 ) .

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

4

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 been reduced. Malate woul 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

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

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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 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 ) .

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

6

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.

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

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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

5 10 15

of Acetaldehyde

(14).

Acetaldehyde nmoles/ml

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 .

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

8

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

*Commercia 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 .

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

1.

BRUEMMER

E T AL.

Vlavor and Appearance of Citrus Products

9

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 high s o l i d s concentrates 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

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

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 (26).

+ 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.

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.

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

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) Brewster, L . C., Hasegawa Food Chem. (1976) 24:21-24.

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

2 Use of Enzymes in the Manufacture of Black Tea and Instant Tea GARY W. SANDERSON and PHILIP COGGON Thomas J. Lipton, Inc., 800 Sylvan Ave., Englewood Cliffs, N.J. 07632

Black tea is manufacture tips of the tea plant (Camellia sinensis, (L), O. Kuntze). These shoot tips are collectively known as the tea flush, and they are comprised of immature plant parts, namely, the terminal bud, the f i r s t 2 or 3 leaves, and the included stem piece. They also contain both the enzymes and the substrates that will react during the black tea manufacturing process. The enzymically catalyzed reactions involving tea flush constituents are essential to the preparation of black tea: These reactions are described further in the following discussion. The black tea manufacturing process i t s e l f begins as soon as the tea flush is plucked (harvested), and it is carried out on, or very near, the plantation where the tea flush is grown. The entire process, from plucking to finished black tea product, requires about 8 to 24 hours. The process is outlined in Table 1, and more details can be found in reviews by Harler ( 1 ) , Eden (2), Hainsworth (3), and Sanderson ( 4 ) . The enzymes known to be involved in black tea manufacture are listed in Table 2 (part A) and they are discussed in the text below. Instant tea may be prepared from either black tea or directly from fresh green tea flush. Processes for the manufacture of instant tea have been summarized by Pintauro ( 5 ) , and by Sanderson ( 6 ) . A few enzymic processes for use in manufacturing i n stant tea have been described in the patent literature. The enzymes involved are listed in Table 2 (part B) and their role in instant tea manufacture is discussed in the following text. Enzymes Involved in Black Tea Manufacture (Table 2 , Part A) Tea Catechol Oxidase. This enzyme is certainly the most important single enzyme in black tea manufacture. As soon as the withered tea flush is macerated (Table 1 ) , an enzymic oxidation of the tea flavanols (I-IV) is i n i t i a t e d . Tea catechol oxidase is the catalyst for this oxidation and the process is called "tea fermentation".

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

2.

SANDERSON A N D COGGON

Table 1 :

Manufacture of Black Tea and Instant Tea

13

O u t l i n e of Black Tea Manufacturing Process.

Steps of Process

Comments

1.

Growth of tea leaves

Tea plants (Camellia s i n e n s i s , [ L . ] 0. Kuntze) are c u l t i v a t e d to encourage the rapid growth of shoot t i p s c o l l e c t i v e l y c a l l e d the f l u s h .

2.

Harvest of tea leaves ( i . e . plucking)

The f l u s h i s picked once every 2 to 4 weeks during the growing season.

3.

Withering

The plucked f l u s h i s caused p a r t i a l l y to d e s s i c a t e from about 80% moisture down to about 65% moisture in 4 to 18 hours

4.

Macerating to cause the endogenous enzymes and tea f l a v a n o l s to come i n t o c o n t a c t .

5.

Fermenting

The macerated tea f l u s h i s allowed to stand f o r 1 to 4 hours while i t oxidizes ( F i g . 1).

6.

Firing

The fermented tea f l u s h i s d r i e d to about 2% moisture in about 20 minutes using hot a i r a t 70° to 95°C.

7.

Sorting

The f i r e d black tea i s s i f t e d i n t o grades according to s i z e of tea particles.

8.

Storing/Shipping

F i r e d black tea may be consumed immediately but i t u s u a l l y takes 2 to 4 months f o r i t to t r a v e l from point of o r i g i n to point of consumption.

9.

Conversion to i n s t a n t tea

Most i n s t a n t tea i s prepared from black tea i n the country i n which i t w i l l be s o l d .

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

14

ENZYMES

Table 2:

IN FOOD AND BEVERAGE

Enzymes with Known Functions in Tea Manufacture.

Name of Enzyme A.

PROCESSING

Function

BLACK TEA MANUFACTURE (These enzymes are endogenous to f r e s h green tea leaves)

1.

Catechol

Oxidase

2.

Peroxidase

3.

Tea M e t a l l o p r o t e i n

Oxidation of tea f l a v a n o l s r e s u l t i n g i n formation o f tea pigments ( F i g . 1) and i n d i r e c t l y i n formation of black tea aroma ( F i g . 2 ) . Removes any peroxide formed during tea fermentation ( F i g . . 1 ) . Can cause oxidat i o n of tea f l a v a n o l s i f hydrogen peroxide i s added

a c i d s , leading to formation of black tea aroma c o n s t i t u e n t s . 4.

Pectin Methyl Esterase

Demethoxylates tea pectins r e s u l t i n g in formation o f hard, glossy covering on black tea p a r t i c l e s when well made.

5.

Alcohol ase

Supposed to play a r o l e in the formation of some of the a l c o h o l s i n black tea aroma by causing reduction of the c o r responding c a r b o n y l s .

6.

Transaminase

Supposed to play a r o l e i n the b i o s y n t h e t i c transformation of amino acids to compounds t h a t are important i n black tea aroma.

7.

Peptidase

Acts to form amino acids from l e a f prot e i n s e s p e c i a l l y during w i t h e r i n g (Table 1): Amino acids are one type of black tea aroma precursor ( F i g . 2 ) .

8.

Phenylalanine Ammonia Lyase

Involved in the biosynthesis of tea flavanols.

9.

5-Dehydroshikimate Dehydrogenase

Involved i n the biosynthesis of tea flavanols.

Dehydrogen-

(Table 2, part B, continued next page)

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

SANDERSON A N D COGGON

Table 2:

Enzymes with Known Functions in Tea Manufacture,(cont.)

Name o f Enzyme B.

Manufacture of Black Tea and Instant Tea

Function

INSTANT TEA MANUFACTURE (These enzymes are from non-tea sources. They have been added to tea preparations f o r s p e c i f i c purposes). Catalyzes h y d r o l y s i s of g a l l o y l groups from tea polyphenolic compounds to produce c o l d water s o l u b l e tea s o l i d s .

1.

Tannase

2.

Polyphenol

3.

Peroxidase

Causes "tea fermentation" when H2O2 i s

4.

Pectinase

Causes breakdown o f water extracted tea pectins to reduce foam forming tendency of i n s t a n t tea powders.

5.

Cellulase

Breaks down c e l l wall material in tea l e a f to increase y i e l d o f i n s t a n t tea solids.

Oxidase

Causes "tea fermentation" (same as A J . above).

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

15

16

ENZYMES

IN FOOD AND BEVERAGE

PROCESSING

Tea catechol oxidase i s an endogenous component of tea f l u s h (7^9), and i t appears to be s o l u b l e i n the cytoplasm of i n t a c t f r e s h green tea leaves (10, 11). The enzyme does not r e a c t with the tea f l a v a n o l s ( I - I V ) , which are present i n the c e l l v a c u o l e s , u n t i l the tea l e a f t i s s u e s are d i s r u p t e d causing the c e l l contents to be mixed. The "Maceration" step o f the Black Tea Manufacturing Process (Table 1) brings about mixing of the tea f l u s h c e l l contents thereby i n i t i a t i n g "Tea Fermentation". The primary r e a c t i o n o c c u r r i n g during tea fermentation i s the o x i d a t i o n o f the tea f l a v a n o l s to t h e i r r e a c t i v e o x i d i z e d intermediates (V-VIII) which then form the black tea pigments (IX-XVI): These r e a c t i o n s are o u t l i n e d i n Figure 1. These same o x i d i z e d tea f l a v a n o l s (VVIII) a l s o act as o x i d i z i n g agents to cause the o x i d a t i v e degradation of other tea f l u s h c o n s t i t u e n t s (Figure 2 ) : Reactions of t h i s type are important in the formation of black tea aroma. The biochemistry of tea fermentation i s discussed f u r t h e r i n reviews by Sanderson (4, 12), Sanderso Tea catecFol oxidas l e a f ( 1 0 , 1 1 , 1 5 , 1 6 ) , but i t i s r e a d i l y i n s o l u b i l i z e d by i n t e r a c t i o n with the tea f l a v a n o l s (17). The r e s u l t i s t h a t tea c a t e chol oxidase i s immobilized w i t h i n the i n s o l u b l e p o r t i o n o f the tea f l u s h during the maceration step of black tea manufacture. One of the s e c r e t s of black tea manufacture i s to obtain c o n d i t i o n s that w i l l bring the tea f l a v a n o l s to the a c t i v e s i t e of the enzymes s i n c e the reverse cannot take p l a c e . In f a c t , w i t h e r i n g , the step o f black tea manufacturing j u s t preceeding maceration, i s c r i t i c a l i n t h a t i t must be c a r r i e d f a r enough to destroy the semipermeable nature of the c e l l membranes and y e t not so f a r as to render the f l u s h so dry t h a t the tea f l a v a n o l s are no longer in s o l u t i o n . F u r t h e r , maceration of the tea f l u s h must be of the kind that w i l l cause mixing of the c e l l contents without reducing the t i s s u e s to a pulp thereby preventing the f r e e entry of oxygen i n t o the "fermenting" macerated tea f l u s h . The l e v e l s of a c t i v i t y of tea catechol oxidase in tea f l u s h appear to vary during the growing season (18-20) and during the black tea manufacturing process (15, 17). "The cause of these changes i s not known but they musT~be~Tmportant s i n c e the enzyme i s e s s e n t i a l to the black tea manufacturing p r o c e s s . C r e d i t f o r e l u c i d a t i o n of the true nature of the tea c a t e chol oxidase enzyme goes to Sreerangachar (21-24). The work, and the c o n t r o v e r s y , that l e d to the i d e n t i f i c a t i o n and charact e r i z a t i o n of tea catechol oxidase i s a most important p a r t o f the h i s t o r y of tea chemistry but one should c o n s u l t Roberts ( 2 5 ) , or Sanderson ( £ ) , f o r more d e t a i l s on t h i s s u b j e c t . The enzyme i s known to contain copper (24). The enzyme has a remarkable s p e c i f i c i t y f o r the ortho-diHydroxy group on the B-ring of the tea f l a v a n o l s ( I - I V ) : Besides the tea f l a v a n o l s , catechol (XVII) and p y r o g a l l o l (XVIII) w i l l serve as substrates f o r t h i s enzyme but g a l l i c a c i d (XIX) and chlorogenic a c i d (XX) do not (26). F i n a l l y , tea catechol oxidase i s s u s c e p t i b l e to p r e c i p i t a t i o n by

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

2.

SANDERSON A N D COGGON

Manufacture of Black Tea and Instant Tea

(Structures

17

Unknown)

Figure 1. Oxidation pathway for teaflavanohduring fermentation. The tea flavanols (I: R = R' = H; II: R = H, R' = galloyl; III: R = OH, R' = H; and IV: R = OH, R' = galloyl) are enzymically oxidized to their respective intermediates (V-VIII) which undergo the following condensation reactions: (A) I or II plus III or IV —» IX—XII; (B) I or II plus gallic acid (XIX) -> XIII or XIV; (Cj I, II, III, or IV XV (by 4,8 linkages); and (D)I, II, III, or IV XVI (by unknown linkages).

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

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

(already present in

(dependent on tea fermentation and firing)

(dependent on tea fermentation only)

MANUFACTURING PROCESS

Black tea aroma constituents and precursors of black tea aroma constituents

flavanols (transitory)

Oxidized tea

Black

BLACK TEA

Black tea aroma constituents

pigments

tea

Figure 2. Proposed scheme for formation of bhck tea aroma. Materials present in fresh (or withered) tea flush are enclosed in tangles. Materials present in dashed enclosures are largely transitory in that they are undergoing change during the black tea man facturing process. Materials present in brackets are those present in the finished black tea product. The amount of any of the ab materials present in the bhck tea produced would be dependent on the conditions of manufacture.

FRESH TEA LEAF

constituents

Volatile aroma

Other precursors

Aroma precursors Amino acids Carotenes Lipids

Tea Flavanols

oxidase

Tea Catechol

2.

SANDERSON A N D COGGON

Manufacture of Bhck Tea and Instant Tea

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the tea f l a v a n o l s ( 2 7 ) , but the a c t i v i t y of t h i s enzyme only decreases slowly when"~the enzyme i s exposed to tea f l a v a n o l s . It i s c l e a r that the tea catechol oxidase enzyme i s well s u i t e d to c a t a l y z e the reactions of "tea f e r m e n t a t i o n . "

XVII

OH

OH

For a d d i t i o n a l information on work done on the e x t r a c t i o n , p u r i f i c a t i o n and p a r t i a l c h a r a c t e r i z a t i o n of tea catechol oxidase, see papers by Takeo (28), Takeo and U r i t a n i (29) and Gregory and Bendall (30). The work that e s t a b l i s h e s the r o l e of tea catechol oxidase as the c a t a l y s t f o r the o x i d a t i o n of the tea f l a v a n o l s during tea fermentation has been reviewed by Roberts ( 2 5 ) , Bokuchava and Skobeleva (31), and Sanderson ( £ ) . Peroxidase, The presence of peroxidase i n tea f l u s h was e s t a b l i s h e d as e a r l y as 1938 by Roberts and Sarma (8, 32). The enzyme i s r e a d i l y extracted from tea f l u s h (26), and^Hike tea catechol oxidase, r e t a i n s i t s a c t i v i t y even a f t e r exposure to 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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tea f l a v a n o l s . The tea peroxidase enzyme has not been c h a r a c t e r ized except that i t i s reported to be composed of 6 or 7 i s o enzymes (33, 34). No r o l e has y e t been described f o r t h i s enzyme in black tea manufacture, but i t w i l l c a t a l y z e the o x i d a t i o n of tea f l a v a n o l s i f hydrogen peroxide i s present (8, 32, 35, 36). Tea M e t a l ! o p r o t e i n . L i n o l e i c and l i n o l e n i c acids a c t as black tea aroma precursors i n t h a t they are o x i d i z e d during f e r mentation to hexanal and 2 ( E ) - h e x e n a l , r e s p e c t i v e l y (37, 38). Recently, a m e t a l l o p r o t e i n present in tea leaves has Been shown (39) to be a non-enzymic f a c t o r that i s r e s p o n s i b l e f o r t h i s oxidation. L i p i d changes in the f i n i s h e d , f i r e d black tea have been i m p l i c a t e d (40) i n q u a l i t y changes t h a t occur during s t o r a g e . L i p o l y s i s has been shown to take place i n teas held under high humidity c o n d i t i o n s . On the other hand, l i p i d o x i d a t i o n was found to be most severe i n teas stored under dry c o n d i t i o n s at elevated temperatures. l i p i d o x i d a t i o n was demonstrate 15 min. showed the same amount of change in the l i p i d s during subsequent storage as unheated control samples of t e a . These r e s u l t s suggest that non-enzymic f a c t o r s , such as the m e t a l l o p r o t e i n c a t a l y z e d o x i d a t i o n of unsaturated f a t t y a c i d s , are important in determining the character of black t e a s . Pectin Methyl E s t e r a s e . The a c t i v i t y of t h i s enzyme i n tea leaves has been i m p l i c a t e d (41, 42) i n the control of the r a t e of fermentation by causing the formation of p e c t i c a c i d from methylated p e c t i n . This h y d r o l y s i s was shown to lead to reduced oxygen uptake when an anaerobic period preceeded fermentation. I t was suggested (42) that the demethylation causes some degree of gel formation anïï t h i s subsequently impedes oxygen d i f f u s i o n i n t o the macerated tea f l u s h , thereby i n h i b i t i n g tea fermentation. A l s o , well made black tea p a r t i c l e s have a dry hard p e c t i n l a y e r on t h e i r s u r f a c e : This p e c t i n coating i s supposed to prot e c t the q u a l i t y o f teas by a c t i n g as a b a r r i e r to water and oxygen, both of which promote r e a c t i o n s t h a t are involved i n loss of tea f l a v o r and the formation o f o f f - f l a v o r s . Alcohol Dehydrogenase. This tea enzyme, which has been studied e x t e n s i v e l y by Hatanaka and his co-workers (43-48), may be determining the amount of the l e a f a l c o h o l s 3(E)-hexenol, and 2(Z)-hexenol, and the corresponding a l d e h y d e s , t h a t are present in f i n i s h e d tea products. These v o l a t i l e compounds are important black tea aroma c o n s t i t u e n t s (1^2, 4 9 ) , which appear to enhance the f l a v o r q u a l i t y at c e r t a i n leveTT (50) but which are a s s o c i a ted with teas of poor q u a l i t y when present at high l e v e l s (51_,52). There appear to be two types of alcohol dehydrogenase i n tea f l u s h (46). The major one requires the co-enzyme n i c o t i n amide adenine dintacleotide (NAD) and the minor one needs n i c o t i n amide adenine d i n u c l e o t i d e phosphate (NADP). This means that 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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l e v e l of these coenzymes, and t h e i r o x i d a t i o n s t a t e during tea fermentation, should be another f a c t o r that determines the l e v e l of l e a f a l c o h o l s , and t h e i r corresponding aldehydes, i n black tea. Transaminase. Wickremasinghe e t a l . (53, 54) have i d e n t i f i e d transaminase a c t i v i t y in macerated tea f l u s h . Wickremasinghe (54) suggests that t h i s enzyme i s involved i n the b i o s y n t h e s i s of terpenoid compounds that are important elements i n the aroma of good q u a l i t y black t e a s . Many f a c t o r s other than transaminase, some of which are a f f e c t e d by weather, are a l s o involved i n the b i o s y n t h e t i c systems that were proposed (54). Peptidase. There i s an increase i n the f r e e amino a c i d content of tea leaves during the withering period (about 18 h r . ) which follows p l u c k i n g A peptidase enzyme has been shown to be present in tea f l u s h (55) in black tea manufactur l e v e l of f r e e amino acids i n tea f l u s h , and f r e e amino acids play a r o l e i n aroma formation ( F i g . 2) (12, 13). Phenylalanine Ammonia Lyase. Tea leaves have been shown (56) to contain phenylalanine ammonia l y a s e . This a c t i v i t y i s highest in the young leaves and decreases as the leaves grow larger. A p o s i t i v e c o r r e l a t i o n was observed between the a c t i v i t y of t h i s enzyme and catechin content as might be expected from an enzyme that i s known to be involved in f l a v a n o l b i o s y n t h e s i s . 5-Dehydroshikimate Reductase. This enzyme has been extracted from tea leaves i n an a c t i v e s o l u b l e s t a t e (57). The a c t i v i t y of t h i s enzyme was p a r t i c u l a r l y high i n tea f l u s h . This i s as would be expected of an enzyme which i s known to be involved in f l a v a nol b i o s y n t h e s i s and which i s endogenous in p l a n t material cont a i n i n g an e x t r a o r d i n a r i l y high l e v e l of polyphenolic compounds. Enzymes Involved In Instant Tea Manufacture (Table 2, Part B ) . Tannase. Cold water s o l u b i l i t y i s an important c h a r a c t e r i s t i c of i n s t a n t tea products s i n c e most i n s t a n t tea i s used to prepare iced t e a . The presence of "tea cream", a c o l d water i n s o l u b l e p r e c i p i t a t e that forms n a t u r a l l y i n brewed tea beverages when they are allowed to stand f o r a few hours at below about 40°C, i s t h e r e f o r e a major problem i n i n s t a n t tea manufacture (16). I t i s known (58-61) that tea cream i s a hydrogen bonded complex formed between the polyphenolic substances of black tea (IX-XVI) and c a f f e i n e (XXI). I t has been found (62, 63) that treatment of tea s o l i d s with the enzyme tannase causes most of the tea cream s o l i d s to become s o l u b l e i n cold water. Tannase i s a fungal enzyme obtained i n g r e a t e s t y i e l d from A s p e r g i l l u s sp. (64-66), and i t i s s p e c i f i c f o r the h y d r o l y s i s

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o f e s t e r s of g a l l i c a c i d (XIX) ( £ 6 - 6 8 ) . The l a r g e s t part of the tea f l a v a n o l s (I-IV) i s e s t e r i f i e d with g a l l i c a c i d ( i . e . II and IV) and these g a l l o y l f l a v a n o l s are transformed i n t o g a l l o y l o x i d a t i o n products (IX-XVI where R=3,4,5-trihydroxybenzoyl) during the tea fermentation process (see Figure 1 ) . Further, i t is known that the preponderance of polyphenolic substances present i n tea cream are e s t e r i f i e d with g a l l i c a c i d (59).

Some work has been done to prepare immobilized tannase (69, 70). These s t u d i e s i n d i c a t e that tannase can be s u c c e s s f u l l y immobilized using v i r t u a l l y a l l of the usual methods and support m a t e r i a l s a v a i l a b l e today. A process f o r the preparation o f cold water s o l u b l e i n s t a n t tea d i r e c t l y from f r e s h green tea f l u s h , which uses tannase in a "pre-conversion" treatment, has been described by Sanderson and Coggon ( 7 j ) . Polyphenol Oxidases. It was pointed out i n the s e c t i o n on the r o l e of tea catechol oxidase i n black tea manufacture t h a t the enzyme was c e n t r a l to the process by which green tea s o l i d s are converted to black tea s o l i d s . S e l t z e r , e t ^ a U (72) were the f i r s t to describe a process f o r the conversion of solTcTs e x t r a c t e d from green tea to black i n s t a n t tea s o l i d s u t i l i z i n g the catechol oxidase enzyme from other tea l e a v e s . In t h i s process green tea e x t r a c t i s obtained from any green tea leaves and macerated f r e s h green tea leaves c o n t a i n i n g a c t i v e tea catechol oxidase i s added to c a t a l y z e the c o n v e r s i o n . Oxygenation of aqueous tea l e a f homogenates has been des c r i b e d (73,74) as a means of preparing i n s t a n t tea products d i r e c t l y from f r e s h tea f l u s h . This kind of process makes use of the catechol oxidase endogenous to the tea f l u s h to e f f e c t the tea fermentation p r o c e s s . Polyphenol oxidases e x t r a c t e d from a number of d i f f e r e n t p l a n t m a t e r i a l s (ex. potato p e e l i n g s ; c a l l u s t i s s u e s of beans, sycamore, and apple p l a n t s ; and c e r t a i n molds) have been shown (75) to be capable of c a t a l y z i n g tea fermentation i n aqueous e x t r a c t s of green tea f l u s h . U n f o r t u n a t e l y , the cost o f these polyphenol oxidases i s probably p r o h i b i t i v e and t h e i r s t a b i l i t y is questionable.

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

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Peroxidase. A process t o prepare i n s t a n t tea d i r e c t l y from f r e s h tea f l u s h has been described (35,36) which u t i l i z e s the peroxidase enzyme that i s endogenous to tea f l u s h to c a t a l y z e the tea conversion p r o c e s s . These processes depend on adding hydrogen peroxide (F^Op) t o s t i r r e d homogenates o f f r e s h tea f l u s h . Of course, the added hydrogen peroxide acts as the o x i d i z i n g agent i n the tea fermentation process and the tea peroxidase serves as an e s s e n t i a l c a t a l y s t f o r the o x i d a t i o n o f the tea flavanols (I-IV). Pectinase. This enzyme has been described as being useful f o r improving the foam forming tendency o f i n s t a n t t e a powders (76). I t i s presumed that pectinase treatment o f t e a e x t r a c t s destroys some o f the natural pectins present which lowers the a b i l i t y o f the s o l i d s present t o form s t a b l e f i l m s . Cellulases. Funga c l a s s i f i c a t i o n have bee s o l i d s which are normally bound t o the tea leaves

(77,78).

Summary The manufacture o f black tea i s e s s e n t i a l l y the mechanical manipulation o f f r e s h green tea f l u s h to cause a conversion o f the green tea s o l i d s t o black tea s o l i d s : This conversion i s c a t a l y z e d by enzymes that are endogenous to the tea f l u s h (Table 2, Part A ) . The enzymology and biochemistry o f the o x i dation o f the tea f l a v a n o l s to black tea pigments (Figure 1) appears to be understood a t l e a s t i n o u t l i n e . On the other hand, the enzymology and the chemistry o f the formation o f black tea f l a v o r , i . e . t a s t e and aroma (Figure 2 ) , i s only beginning to be understood. The d i f f i c u l t y o f e x t r a c t i n g enzymes from t i s s u e s r i c h i n polyphenolic compounds (10, 79-81) such as tea f l u s h has undoubtedly been an o b s t a c l e to more rapid development o f knowledge i n t h i s f i e l d . The manufacture o f i n s t a n t tea o f f e r s many i n t e r e s t i n g p o s s i b i l i t i e s f o r the use o f enzymes (Table 2, Part B ) . It w i l l be i n t e r e s t i n g to watch f u t u r e developments i n t h i s f i e l d . Literature Cited 1. 2. 3.

4.

Harler, C.R. (1963), "Tea Manufacture", Oxford Univ. Press, London, England, 126 pp. Eden, T. (1965), "Tea", 2nd E d . , Longmans, Green and C o . , L t d . , London, England. Hainsworth, E. (1969), "Encyclopedia of Chemical Technology", 2nd. E d . , Editor, A. Standen, pp. 743-755, Wiley (Interscience), New York. Sanderson, G.W. (1972), "Structural and Functional Aspects of Phytochemistry", Runeckles, V . C . , and Tso, T.C., E d . , Academic Press, New York, pp 247-316.

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

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5. 6. 7.

8. 9. 10. 11. 12.

13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34.

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

Pintaro, N. (1970), "Soluble Tea Processes", Park Ridge, N . J . , Noyes Data Corp. Sanderson, G.W. (1972), World Coffee & Tea, A p r i l , 1972, pp 54-57. Bamber, M.K. and Wright, H . , 1902, "Year Book of the Planters Association for 1901-1902", Planters Association, Colombo, Ceylon. Roberts, E . A . H . , and Sarma, S.N. (1938), Biochem. J. 32: 1819-1828. Sreerangachar, H.B. (1939), Current Sci., 8: 13. Sanderson, G.W. (1964), Biochim. Biophys. Acta. 92: 622-624. Buzun, G . A . , Dzhemukhadze, K.M., and Mileshko, L.F. (1970), P r i k l . Biokhim. Mikrobiol. 6: 345-347. Sanderson, G.W. (1975), "Geruch- und Geschmackstoffe", Drawert, F. (editor), Verlag Hans Carl, Nurenburg, W. Germany, pp 65-97. Sanderson, G.W., an Chem. 21: 576-585 Reymond, E. (1976) (ACS Centennial Symposium, New York City, A p r i l , 1976, to be published). Takeo, T. (1966), Agr. B i o l . Chem. 30: 931-934. Buzun, G . A . , Dzhemukhadze, K.M., and Mileshko, L.F. (1966), P r i k l . Biokhim. Mikrobiol. 2 (5): 609-11. Sanderson, G.W. (1964), J. S c i . Fd. Agric. 15: 634-639. Sanderson, G.W. (1964), Tea Quarterly 35: 101-110. Sanderson, G.W. and Kanapathipillai, P. (1964), Tea Quarterly 35: 222-229. Takeo, T., and Baker, J.E. (1973), Agr. B i o l . Chem. 12: 21-24. Sreerangachar, H.B. (1943), Biochem. J. 37: 653-655. Sreerangachar, H.B. (1943), Biochem. J. 37: 656-660. Sreerangachar, H.B. (1943), Biochem. J. 37: 661-667. Sreerangachar, H.B. (1943), Biochem. J. 37: 667-674. Roberts, E.A.H.(1962), "Chemistry of Flavanoid Compounds," Geissman, T . A . , e d . , pp 468-512, London, Pergamon Press. Coggon, P . , Moss, G . A . , and Sanderson, G.W. (1973), Phytochem. 12: 1947-1955. Sanderson, G.W. (1965), Biochem. J. 95: 24-25. Takeo, T. (1965), Agr. Biol. Chem. 29: 558. Takeo, T., and Uritani, I. (1966), Agr. B i o l . Chem. 30: 155-163. Gregory, R . P . F . , and Bendall, D.S. (1966), Biochem. J. 101: 569-581. Bokuchava, M.A., and Skobeleva, N.I. (1969), Advan. Food Res. 17: 215-280. Roberts, E.A.H. (1939), Biochem. J. 33: 836-852. Takeo, T., and Kato, Y. (1971), Plant and Cell Physiol. 12: 217-223. Tirimanna, A . S . L . (1972), J. Chromatogr. 65: 587-588.

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2

SANDERSON

35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64.

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Fairley, C.J., and Swaine, D. (1973), British Patent No. 1,318,035, published May 23, 1975. Fairley, C.J., and Swaine, D. (1975), U.S. Patent No. 3,903,306, published Sept. 2, 1975. Gonzalez, J.G., Coggon, P. and Sanderson, G.W. (1972), J. Food S c i . 37: 797-798. Saijyo, R . , and Takeo, T. (1972), Plant and Cell Physiol. 13: 991-998. Coggon, P . , Romanczyk, L.J., and Sanderson, G.W. (1976), J. Agric. Fd. Chem. (in press). Stagg, G.V. (1974), J. S c i . Fd. Agric. 25: 1015-1034. Lamb, J., and Ramaswamy, M.S. (1958), J. S c i . Fd. Agric. 9: 46-51. Lamb, J., and Ramaswamy, M.S. (1958), J. S c i . Fd. Agric. 9: 51-56. Hatanaka, A. and Harada, T. (1972), Agr. B i o l . Chem. 36: 2033-2035. Hatanaka, A. and Harada Kajiwara, T., Harada, T., and Hatanka, Α . , Agr. B i o l . Chem. 39: 243-247. Sekiya, J., Kawasaki, W., Kajiwara, T., and Hatanka, A. (1975), Agr. B i o l . Chem. 39: 1677-1678. Hatanaka, Α . , Sekiya, J., and Kajiwara, T. (1976), Phytochem. 15: 487-488. Sekiya, J., Numa, S., Kajiwara, T., and Hatanaka, A. (1976), Agr. B i o l . Chem. 40: 185-190. Yamanishi, T. (1975), Nippon Nogei Kagaku Kaishi 49: R1-R9. Tenco Brooke Bond Ltd. (1973), British Patent No. 1,306,017. Yamanishi, T., Wickremasinghe, R . L . , and Perera, K.P.W.C. (1968), Tea Quarterly 39: 81. Gianturco, M.A., Biggers, R . E . , and Ridling, B.H. (1974), J. Agric. Food S c i . 22: 758-764. Wickremasinghe, R . L . , Perera, B.P.M., and deSilva, U . L . L . (1969), Tea Quarterly 40: 26-30. Wickremasinghe, R.L. (7974), Phytochem. 13: 2057-2063. Sanderson, G.W., and Roberts, G.R. (1964), Biochem. J. 93: 419-423. Iwasa, K. (1974), Nippon Nogei Kagaku Kaishi 445. Sanderson, G.W. (1966), Biochem. J. 98: 548-252. Roberts, E.A.H. (1963), J. S c i . Food Agr. 14: 700-705. Wickremasinghe, R.L. and Perera, K.P.W.C. (1966), Tea Quarterly 37: 131-133. Smith, R.F. (1968), J. S c i . Fd. Agric. 19: 530-535. Rutter, P . , and Stainsby, G. (1975), J. S c i . Fd. Agric. 26: 455-463. Tenco Brooke Bond Ltd. (1971), British Patent No. 1,249,932. Takino, Y. (1976), U.S. Patent No. 3,959,497, issued 5/25/76. Yamada, K . , Iibuchi, S., and Mirada, Y. (1967), Hakko Kagaku Zasshi 45: 233-240.

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26

E N Z Y M E S IN F O O D A N D B E V E R A G E PROCESSING

65. I i b u c h i , S., Minoda, Y., and Yamada, K. (1968), Agr. B i o l . Chem. 32: 803-809. 66. Iibuchi, S., Minoda, Y . , and Yamada, K. (1972), Agr. Biol. Chem. 36: 1553-1562. 67. Dyckerhoff, H . , and R. Armbruster (1933), Hoppe S e y l e r ' s Zeitschrift f u r Physiologische Chemie, 219: 38-56. 68. Haslam, E . , Hawortn, R.D., Jones, Κ., and Rogers, H . J . , J. Chem. Soc. (1961), 1829-1835. 69. W e e t a l l , H.H. and Detar, C.C. (1974), B i o t e c h . Bioengineer. 16: 1095-1102. 70. Coggon, P . , Graham, H . N . , and Sanderson, G.W. (1975), British Patent No. 1,380,135. 71. Sanderson, G.W., and Coggon, P. (1974), U.S. Patent No. 3,872,266. 72. S e l t z e r , E . , Harriman, A.J., and Henderson, R.W. (1961), U.S. Patent No. 2,975,057. 73. Millin, D . J . (1970) Sept. 9, 1970. 74. Millin, D . J . (1972), U.S. Patent No. 3,649,297, published March 14, 1972. 75. F a i r l e y , C.J., and Swaine, D. (1970), A u s t r a l i a n Patent No. 21460/70 assigned to Tenco Brooke Bond L t d . 76. Sanderson, G.W., and Simpson, W.S. (1974), U.S. Patent No. 3,787,582. 77. Misawa, Y., Matubara, M., and Inzuka, T. (1968), Nippon Shokuhim Kogyo Gakkaishi 15: 306-309. 78. Toyama, N., and Owatashi, H. (1966), J. Fermentation T e c h n o l . , (Japan) 44: 830-834. 79. Loomis, W.D. (1974), "Methods in Enzymology, V o l . 31, B i o membranes", ( F l e i s c h e r , S., and Packer, L., e d i t o r s ) . Aca demic P r e s s , New York, pp. 528-544. 80. Anderson, J.W. (1968), Phytochem. 7: 1973-1988. 81. Lam, T.H., and Shaw, M. (1970), Biochem. Biophys. Res. Comm. 39: 965-968.

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

3 Coffee Enzymes and Coffee Quality HENRIQUE V. AMORIM and VERA L. AMORIM Department of Chemistry, Sector of Biochemistry, Esc. Sup. Agric, "Luiz de Queiroz," University of São Paulo, 13400 Piracicaba, Sp, Brazil

Coffee beverag coffee seed. I t is the most consumed stimulant beverage i n the world and is second i n i n t e r n a t i o n a l t r a d e . The coffee tree i s a perennial plant and grows in t r o p i c a l and s u b - t r o p i c a l r e g i o n s . The p r i c e of coffee beans (bean, i s a worldwide used name, but the correct form should be coffee seed) depends on the q u a l i t y . I t i s almost impossible to define q u a l i t y because what i s a good coffee f o r one, could be a bad coffee f o r o t h e r s . However, it is important to take i n t o c o n s i d e r a t i o n the acceptance of the product by the majority of the consumers. I t will be considered i n t h i s paper that the best coffee q u a l i t y i s the one that i s accepted as being high q u a l i t y by the majority of people involved in the coffee i n d u s t r y . Coffea a r a b i c a L. f o r example, i s considered the most f l a v o r f u l and i s a l s o the most expensive. I t accounts f o r 3/4 of the world consumption. Following t h i s comes C . Canephora, P i e r r e ex Froehner, known i n the i n t e r n a t i o n a l trade by Robusta, and accounts f o r 1/4. A small percentage comes from C . L i b e r i c a , H i e r n . Within each s p e c i e , coffee i s a l s o c l a s s i f i e d by its q u a l i t y , which includes p h y s i c a l aspects of raw bean ( c o l o r , s i z e , shape and i m p u r i t i e s ) and the f l a v o r q u a l i t y of the beverage ( a c i d i t y , body and aroma) a f t e r r o a s t i n g . The q u a l i t y of the coffee w i t h i n a given specie i s d i c t a t e d by the region where the plant i s grown, by the c u l t u r a l p r a c t i c e s and by h a r v e s t i n g , processing and storage c o n d i t i o n s . In s p i t e of the importance of c o f f e e q u a l i t y to the p r i c e of the commercial bean, little has been done on the chemical and enzymological aspects as coffee seed. Tea i s probably the most studied stimulant beverage. The f l a v o r precursors of black tea and s e v e r a l of the chemical components i n the 27 In Enzymes in Food and Beverage Processing; Ory, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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Figure 1. Cross section of a ripe coffee fruit

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f i n i s h e d i n f u s i o n are w e l l known and t h e i r e f f e c t on q u a l i t y are f a i r l y w e l l e s t a b l i s h e d (1*
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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

about 12 per cent moisture i n the whole f r u i t . The outer l a y e r s are then removed by h u l l i n g . A d e t a i l e d d e s c r i p t i o n of each processing step can be seen i n SIVETZ and FOOTE's book ( 8 ) . Figure 2 shows the main steps of the wet and n a t u r a l processes. WASHED COFFEE

NATURAL COFFEE

Harvesting Harvesting Transportation Transportation Storage Storage Sorting Sorting Pulping Drying Mucilage Removal (Storage) Washing Hulling Drying Storag (Storage) Remova Impuritie Hulling Roasting Storage Grinding Removal o f Impurities I n f u s i o n ( c o f f e e beverage) Roasting Grinding Infusion ( c o f f e e beverage) Figure 2.

Main steps of coffee-handling process

Enzyme A c t i v i t i e s i n D i f f e r e n t Coffee Species and Varieties The f i r s t report of enzyme d e t e c t i o n i n green coffee beans was made by HERNDLHOFER i n B r a z i l i n 1932 ( 9 ) . L i p a s e , protease, amylase, catalase and a "trace""" of peroxidase were the enzymes detected. Polyphenol oxidase could not be confirmed i n the d r i e d seeds. However, the f i r s t to t r y to c o r r e l a t e enzyme a c t i v i t y with the q u a l i t y of c o f f e e was WILBAUX i n 1938 (10). He was s u c e s s f u l l i n measuring the a c t i v i t y of polyphenol oxidase and peroxidase i n the d r i e d seeds using various substrates. In h i s extensive work, WILBAUX compared the a c t i v i t y of l i p a s e , c a t a l a s e , polyphenol oxidase and peroxidase i n seven d i f f e r e n t coffee species and c o r r e l a t e d them with the organoleptic p r o p e r t i e s . Although he d i d not t r y t o c o r r e l a t e these d i f f e r e n c e s i n enzymatic a c t i v i t i e s with the q u a l i t y o f the beverage (because other components such as c a f f e i n e and o i l a l s o d i f f e r e d among the s p e c i e s ) , t h i s pioneering work was not followed by other enzymatic s t u d i e s . Recently, the higher a c t i v i t y of polyphenol oxidase of Robusta

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coffee, i n r e l a t i o n to Arabica species found by WILBAUX ( 1 0 ) , was confirmed i n 1972 by OLIVEIRA (11) and VALENCIA (12). Among the d i f f e r e n t v a r i e t i e s of C. a r a b i c a i n B r a z i l , no d i f f e r e n c e was found i n the q u a l i t y of the beverage i f the coffee i s processed i n the same way. The a c t i v i t y of polyphenol oxidase i s a l s o very similar. Table I shows the a c t i v i t y of polyphenol oxidase i n four d i f f e r e n t species and three d i f f e r e n t varieties. TABLE I. R e l a t i v e a c t i v i t y of polyphenol oxidase i n coffee seeds (DOPA as substrate) of d i f f e r e n t species and v a r i e t i e s ( r e c a l c u l a t e d from OLIVEIRA 1 1 ) COFFEE υ. dewevrei De Wild et D u r a n * var. dewevrei C canephora P i e r r e ex Froehner var. Bukobensis ,C. l i b e r i c a H i e r n C. arabica Linneu var. Bourbon amarelo var. Mundo novo var. Catuai amarelo

,

0 o

'°

2.00 c 23* S g

2

10 1 5

S 0 12^2

The q u a l i t y of the beverage of three C. arabica coffees i s the same, i t i s c h a r a c t e r i s t i c of the specie and i n d i s t i n g u i s h a b l e among them. However, i t i s very d i f f e r e n t from C. canephora, C. l i b e r i c a and C. dewevrei which have t h e i r c h a r a c t e r i s t i c f l a v o r , but i s not much appreciated. Recently, PAYNE et a l . (13) u t i l i z e d malate dehydrogenase and general p r o t e i n banding patterns to d i f f e r e n t i a t e coffee species, v a r i e t i e s and c u l t i v a r s with some success. Although v a r i a t i o n s i n d i f f e r e n t p r o t e i n patterns and enzyme a c t i v i t i e s may contribute to d i f f e r e n t f l a v o r s a f t e r the beans have been roasted, coffees of d i f f e r e n t species also have d i f f e r e n t contents of c a f f e i n e , chlorogenic a c i d , t r i g o n e l l i n e , f a t s , and p o s s i b l y other components (14). For t h i s reason, i t seems reasonable to speculate that the d i f f e r e n c e i n f l a v o r s among species i s given by t h e i r d i f f e r e n t contents of several chemical components. However, the e f f e c t of enzymatic a c t i v i t i e s on f l a v o r can not be excluded.

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Coffee Enzymes on Harvesting, Transport and Storage Natural Coffees. In B r a z i l , coffee i s c l a s s i f i e d with respect t o q u a l i t y of the beverage, by the f o l l o w i n g grades: (from the best t o the poor) Soft (mild), Almost S o f t , Hard ( a s t r i n g e n t ) , S l i g h t l y Rio and Rio ( m e d i c i n a l ) . When the coffee cherry i s harvested i n the f u l l y ripened stage, the moisture content i s about 65 per cent and t h i s kind of f r u i t can produce the best beverage. Immature beans and black beans i f not separated from r i p e beans, depreciate the f i n a l l i q u o r . These d e f e c t i v e beans w i l l not be considered i n t h i s paper. In regions where a l l the f r u i t s reach maturity at almost the same time, the harvesting of only r i p e c h e r r i e s becomes impossible maybe over-ripe i ground. I f the weather i s dry, the q u a l i t y of the beverage of these beans may be f a i r to good. However, i f the climate i s humid, h y d r o l y t i c and o x i d a t i v e reactions caused by the f r u i t enzymes and microorganisms d e t e r i o r a t e the q u a l i t y of the r e s u l t i n g beverage. One experiment u t i l i z i n g f r u i t s c o l l e c t e d i n the same t r e e , but h a r v e s t i n g i n d i f f e r e n t stages of maturity, and d r i e d i n d i f f e r e n t ways showed that the q u a l i t y of the beverage and the a c t i v i t y of polyphenol oxidase d i f f e r , depending upon the process used (Table I I ) . The poorer the l i q u o r q u a l i t y , the lower was the a c t i v i t y o f polyphenol oxidase. TABLE I I . R e l a t i v e a c t i v i t y o f polyphenol oxidase of green coffee and q u a l i t y o f the beverage o f c h e r r i e s harvested and prepared i n d i f f e r e n t ways under wet climate conditions. Beverage Relative PPO a c t . Quality Treatment

%

Ripe cherry, pulped and sun d r i e d 100 Soft Over r i p e , d r i e d i n the tree S l i g h t l y Rio Sample (1) 4-6.9 Sample (2) S l i g h t l y Rio 35-1 Dried i n the ground Rio 15 days 16.9 FRANCO, TEIXEIRA, AMORIM, 1 9 7 1 (unpublished).

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Rio f l a v o r a l s o develops when the bean, i s drying i n the sun and gets wet by r a i n . A f t e r the pulp i s removed, no Rio f l a v o r develops. Working with coffee harvested i n d i f f e r e n t regions, AMORIM and SILVA (15) found a p o s i t i v e c o r r e l a t i o n between the a c t i v i t y of polyphenol oxidase (DOPA as substrate) of the green bean and q u a l i t y of the beverage of C. a r a b i c a . Several papers confirmed t h e i r r e s u l t s (11, , 1 5 » 17% 18, 19)· Polyphenol oxidase, or o-diphenol oxidase, Ts a copper enzyme (EC, 1.10.3.1·) which o x i d i z e s o-diphenols to quinones. Figure 3 shows the r e s u l t s obtained by OLIVEIRA (11) who used 7 coffee samples of each kind of beverage and compared them with the a c t i v i t y of polyphenol oxidase. The c o r r e l a t i o n was s i g n i f i c a n t at the 1 per cent l e v e l . The a c t i v i t y of peroxidase and catalase were a l s However, only peroxidas The Rio coffee had lower a c t i v i t y i n r e l a t i o n to the others. L o c a l i z a t i o n of polyphenol oxidase i n coffee seed has been unsucessful up to now. However, peroxidase was l o c a l i z e d (p-phenylenediamine + ΗρΟ~) (20) c h i e f l y i n the outer l a y e r s of the seed and i n the embryo (Figure 4 ) . The i n t e n s i t y of o x i d a t i o n of p-phenylenediamine i n Soft coffees i s much greater than i n Rio c o f f e e s . In general, Rio coffee i s l i g h t green or yellow-brown, even when i t i s of the current year's crop. The l o c a l i z a t i o n of more intense peroxidase a c t i v i t y i n the outer l a y e r s of the endosperm increases the chance of i t being associated with the d i s c o l o r a t i o n generally found i n Rio coffees. By using a l k a l i n e g e l e l e c t r o p h o r e s i s (21, 22) of crude e x t r a c t s without d i a l y s i s , several polyphenol oxidases and one peroxidase were found i n green coffee beans. However, the number of PPO bands depends on the substrate used (23) (Figure 5)· I t was not p o s s i b l e to d i f f e r e n t i a t e Soft from Rio coffees by the i n t e n s i t y of the bands, because of great v a r i a t i o n s . In B r a z i l , where most of the commercial beans are "natural c o f f e e " , these d i f f e r e n t q u a l i t i e s r e s u l t from d i f f e r e n t methods of harvesting, handling and d r y i n g . I f one harvests only ripened f r u i t or c o l l e c t s green, r i p e and over-ripe f r u i t s , but c l a s s i f i e s them before drying, the r i p e f r u i t always give a good beverage ( S o f t ) . The c l a s s i f i c a t i o n of the Hard coffee i n B r a z i l i s a puzzle. The c h a r a c t e r i s t i c of Hard coffee i s i t s astringency. However, some coffees have no astringency, no Rio f l a v o r and a l s o lack the f u l l t a s t e and aroma of 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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PROCESSING

βΟ

Γ

1

2

3

4 (bttt)

Figure 3. Activity of polyphenol oxidase in raw coffee and the quality of the beverage after roasting and infu sion. Each point represents an average of seven different samples (11).

Figure 4. Localization of peroxidase activity (bhck area in the outer layers) in green coffee seed using p-phenylenediamine + H 0 (20) as substrate. Bubbles are 0 produced by catalase activity. 2

2

2

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

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Soft c o f f e e . These coffees are then c l a s s i f i e d by the p r o f e s s i o n a l t a s t e r s as Hard, considering that they occupy an intermediate p o s i t i o n i n the q u a l i t y s c a l e (A.A.TEIXEIRA, personal communication). The l i t e r a t u r e shows that many kinds of d e f e c t i v e beans, drying temperatures and attack by fungi may give r i s e to t h i s p e c u l i a r f l a v o r (24). Although the a c t i v i t y of polyphenol oxidase seems to be c o r r e l a t e d with the q u a l i t y of B r a z i l i a n "natural" c o f f e e s , other estimates of chemical components of green coffee were made. Soluble carbohydrates, polysaccharides (25)» chlorogenic a c i d s , t o t a l soluble phenolics, hydrolyzable phenols (26), soluble proteins (27)% e l e t r o p h o r e t i c patterns of soluble proteins (24) and a m u l t i p l e l i n e a r regression a n a l y s i s of a l l data were c a l c u l a t e d (28)· The most s i g n i f i c a n had l e s s hydrolyzabl proteins (TCA p r e c i p i t a b l e ) . The d i f f e r e n t patterns of soluble proteins i n agar g e l e l e t r o p h o r e s i s were explained by the f a c t t h a t , i n the e x t r a c t i o n procedure, chlorogenic a c i d was more oxidized by polyphenol oxidase (higher i n the Soft coffees) and i t binds with proteins changing t h e i r e l e c t r i c a l charge (24, 29;· One t e n t a t i v e explanation f o r the lower a c t i v i t y of polyphenol oxidase found i n Rio coffees i s t h i s : i n any step during harvesting and processing the enzyme has a chance to come i n t o contact with the substrates. The oxidized phenolics are very r e a t i v e and bind c o v a l e n t l y with the enzyme, i n h i b i t i n g i t s a c t i v i t y (j£0). Concerning the l e s s e r amount of TCA-precipitable p r o t e i n reported e a r l i e r i n Rio coffee (2£), recent r e s u l t s confirmed t h i s f a c t and shed more l i g h t on the p r o t e i n d i f f e r e n c e s between Soft and Rio coffees (21). I t was found that, although the t o t a l soluble nitrogen i s apparently the same between these two c o f f e e s , SDS (sodium dodecyl s u l f a t e ) g e l e l e t r o p h o r e s i s of the water-soluble p r o t e i n s (treated with 2-mercaptoethanol) shows a d i f f e r e n t pattern between the p r o t e i n s extracted from 5 d i f f e r e n t samples of Rio coffee and 4 samples of Soft coffee (Figure 6 ) . SDS g e l e l e t r o p h o r e s i s separates most of the proteins according to t h e i r molecular weights (51). I f t h i s holds true f o r coffee p r o t e i n s , the gel patterns show that Soft coffee has some proteins of high molecular weight (-64,000 Daltons) that are absent i n Rio c o f f e e . On the other hand, Rio coffee shows some bands of low molecular weight that are

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

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

Figure 5. Alkaline polyacryhmide gel electropho resis (T = 7%) of seed water-soluble proteins: protein banding pattern (1); polyphenol oxidase, DOPA (2), and caffeci acid coupled with m-phenylenediamine (3) as substrates; and peroxidase, ο-dianizidine + H 0 in 10% acetic acid (4) 2

2

Figure 6. SDS polyacryhmide gel electrophoresis (T = 10%) of green coffee, water-soluble proteins (treated with 2-mercaptoethanol) of Soft and Rio samples

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absent i n the Soft c o f f e e . Furthermore, the bands of small polypeptides ( - 9 , 0 0 0 Daltons) are much more intense i n the Rio coffee than i n S o f t c o f f e e s . This suggests that the coffee beans which give Rio f l a v o r undergo h y d r o l y s i s during processing and/or m o d i f i c a t i o n of the t e r t i a r y s t r u c t u r e of t h e i r p r o t e i n s . However, at the present time one cannot be sure that these polypeptides are the cause of the Rio f l a v o r , because other h y d r o l y t i c r e a c t i o n s a l s o take place, probably at the same time: f o r example, the t o t a l extractable l i p i d s (chloroform/methanol) were lower i n Rio coffee i n r e l a t i o n to Soft coffee as shown i n Table I I I . TABLE I I I . Extractable l i p i d s (chloroform/methanol) of Arabica coffees which d i f f e r i n the quality Rio

age

(yrs)

age (.yrs) % lipids 14.84 7 3 3 3 13-51 13.32 3 3 2 2 13.45 mean 1 3 . 7-prbv 8* mean t-i • S t a t i s t i c a l s i g n i f i c a n t at 1% l e v e l .

%

lipids 16.28 14.98 15.44 15.12 5 * c* 11 C5 * ^1:

The density, thickness of c e l l w a l l and volume of c e l l walls are lower i n Rio coffee i n comparison with Soft coffee (j52), as shown i n Table IV. TABLE IV. P h y s i c a l measurements of the whole bean and the hard (outer) endosperm of Arabica coffee beans which d i f f e r i n q u a l i t y of the beverage, (the symbols i n parentheses represent the number of measurements of the c e l l wall) Volume of Wt. 500 C e l l wall Coffee c e l l wall thickness Sample Density beans (mean) .jam % (κ) 6.2 (972) Soft-1 50.7 71.03 1.085 1.014 Soft-2 5.6 (822) 49.7 57.69 37-5 Rio-1 58.21 5-0 (73D 0.967 35.6 49.24 Rio-2 0.761 4.3 (475) AMORIM, SMUCiŒR, PFIoTER ( 3 2 ) Although a l l samples were from the same v a r i e t y (C. arabica L., v a r . Mundo Novo), the the beans were d i f f e r e n t . S o f t - 1 , S o f t - 2 and were kept i n sealed cans f o r three years and was 7 years o l d . (In a l a t e r s e c t i o n we w i l l

coffee age of Rio-1 Rio-2 discuss

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

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the e f f e c t o f storage conditions on p h y s i c a l and chemical aspects of raw c o f f e e ) . Coffee beans contain several glycosidases (33), and three ©i-galactosidases were r e c e n t l y p u r i f i e d by a f f i n i t y chromatography (j54) · BARHAM and coworkers (35) found only one ^-galactosidase (they did not mention the coffee specie) and found a molecular weight of 2 6 , 3 0 0 . SHADAKSHARASWAMY and RAMACHANDRA (56, 37) observed that the a c t i v i t y of <£-galactosidase and ,Ρ-fructofuranosidase incresed when the seed was soaked i n water. This might p a r t l y e x p l a i n the thinner c e l l wall i n Rio c o f f e e s . However, d i f f e r e n c e s i n soluble carbohydrates between Rio and Soft coffee (2£) were not found, probably because the simple sugars were metabolized, g i v i n g r i s e to 002· This accounts f o r part of the dry weight l o s s i n Rio c o f f e e s I t may be i n f e r r e p h y s i c a l analyses, that the Rio f l a v o r i s associated with h y d r o l y t i c and oxidative reactions that take place during harvesting and subsequent processing, while the bean i s s t i l l wet and covered with the pulp. Washed Coffees. The p h y s i o l o g i c a l stage of f r u i t development upon harvesting which gives the best beverage f o r " n a t u r a l " c o f f e e s , a l s o a p p l i e s t o washed c o f f e e s . The r i p e cherry, i f w e l l processed, gives the best beverage. A f t e r harvesting, the coffee cherry should be "pulped" as soon as p o s s i b l e . The time required t o i n i t i a t e the r e a c t i o n s which lead to " o f f " f l a v o r s depends on the temperature of the environment and the storage c o n d i t i o n s . I f the whole beans are kept under a stream of water, the time from harvesting t o pulping may take 48 hours without a f f e c t i n g cup quality. ARCILA and VALENCIA (j58), i n Colombia, studied several f a c t o r s which a f f e c t the q u a l i t y of the beverage of washed coffees and t r i e d t o c o r r e l a t e them with a c t i v i t y of polyphenol oxidase of the d r i e d bean. The enzyme a c t i v i t y i s higher i n coffee pulped immediately; that i s w i t h i n a 12-hour i n t e r v a l from harvesting t o pulping. I t drops i n 24 t o 36 hours and again i n 48 t o 72 hours. S t a t i s t i c a l a n a l y s i s of the data showed that enzymatic a c t i v i t i e s were d i f f e r e n t and i n v e r s e l y c o r r e l a t e d with a c i d i t y , the higher the a c t i v i t y , the lower the a c i d i t y . No d i f f e r e n c e was found i n the body of the beverage, although the 72-hour treatment y i e l d e d a very poor aroma. By considering a s e r i e s of d i f f e r e n t conditions and using m u l t i p l e l i n e a r r e g r e s s i o n a n a l y s i s , these same

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authors found that the a c t i v i t y of PPO was n e g a t i v e l y c o r r e l a t e d with a c i d i t y and p o s i t i v e l y c o r r e l a t e d with the body of the beverage ( 3 8 ) . I t i s b e l i e v e d that upon h a r v e s t i n g , h y d r o l y t i c and o x i d a t i v e r e a c t i o n s i n the pulp and mucilage begin. I f the cherry i s i n j u r e d , attack by microorganisms speeds up these r e a c t i o n s . We have been able to detect a c t i v i t y of p e c t i n esterase, polygalacturonase, d-galactosidase, peroxidase and polyphenol oxidase i n the mucilage of uninjured r i p e f r u i t s (unpublished r e s u l t s ) . Polyacrylamide g e l e l e c t r o p h o r e s i s ( 3 9 ) of mucilage water-soluble p r o t e i n s i n d i c a t e d that there are 2 polyphenol oxidases and four p r o t e i n bands (Figure 7 ) · ^he c o n t r i b u t i o n to " o f f " f l a v o r s developed before pulping by microorganisms and by degradative and oxidative r e a c t i o n pulp/mucilage have WOOTON (40) s t a t e s that brown pigments formed i n the pulp and mucilage l a y e r s depreciate the q u a l i t y of the beverage, as w e l l p h y s i c a l aspects of the bean, i f i t d i f f u s e s through the parchment and reaches the bean. r

Enzymes i n Coffee

Fermentation

Mucilage removal a f t e r the bean i s pulped i s a very important step i n washed coffee processing. I f the mucilage i s not removed, the d r y i n g process becomes d i f f i c u l t . On the other hand, the mucilage i s e a s i l y attacked by microorganisms which add " o f f " f l a v o r i n t o the bean and t a i n t the beverage. The chemical composition of the mucilage has been p a r t i a l y r e s o l v e d . COLEMAN and coworkers (41) determined that p e c t i c a c i d i s one the polysaccharides of the mucilage; i t i s a polymer of p o l y g a l a c t u r o n i c a c i d ( 7 7 · 6 % d. wt. b a s i s ) , with a small amount of arabinose, galactose, xylose and rhamnose. Further work c a r r i e d out by C0RRÊA and h i s group (42, 4£, 44) showed that the p e c t i n i s a l i n e a r polymer containing (1-4) l i n k e d residues of D-galactose. They a l s o i s o l a t e d a h i g h l y branched galactoaraban i n which L-arabinofuranose residues l i n k e d ( 1 - 3 ) and ( 1 - 5 ) apparently occur i n the e x t e r i o r chain, with a core of a branched galactan composed of (1-6) and ( 1 - 3 ) l i n k e d D-galactopyranose r e s i d u e s . Reducing sugars, sucrose, c a f f e i n e , chlorogenic acids (45) and amino a c i d s are a l s o found i n the mucilage (j3) · The water-soluble p r o t e i n patterns are shown i n f i g u r e 7 · Although the mucilage has no

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

40

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

ORIGIN (-)

Figure 7. Water-soluble proteins of coffee fruit mucilage: protein banding pattern, alkaline PAGE, Τ = 7% (1); polyphenol oxidase, DOPA, alkaline PAGE, Τ = 7% (2); and SDS, no 2-mercaptoethanol treat ment, Τ = 10% (3).

(+)

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

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Coffee and Coffee Quality

41

c e l l u a r s t r u c t u r e (46) i t has several h y d r o l y t i c and oxidative enzymes, such as pectinesterase (47), galacturonase, ^.-galactosidase, peroxidase and polyphenol oxidase (catechol and DOPA as substrate) (AMORIM, TEIXEIRA, OLIVEIRA, COSTA - i n p r e p a r a t i o n ) . However, these h y d r o l y t i c enzymes of the mucilage seem to be unable to remove the mucilage when the seed i s kept i n microorganism free environment (48). CARBONNEL and VILANOVA (46) e x p l a i n t h i s f a c t by the e q u i l i b r i u m between the substrate and product of an enzymatic r e a c t i o n . Apparently, the f r u i t s do not have enough enzyme to completely c a t a l y z e s o l u b i l i z a t i o n of the mucilage; conclusive proof f o r this i s s t i l l lacking. The fermentation process which includes the removal of the mucilagenous l a y e r adhering to the parchment, i s achieve mucilage enzymes, followe y fung yeast fermentation (£0). WOSIAK and ZANCAN (£1) i s o l a t e d Pénicillium sp« Fusarium sp, Cladosporium sp and A s p e r g i l l u s sp from B r a z i l i a n coffee f r u i t s and a l l mold s t r a i n s produced i n the c u l t u r e medium enzymes able to break down p e c t i c a c i d and galactoaraban from coffee pulp and mucilage. A decrease i n pH i s caused by exposure of the carboxyl groups of p e c t i n (46), and by the production of a c e t i c and l a c t i c a c i d s (40). There i s a l s o a decrease i n carbohydrates during fermentation (47) and a concurrent production of ethanol (^2)· For low-grown c o f f e e , the fermentation i s f a s t and ethanol production i s very high i n the begining, but then i t disappears. For high-grown c o f f e e s , ethanol increases s t e a d i l y but does not reach 10% of that by low-grown coffee production. A c e t i c a c i d production i s steady and reaches 15/' of a l l v o l a t i l e s . Propionic and b u t y r i c a c i d are a l s o produced, but they increase only a f t e r 20 hours fermentation (52). P r o p i o n i c and b u t y r i c acids give a bad f l a v o r to the coffee beverage. For t h i s reason, fermentation time should be kept as short as p o s s i b l e . On the other hand, i f the coffee i s exposed to the l i q u o r (degradation and oxidized products of mucilage) f o r too long, these products may pass through the parchment, cause a poor appearance of the raw coffee (brown s i l v e r s k i n and center cut) and p o s s i b l y t a i n t the beverage c h a r a c t e r i s t i c s (40). These oxidized products are p o s s i b l y chlorogenic acids which are o x i d i z e d by polyphenol oxidase and peroxidase and l i n k c o v a l e n t l y with amino a c i d s and polypeptides (29)· avoid these browning r e a c t i o n s , W00T0N (4UJ added a

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

42

ENZYMES

IN FOOD A N D B E V E R A G E

PROCESSING

reducing agent, sodium s u l f i t e (1%), with apparent sucess, but the fermentation was delayed; probably because of i n h i b i t i o n of microorganisms. SANINT and VALENCIA (£5) found that long fermentation times of more than 24 hours, depreciate q u a l i t y of the beverage and lower a c t i v i t y of polyphenol oxidase of the d r i e d bean. To avoid a long fermentation time, one can add several enzymes that are commercially a v a i l a b l e , but these are not used on a large scale because of the added c o s t . Benefax (54) i s a material produced from molds and contains 10% of a u s e f u l crude enzyme preparation ( 8 ) . Recently, Ciba-Geigy S.A. marketed ULTRAZIM 100, which i s used by orange j u i c e i n d u s t r i e s . I t has been used to remove coffee mucilage with success i n Kenya (55) and Guatemala (56), and i n B r a z i i n time when 4 mg o mixed with 2 kg of pulped c o f f e e , as demonstrated i n Table V. TABLE V. Fermentation time of pulped coffee under d i f f e r e n t c o n d i t i o n s (temperature 18-20 C ) . Treatment N a t u r a l , dryNatural, under water ULTRAZIM 100 (4 mg - 100 ml/2 kg) Natural + M e t a b i s u l f i t e 500 ppm - 100 ml/2 kg

Hours 16 18

end 4.02 5.29

initial 5.64 5.75

9

5.17

5-69

26

4.54

5.67

preparation) This commercial enzyme may be reused f o r at l e a s t 5 consecutive fermentations by r e c y c l i n g the decanted l i q u i d . To avoid browning of the s i l v e r s k i n , 2 5 0 ppm of m e t a b i s u l f i t e may be added per kg of pulped c o f f e e . No i n h i b i t i o n of the enzyme was noted. Furthermore, m e t a b i s u l f i t e may react with acetaldehyde and other carbonyl compounds, avoiding seed contamination and a f f e c t i n g the q u a l i t y of the beverage (J2£) · I t i s important to note that, a f t e r fermentation, the beans should be washed to remove mucilage and polysaccharide degradation products ( f r e e sugars). Otherwise, these free sugars bind to the s i l v e r s k i n i n the r e g i o n of the center cut and, a f t e r the r o a s t i n g process, the center cut becomes brown (58). This brown center cut i n d i c a t e s that the bean was not

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

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Coffee and Coffee Quality

43

w e l l processed* P o s s i b l e Role of Enzymes During the Drying Process The d r y i n g process i s an important step i n coffee p r e p a r a t i o n . I f drying i s not done properly i t may change the p h y s i c a l aspect of the bean and the q u a l i t y of the beverage. FERRAZ and VEIGA i n 1 9 5 4 C59)y were the f i r s t to c a l l a t t e n t i o n to the importance of drying temperature of n a t u r a l coffee and the q u a l i t y of the beverage. The best temperature was found to be 45°C. Drying between 5 0 to 60°C had the worst e f f e c t s on the q u a l i t y of the beverage. Temperatures above 60°C and up to 90°C gave a f a i r l y good beverage f o r n a t u r a l c o f f e e s . They speculate that temperatures around 55 C a c t i v a t e enzymes which i n t u r n produce compound f l a v o r . On the othe a c t i v a t e (or i n a c t i v a t e ) enzymes which can produce compounds of good or bad f l a v o r s . Although no enzymatic a c t i v i t i e s were measured, the s p e c u l a t i o n of FERRAZ and VEIGA should be taken i n t o c o n s i d e r a t i o n , because temperature may a f f e c t bean enzymatic a c t i v i t i e s . In Colombia, ARCILA and VALENCIA (j>8) studied the e f f e c t of d r y i n g temperature on the a c t i v i t y of polyphenol oxidase and on the q u a l i t y of the beverage. The best temperatures f o r a c i d i t y , body and aroma were found to be 40, 5 0 and 80°C. Temperatures of 5 0 , 60 and 70°C gave a poor beverage, with respect to these three organoleptic c h a r a c t e r i s t i c s . The a c t i v i t y of polyphenol oxidase g e n e r a l l y decreased when temperature increased, with one exception at 40°C. The enzyme a c t i v i t y at 40°C was lower than that at 3 0 and 50°C. From the data presented, i t would appear that there i s s t i l l no d i r e c t r e l a t i o n s h i p between q u a l i t y , temperature and enzymatic activities. In the s e c t i o n on r o a s t i n g the r e s i s t a n c e of some coffee enzymes to high temperatures, probably because of the dry state of the bean, w i l l be discussed. However, i t does seem that both enzymatic and nonenzym&tic r e a c t i o n might be involved i n changing the chemical composition during the d r y i n g process, but t h i s aspect needs f u r t h e r i n v e s t i g a t i o n .

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

44

ENZYMES

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

PROCESSING

Role o f Enzymes During Coffee Storage Coffee storage i n humid and warm regions i s a serious problem, because the coffee bean becomes white, or yellow to brown, depending on the degree o f moisture i n the a i r and the time of storage. T h i s change i n c o l o r i s accompanied by a decrease i n flavor quality. MULTON and h i s group (60, 61, 62) have done a s e r i e s o f s t u d i e s on c o f f e e s t o r a g e T n d i f f e r e n t environment c o n d i t i o n s , analyzing microorganisms, water absorption, and enzymatic a c t i v i t i e s , and comparing them with the q u a l i t y o f the beverage. T h e i r major f i n d i n g s may be summarized as follows: above 75% r e l a t i v e humidity, the coffee bean absorbs a s i g n i f i c a n t amount of water and the increase i n moisture content i growth, although th The q u a l i t y o f the beverage i s completely changed and a f t e r 3 0 days a t 9 5 % r e l a t i v e humidity, the c o f f e e beans are completely r o t t e n . At 9 5 % r e l a t i v e humidity there i s a l s o an increase of l i p a s e a c t i v i t y , with a concurrent increase i n free f a t t y a c i d s . In a d d i t i o n , ribonuclease and protease a c t i v i t i e s seem to increase under these c o n d i t i o n s . The dry weight l o s s may reach 2 5 % i n 2 0 0 days i f the r e l a t i v e humidity of the a i r i s above 9 0 % . In B r a z i l , JORDÂO et a l . (6£) a l s o found an increase i n f r e e f a t t y a c i d s during three years storage o f green c o f f e e . The peroxide value increased only a f t e r 2 years storage. In A f r i c a , ESTEVES (64) studying a c i d i t y i n the o i l of Robusta and Arabica c o f f e e s a l s o found an increase i n t i t r a t a b l e a c i d i t y with i n c r e a s i n g time of storage i n both types of c o f f e e s . PEREIRA (6£) i n Portugal was the f i r s t to report a decrease i n polyphenol oxidase a c t i v i t y i n green coffee with storage time. H i s r e s u l t s were confirmed l a t e r by OLIVEIRA ( 1 1 ) , VALENCIA ( 1 2 ) and AMORIM et al. (19). Figure 8 shows the polyphenol oxidase a c t i v i t i e s obtained by OLIVEIRA ( 1 1 ; with d i f f e r e n t coffee species a f t e r d i f f e r e n t times o f storage. The value f o r Arabicas represents the average o f four d i f f e r e n t v a r i e t i e s grown i n d i f f e r e n t regions; a l l gave similar patterns. Stored green c o f f e e s that give d i f f e r e n t q u a l i t i e s of beverage a l s o show decreases i n polyphenol oxidase a c t i v i t y and t o t a l carbonyls with time of storage (Table V I ) .

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

A M O R I M AND A M O R I M

Coffee and Coffee Quality

Ε

Time (in months) of storage Figure 8.

Polyphenol oxidase activity during raw coffee bean storage (11)

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

46

E N Z Y M E S IN FOOD AND

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TABLE VI. T o t a l carbonyls i n coffee o i l and polyphenol oxidase (PPO) a c t i v i t y of Arabica green coffee beans (each symbol i s the average Coffee Sample Soft Rio Soft Rio

(stored (stored (stored (stored

1 1 2 2

yr) yr) yr) yr)

Total carbonyls umol/g o i l 113-7 a 88.8 b 93-0 b 87.6 b

PPO A c t i v i t y Abs. 10 min/g powder 1.72 a 0.72 b 0.97 c ρ-; ^ ^ Λ

Λ Λ

« 4 - zx>r

level. AKORIM et a l . (Γ9). The higher amount of t o t a l carbonyls found i n the best coffees agree CALLE ( 6 6 ) i n Colombi i n d i a , who found more aldehydes i n the best c o f f e e s . In a preliminary experiment with Arabica c o f f e e s , MELO and coworkers ( 6 8 ) and TEIXEIRA et a l . ( 6 9 ) observed that green coffee stored i n sealed cans or p l a s t i c bags f o r 21 months were s t i l l green, whereas coffee stored f o r the same period of time i n bags made of paper, cotton or vegetable f i b e r were white-yellow and y i e l d e d a poor beverage. These bleached beans showed concurrent lower d e n s i t i e s because of an increase i n s i z e (swelling) (Table VII). The a c t i v i t i e s of polyphenol oxidase and peroxidase were a l s o lower i n the s p o i l e d c o f f e e s . I t i s i n t e r e s t i n g to note that the beans kept i n cans and p l a s t i c had p r a c t i c a l l y a constant moisture content ( ~ 1 0 % ) , but coffee stored i n paper, cotton and vegetable f i b e r containers changed moisture contents from 9 to 13%* depending upon the season. I t i s well known t h a t , upon storage green coffee gradually becomes white. The d i s c o l o r a t i o n s t a r t s i n the outer l a y e r s of c e l l s and goes towards the center. The c o l o r of green coffee of a new crop, i f well processed, i s dark green or greenish-blue. With increased time of storage i t becomes l i g h t green and white. Depending upon the environmental conditions (humidity and temperature), the bean may change to yellow or brown. This change i n c o l o r of green coffee with storage was e x t e n s i v e l y studied by BACCHI ( 7 0 ) i n B r a z i l . By using two v a r i e t i e s of C. a r a b i c a , d i f f e r e n t methods of processing (wet and n a t u r a l ) , d i f f e r e n t pulping and h u l l i n g processes, and d i f f e r e n t storage c o n d i t i o n s , BACCHI concluded that

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

CD

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

2 1.35

0.905

=3-

C5

0.901

119.6 52

2

2

83

88

WELO e t . a l . (68).

cans.

yellowish

yellowish

0.28 0.05 1.9 • R e l a t i v e a c t i v i t y i n percentage r e l a t i v e to a c t i v i t y i n sealed ( s u b s t r a t e : PPO = DOPA; PER = o - d i a n i z i d i n e + H 0 ) .

l . s . d . 5%

Vegetable fiber

51

53

1.28

0.875

118.7 118.8

Cotton

yellowish

81

1.28

120.9

5· P l a s t i c Paper

cr : S 3

§3

1

-

green green

105

96

Color

1.32

PER0X.* Activity 100

1.118

PPO* Activity 100

1.46

1.124-

120.5

Can

Density

Dry wt. 1000 beans

Container type

Soluble Ν % d.wt.

Hard

Hard

Hard

Almost Soft

Almost Soft

Quality of beverage

TABLE VII· P h y s i c a l and chemical changes of green coffee stored i n d i f f e r e n t containers during 21 months ( a l l estimations were made with four replications).

48

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

the most important f a c t o r which causes the bean to become white i s mechanical i n j u r y , from harvesting to h u l l i n g , regardless of whether the coffee i s processed by the wet or the n a t u r a l method. The environmental f a c t o r which enhances most of the change i n c o l o r i s the moisture of the a i r . The chemical mechanism of coffee d i s c o l o r a t i o n has not y e t been establhished. However, from the data a v a i l a b l e i n the l i t e r a t u r e , and by comparison with d i s c o l o r a t i o n i n vegetables and f r u i t s (71), i t seems that green coffee bean d i s c o l o r a t i o n i s due to enzymatic o x i d a t i o n of phenolic compounds by polyphenol oxidase and peroxidase, with a concurrent oxidation o f ascorbic a c i d (£2). The ascorbic a c i d o x i d a t i o n might be coupled with the reduction of quinones produced by the a c t i o n of PPO and PER on phenolics (21)· f r u i t s i s followed y phenolics ascorbic a c i d and polyphenol oxidase a c t i v i t y (71% 2 2 ) . In c o f f e e , AMORIM*et a l . (26) found l e s s hydrolyzable phenolics i n Rio coffee than i n Soft c o f f e e , but the soluble chlorogenic acids (A.O.A.C. procedure (7^)) content i n the best coffee was s i g n i f i c a n t l y lower, i n comparison with the other q u a l i t i e s (26)· Chlorogenic a c i d determination i n green coffee should be r e i n v e s t i g a t e d because d i f f e r e n t types of coffee may give d i f f e r e n t r e s u l t s , depending upon the method used (75)· There i s a p o s s i b i l i t y that phenolics bound t o c e l l wall are responsible f o r the d i s c o l o r a t i o n r e a c t i o n s i n coffee. I t i s well documented i n the l i t e r a t u r e , that i n j u r y causes d i s c o l o r a t i o n or/and browning reactions i n plant t i s s u e s . Coffee beans seem t o be no exception. However, b e t t e r methods f o r a n a l y s i s should be devised i n order t o e x p l a i n many of the chemical and p h y s i c a l changes which occur i n coffee deterioration. a

Enzyme A c t i v i t i e s and P r o t e i n S o l u b i l i t y During the Roasting Process Coffee aroma and t a s t e develop only a f t e r the bean i s roasted. The r o a s t i n g temperature i s 24-0°C or higher and i t requires 3 t o 15 min., depending upon the temperature,load, machinery, e t c . Coffee aroma begins to form when the bean reaches 180-190°C (14, 76» 77)· The p r i n c i p a l precursor of coffee aroma seems t o be t r i g o n e l l i n e , sucrose, f r u c t o s e , glucose, f r e e amino a c i d s and peptides (£)· Carboxylic and

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

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phenolic acids play an important r o l e i n the t a s t e o f the beverage (6, 77)· The a c t i v i t i e s of s e v e r a l enzymes i n the green bean must play an important r o l e i n modifying the q u a l i t y of the f i n a l beverage, as has been discussed i n t h i s paper. I n s e v e r a l processing steps there are occasions t o modify the chemical composition of the bean. I t i s not probable that enzyme a c t i v i t i e s , per se, play a major r o l e i n coffee f l a v o r during the r o a s t i n g process, because the coffee bean i s dehydrated and the high temperatures would i n a c t i v a t e a l l enzymes. However, due t o the f a c t that 20% o f the a c t i v i t y o f polyphenol oxidase could be retained a f t e r 5 hours exposure of green coffee beans t o 125°C (MELO and AMORIM, 1972, unpublished), i t was necessary to look a t the a c t i v i t y o f s e v e r a l h y d r o l y t i c ( β-galactosidase phosphatase, ( 7 7 ) ) peroxidase) enzymes during the r o a s t i n g process. Figure 9 shows the r e l a t i v e a c t i v i t i e s of several enzymes plus the water-soluble p r o t e i n s (TCA p r e c i p i t a t a b l e ) during r o a s t i n g . As can be seen, the degree o f i n a c t i v a t i o n of the enzymes with i n c r e a s i n g temperature, d i f f e r s depending on the enzyme. At 5 min. ( 1 7 0 ° C ) , the powder has no coffee aroma. Instead, an odor reminiscent of roasted peanut aroma was observed. At 8 min. (187°C), a c l e a r l y defined coffee aroma was detected, which increases a t 12 and 15 min. of r o a s t i n g . The i n s o l u b i l i z a t i o n of p r o t e i n s coincides with the i n a c t i v a t i o n of enzymes and coffee aroma formation. Between the 5 t h t o 8th minute, the free amino a c i d s , peptides, t r i g o n e l l i n e and f r e e sugars should be r e a c t i n g to produce the coffee aroma· I f the f r e e amino acids and polypeptides are important i n coffee aroma formation (j?, 76), the r a t i o o f these amino a c i d s , as w e l l as t h e i r t o t a l amounts should be important to the f l a v o r o f the f i n a l beverage. A l s o , the amino a c i d composition o f the proteins and perhaps t h e i r t e r t i a r y s t r u c t u r e s also play a r o l e i n coffee f l a v o r . I t i s w e l l known that b a s i c and s u l f u r amino acids are destroyed by the r o a s t i n g process (22.* 81). Furthermore, POKORNY et a l . (81) observed that f r e e s u l f u r and b a s i c amino acids i n green coffee decrease with storage. I n B r a z i l , DOMONT et a l . (82), comparing the behavior o f amino acids during the r o a s t i n g of Soft and Rio c o f f e e s , observed that Rio coffees had 2 5 % more free amino acids than Soft c o f f e e s , and the speed of p y r o l y s i s of s e r i n e , threonine and c h i e f l y

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

50

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

PROCESSING

O

O BETA GALACTOSIDASE

Δ

Δ POLYPHENOL OXIDASE

Figure 9. Water-soluble proteins and activity of β-galactosidase (p-nitrophenyl-βΌ-galactopyranoside), polyphenol oxidase (DOPA), peroxidase (pyrogallol + H 0 ), β-glucosidase (p-nitrophenyl-$-\)-g]LUCopyranoside), and acid phosphatase (p-nitrophenyl phosphate) during the roasting process. The temperature indicated is that of the air surrounding the bean. 2

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

2

3.

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a r g i n i n e , i s much f a s t l y i n Rio than i n Soft coffee samples. These r e s u l t s agree i n part with the observation that Rio coffee has more low molecular weight polypeptides than Soft c o f f e e s , as seen i n Figure 6· Thus, there i s a p o s s i b i l i t y that these polypeptides may a f f e c t the precursors of aroma formation. Conclusion From a review of the l i t e r a t u r e and the data presented i n t h i s r e p o r t , there i s only one enzyme that i s c e r t a i n to depreciate coffee q u a l i t y . This i s the polyphenol oxidase of the pulp and mucilage (two enzymes detected), which o x i d i z e s chlorogenic a c i d and other phenolics to quinones Thes quinone the for complexes with amin brown c o l o r , that bind to the cente cut o the s i l v e r s k i n . This brown c o l o r of the center cut and s i l v e r s k i n depreciate the q u a l i t y of raw coffee and i t s appearance a f t e r r o a s t i n g . However, whether these pigments p o s i t i v e l y a f f e c t the q u a l i t y of the beverage, i s s t i l l disputed. Commercial p e c t i n o l y t i c enzymes may be used i n conjunction with reducing agents to speed up mucilage removal and avoid browning r e a c t i o n s . Although polyphenol oxidase and peroxidase a c t i v i t i e s i n many instances are c o r r e l a t e d with q u a l i t y of the beverage, proof of the mechanism and the compounds which take part i n these changes and a f f e c t cup q u a l i t y i s s t i l l l a c k i n g . Because of the f a c t that peroxidase i s located c h i e f l y i n the outer l a y e r s of the seed, i t seems probable that t h i s enzyme i s associated with coffee d i s c o l o r a t i o n upon storage. Considering a l l h y d r o l y t i c r e a c t i o n s catalysed by coffee seed enzymes, the l i p a s e s and other enzymes of l i p i d metabolism should be important, because o i l i s the aroma c a r r i e r . Also, the p r o t e i n hydrolases deserve a t t e n t i o n because they a f f e c t a known important coffee aroma precursor: the amino acids and polypeptides. By a f f e c t i n g the r a t i o of these aroma (and probably t a s t e ) precursors, i t i s reasonable to believe that both aroma and taste should be a l t e r e d . From the a v a i l a b l e l i t e r a t u r e on coffee chemistry i t seems that the c h a r a c t e r i s t i c f l a v o r of each coffee specie i s given c h i e f l y by the i n h e r i t e d chemical composition of the bean. However, the v a r i a t i o n i n q u a l i t y ( p h y s i c a l and organoleptic) whithin each specie, seems to be associated with

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v a r i a t i o n s i n chemical composition, caused "by the a c t i o n of h y d r o l y t i c and o x i d a t i v e enzymes of the bean and/or micr oorganisms. The d e l e t e r i o u s e f f e c t s of enzymes on the q u a l i t y of the r e s u l t i n g beverage may be prevented by using more s u i t a b l e methods f o r coffee processing and storage. Acknowledgment Research was c a r r i e d out with grants from the I n s t i t u t o B r a s i l e i r o do Cafe, Conselho Nacional do Desenvolvimento C i e n t i f i c o e Tecnologico e Fundaçào de Amparo a Pesquisa do Estado de Sao Paulo. We are g r a t e f u l to Dr. A. A. T e i x e i r a and Dr. C. M. Franco f o r h e l p f u l d i s c u s s i o n s and to Dr. Robert L. Ory f o r reviewing the manuscript. Literature Cited

9.

1. Sanderson, G.W. " S t r u c t u r a l and Functional Aspects of Phytochemistry". V. C. Runeckless and T. C. Τso E d i t o r s , v o l . 5, p. 24-7-316. Academic Press, New York and London, 1972. 2. Sanderson,G.W., Ranadive, A.S., Eisenberg, L.S., F a r r e l , F.J., Simons, R., Manley, C. H., Coggon, P., " P h e n o l i c , S u l f u r , and Nitrogen Compounds i n Food F l a v o r s " . ACS Symposium S e r i e s , 26, p. 14-46 (1976). 3. B i e h l , B., Ann. Technol. A g r i c . (1972) 21: 435-455. 4. Purr, Α., Ann. Technol. A g r i c . (1972) 21: 457-472. 5. Russwurm, H., 4ème Coll oque I n t e r n a t i o n a l sur, la Chemie du Cafè Vert, T o r r e f i e s and l e u r Derives. (ASIC), p. 103-109, Amsterdam, (1969). 6. Feldman, J.R., Ryder, W. S., Kung, J. T., J. Agr. Food Chem. (1969) 17: 733-739. Vitzthum, O. G., "Kaffee und coffein". p. 1-77. Springer-Verlag, Berlin, Heildelberg, New York, 1975. 8. S i v e t z , M., H. E. Foote, "Coffee Processing Technology", v o l . I, p. 4 8 - 9 9 . The A 10. Wilbaux, R., Repport annual. 2ème p a r t i e , p. 3-45. Congo, I n s t . Nat. E t . Agron. 1938. 11. O l i v e i r a , J.C., Relacão da a t i v i d a d e da p o l i f e n o l oxidase, peroxidase e catalase dos grãos de café e a qualidade da bebida. Doctor T h e s i s . Esc. Sup. Agr. "Luiz de Queiroz". U n i v e r s i t y of Sao Paulo. (1972), P i r a c i c a b a , SP, B r a z i l , 80 pages.

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

3.

AMORIM

12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31.

32. 33.

AND

AMORIM

Coffee and Coffee Quality

53

Valencia, G.A., Cenicafé (Colombia) (1972) 2 3 : 3-18. Payne, R.C., Oliveira, A.R., F a i r b r o t h e r s , D.E., Biochem. Systemat. (1973) 1: 59-61. S t r e u l i , H., 6ème Colloque I n t e r n a t i o n a l sur la Chemie des Cafés (ASIC) p. 6 1 - 7 2 , Bogota, Colombia. 1973. Amorim, H.V., S i l v a , D.M., Nature (1968) 219: 381-382. Rotemberg, B., Iachan, Α., Rev. Bras. Tecnol. (1971) 2 : 67-69. Rotemberg, Β., Iachan, Α., Rev. Bras. Tecnol. (1972) 3 : 155-159. Gopal, N.H., Annual Detailed Technical Report, Coffee Board (India) (1974-75) 28: 109-139. Amorim, H.V., Legendre, M.G., Amorim, Vera L., St. Angelo, Α., (1976) 193-195. Raa, J., P h y s i o l . P l a n t . (1973) 28: 1 3 2 - 1 3 3 · Amorim, H.V., Josephson, R.V., J. Food Sci. (1975) 40: 1179-1184. Haard, N.F., Tobin, C.L., J. Food S c i . (1971) 36: 854-857. Van Loon, L.C., Phytochem. (1971) 10: 503-507. Amorim, H.V., T e i x e i r a , Α.Α., M.Melo, V.F. Cruz, E. Malavolta, T u r r i a l b a (1975) 25: 18-24. Amorim, H.V., T e i x e i r a , A.A., Breviglieri, O., Cruz, V.F., Malavolta, Ε., T u r r i a l b a (1974) 24: 214-216. Amorim, H.V., T e i x e i r a , Α.Α., Guercio, M.A., Cruz, V.F., Malavolta, E., T u r r i a l b a (1974) 24: 217-221. Amorim, H.V., T e i x e i r a , Α.Α., Melo, Μ., Cruz, V. F., Malavolta, E., T u r r i a l b a (1974) 24: 304-308. Amorim, H.V., Cruz, V.F., T e i x e i r a , Α.Α., Malavolta, E., T u r r i a l b a (1975) 2 5 : 25-28. Melo, Μ., Amorim, H.V., T u r r i a l b a (1975) 25: 243-248. Loomis, W.D., B a t t a i l e , J., Phytochem. (1966) 5: 423-438. Weber, K., Pringe, J.R., Osborn, M., "Methods Enzymol." v o l . 26, Part C. Ed. H i r s , C. H. W. and Timasheff, S. N. p. 3-27. Academic Press, New York, London ( 1 9 7 2 ) . Amorim, H.V., Smucker, R., Pfister, R., T u r r i a l b a (1976) 26: 24-27. Courtois, J.Ε., Petek, F., "Methods Enzymol." v o l . 8 Ed. Neufeld E., Ginsburg, V. p. 565-571. Academic Press, New York and London (1966).

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

5434.

Harpaz, Ν., Flowers, H.M., Sharon, Biochem. ENZYM E S IN FOOD A N D B E V E RN., A G E PROCESSING Biophys. Acta ( 1 9 7 4 ) 341: 2 1 3 - 2 2 1 . 35. Barham, D., Dey, P.M., Griffiths, D., Pridham, J . B., Phytochem. ( 1 9 7 1 ) 1 0 : 1 9 5 9 - 1 9 6 3 . 36. Shadaksharaswamy, M., Ramachandra, G., Phytochem. (1968) 7: 7 1 5 - 7 1 9 . 37. Shadaksharaswamy, M., Ramachandra, G., Enzymologia (1968) 35: 93-99. 38. A r c i l a , P.J., V a l e n c i a , A.G., Cenicafé (Colombia) (1975) 26: 55-71. 39. Davis, B.J., Ann. New York Acad. Sci. (1964) 1 2 1 : 404-427. 40. Wooton, A.E., Report C. R. 1 2 . East A f r i c a n Research Organization ( 1 9 6 3 ) . 41. Coleman, R.J., Lenney, J.F., Coscia, A.T., D i Carlo, F.J., Arch. Biochem. Biophys. (1955) 59: 157-164. 42. Corrêa, J.B.C., Ciênc. (1971 43. Corrêa, J.B.C., Odebrecht, S., Fontana, J.D., An. Acad. b r a s . Ciênc. ( 1 9 7 4 ) 46: 349-356. 44. Corrêa, J.B.C., Coelho, E.O., Fontana, J.D., An. Acad. bras. Ciênc. ( 1 9 7 4 ) 46: 3 5 7 - 3 6 0 . 45. Franco, C.M., J . Agronomia ( B r a s i l ) (1939) 2 : 131-139. 46. Carbonnel, R.J., Vilanova, T.M., El café de El Salvador (1952) 22: 4 1 1 - 5 5 6 . 47. Rolz, C., Menchu, J.F., Espinosa, R., G a r c i a -Prendes, Α., 5ème Colloque I n t e r n a t i o n a l sur la Chemie des Cafes V e r t s , Torréfiés et l e u r Dérivés, Lisbonne (ASIC) p. 259-268, Lisbonne (1971). 48. Franco, C.M., B o l . Supta. Serv. Café, São Paulo (1944) 19: 250-256. 49. Franco, C.M., Bragantia (1960) 19: 621-626. 50. C a l l e , V.H., Cenicafé (Colombia) ( 1 9 6 5 ) 16: 3-11. 51. Wosiaki, G., Zancan, G.T., Arq. Biol. Tecnol. ( 1 9 7 3 ) 16: 129-134. 52. Menchu, J.F., Rolz, C., Café, Cacao, The (1973) 17: 53-61. 53. Sanint, O.B., V a l e n c i a , G.A., Cenicafé (Colombia) ( 1 9 7 0 ) 21: 59-71. 54. Johnston, W.R., Foote, H.E., U.S.A. patent 2, 526, 8 7 3 ( 1 0 / 2 4 / 1 9 5 0 ) . 55. Arunga, R.O., Kenya Coffee ( 1 9 7 3 ) 38: 354-357. 56. Kupferschmied, B., Revista C a f e t a l e r a ( 1 9 7 5 ) 42. 57. Rodriguez, D.B., Frank, H.Α., Yamamoto, H.Y., J . S c i . Fd. A g r i c . ( 1 9 6 9 ) 2 0 : 1 5 - 1 9 . 58. T e i x e i r a , Α.Α., Ferraz, M.B., A Rural ( 1 9 6 3 ) Maio: 28-29.

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

3.

AMORIM

AND

AMORIM

Coffee and Coffee Quality

55

5 9 · F e r r a z , M.B., Veiga, Α.Α., B o l . Supta. Serv. Café (1954) 29: 5-6. 60. Multon, J.L., Poisson, J., Cahagnier, Β., Hahn,D., B a r e l , M., Santos, A.C., Café, Cacao, The (1974) 18: 121-132. 61. Santos, A.C., Hahn, D., Cahagnier, B., Drapron,R., G u i l b o t , Α., Lefebvre, J., Multon, J.L., Poisson, J., Trentesaux, E., 5eme Colloque I n t e r n a t i o n a l sur la Chemie des Cafés (ASIC), 304-315, Lisbonne ( 1 9 7 1 ) . 62. Poisson, J., Cahagnier, Β., Multon, J.L., Hahn,D., Santos, A.C., 7ème Colloque I n t e r n a t i o n a l sur l a Chemie des Cafés (ASIC), Hamburg ( 1 9 7 5 ) ( i n press). 63. Jordão, B.A., G a r r u t t i , R.S., A n g e l u c c i , Ε., Tango, J.S., T o s e l l o , Υ., C o l e t . I n s t . Tec. Alim. (1969-70) 3: 253 64. Esteves, A.B., Estud 297-317. 65. P e r e i r a , M.J., Estud. Agron. (Lisboa) (1962) 3: 153-156. 66. C a l l e , H.V., Cenicafe (Colombia) (1963) 14: 187-194. 67. Gopal, N.H., Venkataramana, D., Ratna, N.G.N., Indian Coffee (1976) 40: 2 9 - 3 3 . 68. Melo, M., F a z u o l l i , L.C., T e i x e i r a , Α.Α., Amorim, H.V., Resumos Soc. Bras. Progr. Ciênc. Doc. 1 3 3 - 6 . 3 , p. 846., ( B r a s i l i a ) (1976). 69. T e i x e i r a , Α.Α., F a z u o l i , L.C., Carvalho, Α., Resumos Soc. Bras. Progr. Ciênc. Doc. 72-601, p. 777 (Brasília) (1976). 70. Bacchi, O., Bragantia (1962) 21: 467-484. 71. Ponting, J.D., J o s l y n , M.A., Arch. Biochem. (1948) 19: 47-63. 72. Vasudeva, Ν., Gopal, N.H., J . Coffee Res. (1974) 4: 25-28. 73. Weaweaver, C.; Charley, H., J . Food. Sci. (1974) 39: 1200-1202. 74. Official Methods of A n a l y s i s of The A.O.A.C. p. 217. 10th E d i t i o n , Washington (1965). 75. Amorim, H.V., G u e r c i o , M.A., Cortez, J.G., Malavolta, E., An. Esc. Sup. Agr. " L u i z de Queiroz", USP (1973) 30: 281-291. 76. B a l t e s , W.,7ème Colloque I n t e r n a t i o n a l sur l a Chemie du Cafè (ASIC), Hamburg (1975) (in press). 77. Reymond, D., 171st A.C.S. meeting, New York (1976). 78. Berndt, W., Meier-Cabell, E., 7ème Colloque I n t e r n a t i o n a l sur la Chemie des Cafés (ASIC), Hamburg (1975) (in press).

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79. Thaler, H., G a i g l , R., Z. Lebensm. Untersuch. Forsch. (1963) 120: 357-363. 80. O l i v e i r a , J.S., 5ème Colloque I n t e r n a t i o n a l sur la Chemie des Cafés (ASIC), p. 115-119, Lisbonne (1971).

81. Pokorny, J., Con, N.H., Bulantova, H., Janiced,G., Nahrung (1974) 18: 799-805. 82. Domont, G.B., Guimaraes, V., Solewicz, Ε., Perrone, J.C., An. Acad, brasil. Ciênc. (1968) 40: 2 5 9 R.

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

4 Enzymatic Modification of Milk Flavor W. F. SHIPE Department of Food Science, Cornell University, Ithaca, N.Y. 14853

Until r e c e n t l y enzymati undesirable. So most of the a t t e n t i o n has been given to inactivating the enzymes, r a t h e r than u s i n g them. But research in the l a s t few years i n d i c a t e that enzymatic m o d i f i c a t i o n of m i l k may be f e a s i b l e i n some cases. Enzymes reported to a f f e c t m i l k f l a v o r are e i t h e r o x i d a t i v e or h y d r o l y t i c . Table I shows the o x i d a t i v e enzymes and t h e i r r e a l or p o t e n t i a l e f f e c t . Table I.

O x i d a t i v e Enzymes Reported

1. 2. 3.

"Oleinase" Xanthine oxidase Lactoperoxidase

4. 5. 6.

S u l f h y d r y l oxidase Hexose oxidases Lactose dehydrogenase

to Have F l a v o r Impact

Enhances oxidation? Enhances oxidation? Enhances o x i d a t i o n (nonenzymatically). Reduces cooked f l a v o r . Increases a c i d i t y . Increases a c i d i t y .

In 1931 Kende (1) reported that an enzyme which he c a l l e d o l e i n a s e was i n v o l v e d in o x i d i z e d f l a v o r development. T h i s conc l u s i o n was based p r i m a r i l y on the f a c t that o x i d i z e d f l a v o r development was i n h i b i t e d by heat treatment. I t was assumed that the i n h i b i t i o n was a r e s u l t of the i n a c t i v a t i o n of " o l e i n a s e " . I t was not recognized at that time that heat treatment tends to inhibit o x i d a t i o n , presumably by producing a c t i v e s u l f h y d r y l groups. The existence of an o l e i n a s e was never confirmed but people are still searching f o r it. I t has been reported that xanthine oxidase c o n t r i b u t e s to spontaneous o x i d i z e d f l a v o r development (2). Evidence has been presented which shows a good c o r r e l a t i o n between xanthine oxidase activity and production of TBA r e a c t i v e components. Furthermore, thermal and chemical treatments known to i n a c t i v a t e xanthine oxidase a l s o i n h i b i t e d the development of o x i d i z e d f l a v o r . However, there is no d i r e c t evidence to show that m i l k contains a

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s u b s t r a t e that i s acted on by xanthine oxidase. Recently, i t has been p o s t u l a t e d (3) that xanthine oxidase produces superoxide anion i n milk which may undergo non-enzymatic dismutation t o form s i n g l e t oxygen which could c a t a l y z e l i p i d o x i d a t i o n . Furthermore, i t was p o s t u l a t e d that the superoxide dismutase could i n h i b i t t h i s o x i d a t i o n by c a t a l y z i n g the conversion of superoxide anion to t r i p l e t oxygen and hydrogen peroxide. Superoxide anion has a l s o been detected (4) i n m i l k serum that had been exposed to f l u orescent l i g h t . Regardless of the source of the superoxide anion, superoxide dismutase could p o s s i b l y c o n t r i b u t e to the m i l k a n t i oxidant system. However, more research i s needed to determine i f e i t h e r xanthine oxidase or superoxide dismutase have any impact on m i l k f l a v o r . I n c i d e n t a l l y , i f xanthine oxidase does c a t a l y z e o x i d a t i o n the enzyme can be i n a c t i v a t e d by heating to 94°C f o l l o w i n g normal p a s t e u r i z a t i o n . M i l k p l a n t s with steam i n j e c t i o n - v a c uum p r o c e s s i n g equipment can e a s i l y produce xanthine oxidase f r e e m i l k with only a s l i g h E r i k s s o n (5) reporte d a t i o n by v i r t u a l of the heme i r o n that i t c o n t a i n s . In other words, i t i s a non-enzymatic c a t a l y s t . I mention t h i s because there may be other enzymes that have s i g n i f i c a n t non-enzymatic effects. Consequently, when we use enzymes we must not ignore p o s s i b l e non-enzymatic e f f e c t s of enzymes. In 1967 Kiermeier and Petz (6) i n Germany i s o l a t e d a s u l f h y d r y l oxidase from milk. T h i s enzyme o x i d i z e s s u l f h y d r y l groups to d i s u l p h i d e s . S u l f h y d r y l oxidase s t u d i e s have a l s o been conducted at North C a r o l i n a State and C o r n e l l . Table I I shows the e f f e c t of t h i s enzyme on s u l f h y d r y l groups and cooked f l a v o r as reported by Siewright (7). As w i l l be noted t h i s enzyme can s i g n i f i c a n t l y lower cooked f l a v o r . I t has been suggested that s u l f h y d r y l oxidase might be used to e l i m i n a t e the strong cooked f l a vor i n m i l k that i s produced by u l t r a h i g h temperature (UHT) Table I I . E f f e c t of S u l f h y d r y l Oxidase on S u l f h y d r y l Concentrat i o n and Cooked F l a v o r . Sulfhydryl Flavor Sample Level Intensity Control 0.18 3.4 1% enzyme s o l u t i o n 0.09 1.6 2% enzyme s o l u t i o n 0.05 1.1 a

^Expressed as o p t i c a l d e n s i t y . Ellman - DTNB Method Flavor intensity: 1 = s l i g h t -> 4 = very strong cooked treatment. However, one needs to determine whether the s u l f h y d r y l oxidase treatment w i l l make the m i l k more s u s c e p t i b l e to o x i d a t i o n by e l i m i n a t i n g s u l f h y d r y l groups which have anti-oxygenic properties. Since UHT treatment i s u s u a l l y given to prolong storage l i f e and the r i s k of o x i d i z e d f l a v o r reaching the d e t e c t a b l e l e v e l i n c r e a s e s with age, one should avoid lowering the o x i d a t i v e s t a b i l i t y of the UHT milk. To preserve the anti-oxygenic b e n e f i t

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and s t i l l e l i m i n a t e cooked f l a v o r , one might wait to add the s u l f h y d r y l oxidase u n t i l a f t e r storage, i . e . j u s t before consumption. Rand (8) has s t u d i e d two methods f o r conversion of l a c t o s e i n m i l k to a c i d . In one method he f i r s t hydrolyzed the l a c t o s e to glucose and galactose w i t h l a c t a s e and then converted the hexoses to the corresponding a l d o b i o n i c a c i d s with hexose oxidases. In the second method he used l a c t o s e dehydrogenase to produce l a c t o b i o n i c d e l t a l a c t o n e which subsequently hydrolyzed to l a c t o b i o n i c a c i d . He reported that t h i s l a t t e r method was l e s s e f f i c i e n t than the f i r s t method. The r a t e of a c i d production with hexose o x i dases was increased by the a d d i t i o n of c a t a l a s e and I^O^ which s u p p l i e d oxygen f o r regenerating the reduced enzymes. The enzymatic process changes the f l a v o r and a l s o gets r i d of the l a c t o s e which i s an a d d i t i o n a l advantage f o r those with l a c t o s e i n t o l e r ance. Glucose o x i d a s e - c a t a l a s oxygen scavengers i n product mixes, c o f f e e and a c t i v e dry yeast (9). Since these products do not c o n t a i n enough moisture f o r the enzymatic r e a c t i o n i t i s necessary to provide water. T h i s problem has been solved by p u t t i n g glucose, glucose o x i d a s e - c a t a l a s e and s u f f i c i e n t moisture i n a p l a s t i c envelope. T h i s envelope should r e a d i l y transmit gaseous oxygen but should r e s t r i c t moisture passage. I t has been reported that these enzymatic oxygen scavenger packets produced lower l e v e l s of r e s i d u a l oxygen than vacuum treatment followed by f l u s h i n g with n i t r o g e n . Although these packages have had only l i m i t e d comm e r c i a l use they do i l l u s t r a t e an ingenious approach to f l a v o r control. Table I I I l i s t s the h y d r o l y t i c enzymes that have a f l a v o r Table I I I . 1. 2. 3. 4. 5. 6.

H y d r o l y t i c Enzymes with F l a v o r Impact

Lipase Lactase M i l k proteases Phospholipase C Phospholipase D Trypsin

Causes h y d r o l y t i c r a n c i d i t y . Increases sweetness. Increases b i t t e r n e s s Inhibits oxidation. Inhibits oxidation. Inhibits oxidation.

impact. Of course, l i p a s e heads the l i s t because i t i s the natur a l l y o c c u r r i n g enzymes that appears to have the g r e a t e s t f l a v o r impact. In f a c t two survey taken i n the past year i n New York C i t y i n d i c a t e s that h y d r o l y t i c r a n c i d i t y was the p r i n c i p a l o f f f l a v o r i n commercial m i l k samples. Therefore, the dairymen needs to know how to c o n t r o l l i p o l y s i s . C o n t r o l i n t h i s case p r i m a r i l y i n v o l v e s keeping the enzyme away from the s u b s t r a t e . At the time of m i l k s e c r e t i o n the f a t globule i s f a i r l y w e l l p r o t e c t e d from the l i p a s e by the f a t g l o b u l e membrane. T h i s membrane can be a l t e r e d by thermal or mechanical abuse. Obviously the a l t e r a t i o n i s increased by i n c r e a s i n g the abuse. The mere c o o l i n g of some

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

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milk from 37 to 5°C i s enough to cause l i p o l y s i s presumably by l i p a s e l o c a t e d i n the membrane. Heating cooled m i l k up to 30°C and r e c o o l i n g to 5°C causes even greater i n c r e a s e s i n l i p o l y s i s . The e f f e c t of t h i s s o - c a l l e d temperature a c t i v a t i o n i s shown i n Table IV. Apparently, these temperature changes causes d i s r u p t i o n Table IV.

E f f e c t of " A c t i v a t i o n

Temperature °C A c i d degree v a l u e s As determined

3

15 1.0

20 1.3

11

Temperature on L i p a s e A c t i o n 25 2.8

30 3.9

35 2.2

40 0.9

by method of Thomas, N i e l s e n and Olson (10).

of the f a t globule membrane so as to i n c r e a s e the contact between f a t and l i p a s e . Mechanical a g i t a t i o n such as simple s t i r r i n g or pumping can a l s o expose the f a t to the enzyme. The magnitude of t h i s e f f e c t i s dependent on the temperature as i s shown i n Table V. Obviously, the e f f e c i s kept c o o l . Vigorou n i z e r produces even more d r a s t i c i n c r e a s e s . The increase produced Table V.

E f f e c t of Temperature and Shaking on L i p o l y s i s Temperature 5°C 26°C % Increase i n l i p o l y s i s on shaking 17 370 As determined

by Kurkovsky and Sharp (11).

by homogenization exceeds the amount of i n c r e a s e of f a t g l o b u l e surface area. Apparently, p a r t of the i n c r e a s e i s due to changes i n the nature of the f a t g l o b u l e membrane. F o r t u n a t e l y , most of the l i p a s e i n m i l k i s i n a c t i v a t e d by p a s t e u r i z a t i o n which i s done e i t h e r j u s t before or a f t e r homogenization. Most l i p o l y s i s can be avoided i f m i l k i s not subjected to thermal or mechanical abuse and i s p a s t e u r i z e d soon a f t e r production. However, bulk handling of m i l k and t r a n s p o r t i n g i t over as much as 300 or 400 m i l e s makes i t d i f f i c u l t to f o l l o w simple c o n t r o l measures. Furthermore, p a s t e u r i z a t i o n does not completely i n a c t i v a t e a l l of the l i p a s e (12) and i n the case of homogenized milk, t h i s can cause f l a v o r problems. Consequently, other methods of c o n t r o l l i n g l i p o l y s i s are being sought. Lipase can be i n a c t i v a t e d by adding H^O^, but such a d d i t i o n s are not l e g a l . Exposure to f l u o r e s c e n t l i g h t can i n h i b i t l i p o l y s i s but t h i s would be a questionable procedure because of the r i s k of producing l i g h t induced o f f - f l a v o r s . Shahani and Chandan (13) observed that non-fat m i l k s o l i d s i n h i b i t e d p u r i f i e d m i l k l i p a s e , presumably by absorbing on the surface of the substrate. The a d d i t i o n of s o l i d s would a l s o i n c r e a s e the n u t r i t i v e value of the product and thus would have a double b e n e f i t . A d d i t i o n a l evidence on the e f f e c t of a d d i t i v e s was revealed i n a recent study (14) on the e f f e c t of l e c i t h i n , c a s e i n and t r y p s i n on l i p o l y s i s (Table V I ) . In t h i s study, emulsions of 2% m i l k f a t were s t a b i l i z e d with 0.5% c a s e i n + 0 . 5 % bovine l e c i t h 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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Table VI.

I n t e r a c t i o n of L i p a s e and T r y p s i n i n D i f f e r e n t Emulsions of M i l k Fat Free F a t t y Acids (peg/ml) Additives Without T r y p s i n With T r y p s i n 1

1

,

1

8

Lecithin h h L e c i t h i n + Casein 3.40 2.50^ Casein 7.44° 15.51 Casein + L e c i t h i n 0.65* 0.87 Numbers with same s u p e r s c r i p t are not s i g n i f i c a n t l y d i f f e r e n t (P < 0.05) a

1

or 0.5% of each. The emulsions were then incubated a t 21°C f o r 60 min. with l i p a s e or l i p a s e and t r y p s i n . Casein enhanced l i p o l y s i s when added alone or a f t e r l e c i t h i n , but i t appeared to i n h i b i t when added p r i o r t o l e c i t h i n . Since m i l k l i p a s e i s known to a s s o c i a t e with c a s e i n " a t t r a c t " l i p a s e to th a f t e r the c a s e i n may block the absorption of the l i p a s e . The f a c t that t r y p s i n enhances l i p o l y s i s when c a s e i n alone was used, suggest that the unhydrolyzed c a s e i n does p r o t e c t the f a t to some extent. Regardless of the explanation of these r e s u l t s , they c l e a r l y i l l u s t r a t e that l i p o l y s i s i s a f f e c t e d by the m a t e r i a l that i s absorbed on the f a t globule. So f a r I have been t a l k i n g about i n a c t i v a t i o n or i n h i b i t i n g l i p o l y s i s but i n many products f r e e f a t t y a c i d s are d e s i r a b l e . Even good f r e s h m i l k contains some f r e e f a t t y a c i d s . But by comp a r i s o n good b u t t e r contains 6 to 7 times more f r e e f a t t y a c i d s , cheddar cheese 4 t o 5 times more and blue cheese 50 t o 100 times more. Lipases play a dual r o l e i n blue cheese f l a v o r production by r e l e a s i n g v o l a t i l e f a t t y a c i d s , some o f which c o n t r i b u t e d i r e c t l y t o f l a v o r and some serve as precursors of methyl ketones. The methyl ketones are major c o n t r i b u t o r s to blue cheese f l a v o r . Richardson and Nelson (15) have i n d i c a t e d that cheddar cheeses were o r g a n o l e p t i c a l l y p r e f e r r e d when g a s t r i c l i p a s e preparations were included i n t h e i r manufacture. So l i p o l y s i s i s o f t e n d e s i r able i f we can c o n t r o l the amount and kinds of f a t t y a c i d s r e leased. M i l k and p a n c r e a t i c l i p a s e s are f a i r l y n o n - s p e c i f i c i . e . the r a t i o of f a t t y a c i d s i n the hydrolysate and the unhydrolyzed f a t a r e e s s e n t i a l l y the same. By c o n t r a s t p r e - g a s t r i c ( i . e . o r a l ) l i p a s e s from calves and k i d s and fungal sources p r e f e r e n t i a l l y r e l e a s e high proportions of short chain f a t t y a c i d s . But even the fungal enzymes e x h i b i t considerable d i f f e r e n c e s as i s shown i n Table V I I . Arnold et a l . (16) have reviewed the d i f f e r e n c e s i n s p e c i f i c i t y of v a r i o u s l i p a s e s and how d i f f e r e n t l i p a s e s can be used t o produce l i p o l y z e d milk f a t with s p e c i f i c f l a v o r characteristics. They pointed out that s e l e c t i v e l y hydrolyzed f a t s are added to a v a r i e t y of foods i n c l u d i n g c e r e a l products, margarines, s a l a d d r e s s i n g s , c o f f e e whiteners, soups, snack foods and c o n f e c t i o n a r y products such as m i l k chocolates, creams and

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Table V I I .

R e l a t i v e Release of Free F a t t y Acids by Two Fungal Lipases Source of L i p a s e Short chain Total free f a t t y acids fatty acids (μ Equiv.) (%) Pénicillium r o q u e f o r t i 38 110 2 Achromobacter 96 fudges. Lactase i s the next h y d r o l y t i c enzyme on the l i s t but s i n c e t h i s t o p i c i s covered i n the next chapter, I w i l l only take time to p o i n t out a unique use of t h i s enzyme. A commercial company i s c u r r e n t l y producing packets of l a c t a s e which bears the f a n c i f u l name " L a c t i - A i d " . The consumer i s i n s t r u c t e d to add the L a c t Aid to a quart of m i l k and to s t o r e i t i n the r e f r i g e r a t o r f o r 24 hours. During t h i s time the l a c t o s e w i l l be hydrolyzed and the sweetness w i l l be i n c r e a s e d the consumer c a r r y out th t e n t i a l use of enzymes. Since consumers are a l r e a d y u s i n g enzymatic meat t e n d e r i z e r s , home use of enzymes may become q u i t e commonplace. Perhaps, we should consider preparing enzyme packets for use by the farmers. For example, we might be able to e l i m i nate the l i p a s e problem by having the farmer add the r e q u i r e d amount of ^2®2 l i p a s e and then have him add s u f f i c i e n t c a t a l a s e to get r i d of the excess ^2^2' l a r i l y , app r o p r i a t e amounts of s u l f h y d r y l oxidase might be packaged f o r use i n bulk-dispensed m i l k such as i s used i n c a f e t e r i a s . The s u l f h y d r y l oxidase could be added f a r enough i n advance of s e r v i n g time to allow the enzyme to e l i m i n a t e cooked f l a v o r . t

0

i

n

a

c

t

i

v

a

t

e

t

n

e

s i m i

M i l k has been known to c o n t a i n proteases f o r some time, but there i s no d i r e c t evidence to i n d i c a t e that they c o n t r i b u t e to f l a v o r i n f l u i d milk. Some s c i e n t i s t s b e l i e v e that they c o n t r i b ute to the f l a v o r of raw m i l k cheeses. In f a c t , a recent r e p o r t (17) i n d i c a t e d that m i l k proteases survived p a s t e u r i z a t i o n and t h e r e f o r e , was a l s o important i n p a s t e u r i z e d products. S i m i l a r proteases from b a c t e r i a l o r i g i n are known to produce b i t t e r f l a v o r s , and perhaps i n some cases b i t t e r f l a v o r i n m i l k may be produced by them. T h i s might p a r t i c u l a r l y be true i n m i l k that has been stored f o r s e v e r a l days. The l a s t three h y d r o l y t i c enzymes on the l i s t have been shown to i n h i b i t o x i d a t i o n . The a c t i o n of these enzymes are of e s p e c i a l i n t e r e s t because they i l l u s t r a t e how r a t h e r l i m i t e d enzymatic a c t i o n can have a s i g n i f i c a n t f l a v o r impact. Furthermore, i t i s worth n o t i n g that these three enzymes c a t a l y z e three d i f f e r e n t h y d r o l y t i c r e a c t i o n s y e t a l l i n h i b i t o x i d a t i o n . T h i s immediately r a i s e s two questions: F i r s t , why should h y d r o l y s i s i n h i b i t oxidation? and second, do they have some common i n h i b i t o r y mechanism? Before t r y i n g to answer these two question, l e t s consider the i n d i v i d u a l enzymes, s t a r t i n g w i t h phospholipases C and D.

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 f phosphotidylcholine i s the s u b s t r a t e , phospholipase C y i e l d s a d i g l y c e r i d e plus phosphorycholine whereas D y i e l d s phosphatidic a c i d and c h o l i n e . Skukla and Tobias (18) claimed that the phosp h a t i d i c a c i d produced by phospholipase D was r e s p o n s i b l e f o r i t s anti-oxygenic e f f e c t . On the b a s i s of our r e s u l t s we have assumed that the anti-oxygenic e f f e c t of phospholipase C was due to a r e arrangement of one or more of the f a t globule membrane components. Presumably t h i s rearrangement decreases the s u s c e p t i b i l i t y of the l i p i d s to o x i d a t i o n by reducing the exposure of the substrate to pro-oxidants such as copper or i r o n . The i n h i b i t i o n of copper induced o x i d a t i o n by phospholipase C, as reported by Young (19), i s shown i n Table V I I I . Whereas, phospholipase C i n h i b i t s oxidat i o n , Chrisope and M a r s h a l l (20) reported that i t enhances l i p o l y sis. Therefore, t h i s enzyme should not be used i n raw m i l k unless Table V I I I .

E f f e c t of Phospholipase C A c t i o n TBA Reactin

Sample I d e n t i f i c a t i o n Control Milk M i l k +0.05 ppm Copper M i l k + Copper + Enzyme

326 .02 .07 .03

362 .03 .09 .04

3

on Development of

377 .04 .09 .04

Incubated f o r 1 hr at 37°C, followed by thermal i n a c t i v a t i o n and ^storage f o r 3 days at 5°C. TBA values expressed as absorbance at 532 nm. Samples w i t h TBA values greater than 0.04 u s u a l l y have detectable o x i d i z e d f l a v o r s . one

i s i n t e r e s t e d i n i n c r e a s i n g the f r e e f a t t y a c i d content. The use of t r y p s i n to i n h i b i t o x i d a t i o n i s perhaps the most i n d i r e c t method f o r c o n t r o l l i n g milk f l a v o r . Ever s i n c e the use of t r y p s i n f o r t h i s purpose was f i r s t reported (21) i n 1939, s c i e n t i s t s have been t r y i n g to e l u c i d a t e the mechanism of i t s a c t i o n . Since p r e l i m i n a r y s t u d i e s had i n d i c a t e d that t r y p t i c a c t i o n a f f e c t e d the f a t globule membrane we i s o l a t e d the membrane using the method of B a i l i e and Morton (22). Our r e s u l t s i n d i c a t e d that approximately 25% of the membrane m a t e r i a l was released i n t o the serum phase by the enzymatic a c t i o n . Furthermore, SDS-polyacrylamide e l e c t r o p h o r e t i c separations revealed (23) that the membrane m a t e r i a l had been p a r t i a l l y hydrolyzed by the t r y p s i n . This suggests that the t r y p s i n e f f e c t may be due to both a r e l e a s e of membrane m a t e r i a l and p r o t e i n h y d r o l y s i s . I t has been suggested that p r o t e i n h y d r o l y s i s i n h i b i t s o x i d a t i o n by i n c r e a s i n g the copper binding c a p a c i t y of the milk. Results of our s t u d i e s (24) i n d i c a t e d that t r y p t i c a c t i o n does increase the copper b i n d i n g c a p a c i t y of the milk. Although there was some v a r i a b i l i t y i n the amount bound, a l l samples showed s i g n i f i c a n t increases. Heat i n a c t i v a t e d t r y p s i n d i d not cause any increase i n copper b i n d i n g thus i n d i c a t i n g that the e f f e c t was due to h y d r o l y t i c a c t i o n r a t h e r than d i r e c t binding to the enzyme.

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Results by Babish (25) showed that t r y p t i c a c t i o n reduced oxygen uptake i n samples c o n t a i n i n g added copper (Table IX). The l i n e a r i t y of the data r e v e a l s the l a c k of an i n d u c t i o n p e r i o d which suggests a heme c a t a l y z e d o x i d a t i o n . T h i s theory i s Table IX.

E f f e c t of T r y p s i n Treatment on Oxygen Intake Time (hours)

Control Milk M i l k + Copper M i l k + T r y p s i n + Copper

0 6.4 6.2 5.7

12

24

36

5.5 5.4

4.0 4.9

3.4 4.7

48 5.2 2.7 4.4

supported by the observation that both h i s t i d i n e and antimycin A i n h i b i t e d o x i d a t i o n i n milk. These two substances are capable of complexing w i t h heme p r o t e i n T r y p t i c a c t i o n may a l s o a f f e c t the c a t a l y t i c a c t i o n of hem a c t i o n t r y p s i n does i n h i b i r e s u l t s of a t y p i c a l experiment i s shown i n Table X. Our r e s u l t s (23) a l s o i n d i c a t e that t r y p s i n treatment increased Vitamin A stability. T h i s i n d i c a t e s a f r i n g e b e n e f i t of the enzymatic treatment. Table X. Sample

E f f e c t of T r y p s i n Treatment on the O x i d a t i v e of m i l k TBA V a l u e s Trial 1 Trial 2 3

Control Milk M i l k + Copper M i l k + T r y p s i n + Copper a

Stability

0.03 1.00 0.03

V a l u e s represent absorbance at 532 nm,

0.03 0.70 0.02 3 days a f t e r treatment

The foregoing r e s u l t s were obtained with s o l u b l e t r y p s i n but we have a l s o used immobilized t r y p s i n (26,27). Inasmuch as we only want to hydrolyze a small f r a c t i o n of the p r o t e i n i n t h i s treatment, the immobilized enzyme has the advantage of enabling us to c a r e f u l l y c o n t r o l the extent of h y d r o l y s i s . We have bound t r y p s i n to both porous g l a s s and tygon tubing. Although porous g l a s s has much greater s u r f a c e area f o r b i n d i n g enzymes, the tygon tubing i s e a s i e r to s a n i t i z e and does not plug up. Incid e n t a l l y , we have used 10% ethanol to s a n i t i z e the r e a c t o r or tubing a f t e r each use. By bubbling a i r or n i t r o g e n i n t o the flow stream through the tygon tubing, turbulence i s increased and b e t t e r substrate-enzyme contact i s obtained (26). The tygon tubing s t i l l does not provide as much contact but i t i s l e s s l i k e l y to be plugged by the f a t i n whole milk. Skim milk i s l e s s apt to plug a column r e a c t o r and whey passes through q u i t e readily. So i t i s more f e a s i b l e to t r e a t these two products with immobilized enzymes than whole milk. Swaisgood and a s s o c i a t e s (28,29) a t North C a r o l i n a State have used immobilized s u l f h y d r y 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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oxidase, rennin and a fungal protease to t r e a t skim milk. A number of workers have prepared immobilized l a c t a s e f o r t r e a t i n g skim m i l k and whey. In c o n c l u s i o n , enzymes can be used to improve m i l k f l a v o r i n three ways. Namely 1. By producing or i n c r e a s i n g d e s i r a b l e f l a v o r s such as sweetness. 2. By removing o f f - f l a v o r s such as strong cooked f l a v o r . 3. By preventing development of o f f - f l a v o r s , such as o x i dized f l a v o r . In most cases o x i d i z e d f l a v o r development i s not a c r i t i c a l problem at the present time. However, i t could become one i f we adopt wholesale f o r t i f i c a t i o n of m i l k with i r o n or i n c r e a s e the polyunsaturated f a t t y a c i d content by feeding encapu l a t e d unsaturated o i l s . Whether any of these treatment are used w i l l depend on the seriousness of the f l a v o r problem and the cost of the treatment. Literatur 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

Kende S. Proc. IX Intern. Dairy Congr. (1931), Subject 3, paper 137. Aurand, L. W. and Woods, A. E. J . Dairy S c i . (1959) 42, 1111. Hicks, C. L., Korycka-Dahl, M. and Richardson, T. J . Dairy S c i . (1975) 58, 796 (Abstr.). Korycka-Dahl, M. and Richardson, T. Abstr. 71st Ann. Mtg. Amer. Dairy S c i . Assn. (1976) p54,D49. E r i k s s o n , C. E. J . D a i r y S c i . (1970) 53, 1649 (Abstr.). Kiermeier, F. and Petz, Ε. Z. Lebensmittelunters u-Forsch. (1967) 132, 342. Sievwright, C. A. M.S. T h e s i s , C o r n e l l Univ., Ithaca, NY (1970). Rand, A. G., J r . and Hourigan, J . A. J . D a i r y S c i . (1975) 58, 1144. Reed, G. "Enzymes i n Food P r o c e s s i n g " pp386-390. Academic Press, New York (1966). Thomas, E. L., N i e l s e n , A. J . and Olson, J . C., J r . Amer. M i l k Rev. (1955) 17, 50. Krukovsky, V. N. and Sharp, P. F. J . D a i r y S c i . (1938) 21, 671. Harper, W. J . and Gould, I. Α. XV I n t e r n . D a i r y Congr. (1959) 1455. Shahani, Κ. M. and Chandan, R. C. J . D a i r y S c i . (1963) 46, 597. M a r s h a l l , R. T. and Charoen, C. Abstr. 71st Ann. Mtg. Amer. D a i r y S c i . Assn. (1976) p51,D40. Richardson, G. H. and Nelson, J . H. J . D a i r y S c i . (1967) 50, 1061. Arnold, R. G., Shahani, Κ. M. and Dwivedi, Β. K. J . Dairy S c i . (1975) 58, 1127.

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

66 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.

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Noomen, A. Netherlands M i l k Dairy J . (1975) 29, 153. Shukla, T. P. and Tobias, J . J . D a i r y S c i . (1970) 53, 637. Young, M. H. M.S. T h e s i s , C o r n e l l Univ., Ithaca, NY (1969). Chrisope, G. L. and M a r s h a l l , , R. T. J . D a i r y S c i . (1975) 58, 794 (Abstr.). Anderson, E. O. M i l k Dealer (1939) 29(3)32. Bailie, M. J . and Morton, R. K. Biochem. J . (1958) 69,35. Gregory, J . F. and Shipe, W. F. J . D a i r y S c i . (1975) 58, 1263. Lim, Diana and Shipe, W. F. J . Dairy S c i . (1972) 55, 753. Babish, J . E. M.S. T h e s i s , C o r n e l l Univ., Ithaca, NY. (1974). Senyk, G. F., Lee, E. C., Shipe, W. F., Hood, L. F. and Downes, T. W. J . Food S c i . (1975) 40, 288. Lee, E. C., Senyk, G. F. and Shipe, W. F. J . Dairy Sci. (1975) 58, 473. Brown, R. J., Poe Abstr. 71st Ann. Mtg. J a n o l i n o , V. G. and Swaisgood, H. E. Abstr. 71st Ann. Mtg. Amer. Dairy S c i . Assn. (1976) p50,D37.

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

5 Use of Lactase in the Manufacture of Dairy Products J. H . WOYCHIK and V. H . HOLSINGER Eastern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, Philadelphia, Pa. 19118

The p o t e n t i a l f o r l a c t a s d a i r y products has been recognized f o r many years. Lactase (β-D -galactosidase) hydrolyzes m i l k l a c t o s e i n t o its c o n s t i t u e n t mono saccharides, glucose and galactose. Chemical and p h y s i c a l changes that occur as a r e s u l t of l a c t o s e h y d r o l y s i s provide the r a t i o n a l e f o r its a p p l i c a t i o n . The p r i n c i p a l changes are reduced l a c t o s e content, increased carbohydrate solubility, increased sweetness, higher osmotic pressure, reduced viscosities, and more r e a d i l y fermentable sugar. Enzymatic h y d r o l y s i s o f l a c t o s e i n d a i r y foods would improve product q u a l i t y and provide low-lactose products f o r the l a c t o s e i n t o l e r a n t segment o f the population. A v a i l a b l e Lactases E f f o r t s to utilize l a c t a s e s f o r the manufacture o f hydrolyzed l a c t o s e (HL) products have been r e s t r i c t e d p r i m a r i l y by the l a c k of s u i t a b l e , commercially a v a i l a b l e , enzymes. Lactases a r e found i n p l a n t s , animals and microorganisms (Table I ) , but only micro bial sources can be used commercially. The pH optima of the m i c r o b i a l l a c t a s e s are q u i t e v a r i e d . The two enzymes of commer cial value a r e i s o l a t e d from the fungus, A s p e r g i l l u s n i g e r (1), and the yeast, Saccharomyces lactis (2), and differ widely i n t h e i r p r o p e r t i e s , p a r t i c u l a r l y in pH optimum (Table I I ) . The S. lactis enzyme (pH optimum o f 6.8-7.0) i s i d e a l l y s u i t e d f o r the h y d r o l y s i s o f l a c t o s e i n m i l k and sweet whey (pH 6.6 and 6.1, r e s p e c t i v e l y ) ; l a c k o f stability below pH 6.0 precludes its appli cation to a c i d whey (pH 4.5). The A. n i g e r l a c t a s e (pH optimum 4.0-4.5) has good pH stability, but the fall-off in r e l a t i v e activity at pH values above 4.5 l i m i t s i t s usefulness p r i m a r i l y to a c i d whey. Both o f these l a c t a s e s are a v a i l a b l e commercially i n p u r i f i e d form.

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

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Table I Lactase D i s t r i b u t i o n

Plants Phaseolus v u l g a r i s , k e f i r g r a i n s , almonds, t i p s of w i l d r o s e s , and seeds of soybeans, a l f a l f a and

coffee.

Animal I n t e s t i n a l brush border

(pH

optimum 5.5-6.0).

Microbial B a c t e r i a (pH optimum 6.5-7.5) Fungi (pH optimum 2.5-4.5) Yeasts

(pH optimum 6.0-7.0)

Table I I P r o p e r t i e s of A. n i g e r and S^. l a c t i s Lactases

A. pH Optimum

4.0-4.5

Temperature Optimum pH

Stability

Half-life

niger

55°C 3.0-7.0

S.

lactis

6.8-7.0 35°C 6.0-8.5

(Days) at 50°C

Whole A c i d Whey 5% Lactose

8 100

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

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P u r i t y , a v a i l a b i l i t y , and cost of l a c t a s e s are important c o n s i d e r a t i o n s i n any f u l l - s c a l e enzymatic process f o r l a c t o s e h y d r o l y s i s . Consequently, although l a c t o s e h y d r o l y s i s can be achieved most simply by the a d d i t i o n of s o l u b l e enzyme to milk or whey, immobilized enzyme technology has been evaluated with l a c tases i n an e f f o r t to improve the economics of l a c t o s e h y d r o l y s i s 0,-4,5,6). While e f f o r t s have been made to develop a s a t i s f a c t o r y immobilized process f o r the S^. l a c t i s l a c t a s e (2), i t s s t a b i l i t y f o l l o w i n g immobilization has not been s u f f i c i e n t to warrant i t s use. Problems a s s o c i a t e d w i t h p r o t e i n adsorption to a v a r i e t y of enzyme support systems and maintaining acceptable column s a n i t a t i o n l e v e l s working with n u t r i t i v e substrates, such as m i l k or sweet whey, f u r t h e r l i m i t i t s usefulness. I t would appear that batch treatment with s o l u b l e enzyme would be the method of choice f o r HL d a i r y products made with S_. l a c t i s l a c t a s e . In c o n t r a s t , the A to use i n immobilized form of s o l i d supports ( 3 , 4 , 2 î i , £ ) · Depending on the substrates used and operating c o n d i t i o n s , o p e r a t i o n a l h a l f - l i v e s from 8 to 100 days have been obtained. Although some o p e r a t i o n a l problems s t i l l e x i s t , there i s l i t t l e doubt that the A. n i g e r l a c t a s e can be used s u c c e s s f u l l y i n immobilized systems. Applications The h y d r o l y s i s of l a c t o s e i n whole or skim milk, best accomp l i s h e d by batch treatment with S. l a c t i s l a c t a s e , can be achieved by incubation with 300 ppm l a c t a s e at 32°C f o r 2.5 hr (10), or at 4°C f o r 16 hr (11). H y d r o l y s i s l e v e l s of 70-90% are obtained and the m i l k can be processed i n t o a v a r i e t y of products or used d i r e c t l y as a beverage. Some of the advantages and d i s advantages i n a p p l i c a t i o n of l a c t a s e s i n the manufacture of d a i r y products and processes f o l l o w . F l u i d and Dried M i l k Products Beverage Products. In recent years, numerous s t u d i e s (12, 13,14) have e s t a b l i s h e d a p a t t e r n of l a c t o s e i n t o l e r a n c e among nôh^aucasian c h i l d r e n and a d u l t s that i s a t t r i b u t a b l e to low i n t e s t i n a l l a c t a s e l e v e l s . F l a t u l e n c e , cramps, d i a r r h e a , and p o s s i b l y a general impairment i n normal d i g e s t i v e processes may accompany l a c t o s e i n t o l e r a n c e . The i n a b i l i t y of i n d i v i d u a l s to hydrolyze l a c t o s e can have serious i m p l i c a t i o n s i n n u t r i t i o n programs based on milk products. The HL m i l k prepared i n our l a b o r a t o r y f o r beverage use was evaluated i n terms of i t s p h y s i c a l and o r g a n o l e p t i c p r o p e r t i e s . The f a c t that HL m i l k i s sweeter than normal milk posed some problems i n d e f i n i n g i t s f l a v o r i n t a s t e panel t e s t i n g . Based on f l a v o r scores (Table I I I ) i t was evident that a r e c i p r o c a l r e l a t i o n s h i p e x i s t e d between the amount of l a c t o s e hydrolyzed i n the

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product and i t s f l a v o r score. I t was c l e a r that judges t r e a t e d the marked sweetness as a " f o r e i g n " f l a v o r . A d d i t i o n a l t a s t e panel s t u d i e s showed that h y d r o l y s i s of 30, 60 and 90% l a c t o s e had the same e f f e c t on f l a v o r of m i l k as the a d d i t i o n of 0.3, 0.6, and 0.9% sucrose. Paige et a l . (15) reported that Negro adolescents found m i l k with 90% of i t s l a c t o s e hydrolyzed q u i t e acceptable to d r i n k , although the m a j o r i t y of the group surveyed judged i t sweeter than c o n t r o l m i l k .

Table I I I F l a v o r Scores of Hydrolyzed-Lactose

Milk

% Lactose Hydrolyzed

F l a v o r Score

0

37.

30

37.0

60

36.7

90

36.2

In a d d i t i o n to overcoming the problems of the l a c t o s e i n t o l erant, h y d r o l y s i s of l a c t o s e i n m i l k i s advantageous f o r the p r e p a r a t i o n of m i l k concentrates. A h i g h l y a t t r a c t i v e method of p r e s e r v i n g whole m i l k i s f r e e z i n g a 3:1 concentrate. The products keep much longer than f l u i d m i l k under normal r e f r i g e r a t i o n and, when r e c o n s t i t u t e d , have a f l a v o r v i r t u a l l y i n d i s t i n g u i s h able from f r e s h milk. However, concentrates prepared from normal m i l k have a tendency to t h i c k e n and coagulate on standing. T h i s p r o t e i n d e s t a b i l i z a t i o n r e s u l t s from c r y s t a l l i z a t i o n of l a c t o s e which has been brought to i t s s a t u r a t i o n p o i n t i n the concentrat i o n process. E a r l y research (16) showed that l a c t o s e h y d r o l y s i s l e d to improvement i n the p h y s i c a l s t a b i l i t y of the concentrates during storage. However, h y d r o l y s i s of 90% of l a c t o s e alone d i d not i n c r e a s e storage s t a b i l i t y by more than one month over the c o n t r o l (Figure 1 ) . HL samples heat t r e a t e d at 71°C f o r 30 min a f t e r canning showed only a moderate r i s e i n v i s c o s i t y a f t e r 9 months storage (lower c u r v e ) . Organoleptic e v a l u a t i o n showed no d i f f e r e n c e i n f l a v o r score of the r e c o n s t i t u t e d concentrate with 90% of i t s l a c t o s e hydrolyzed and a f r e s h whole m i l k c o n t r o l with sucrose added. S i m i l a r l y , l a c t o s e c r y s t a l l i z a t i o n was avoided when skim m i l k concentrates were prepared from HL m i l k and stored at c o l d temperatures.

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

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Dried Products. No problems were encountered i n the manuf a c t u r e of m i l k powder from HL whole m i l k . However, HL skim m i l k powder, with a major p o r t i o n of i t s l a c t o s e hydrolyzed, had a tendency to s t i c k to the hot metal s u r f a c e s of the dryer at temperatures s i g n i f i c a n t l y lower than r e g u l a r skim m i l k s t i c k s (60 vs 75°C) at comparable moisture l e v e l s . Thus, the surfaces of the powder c o l l e c t i n g apparatus need to be h e l d below 60°C to avoid the s t i c k i n g problem. Cultured Products. C u l t u r e d products have long been thought to c o n t a i n low l e v e l s of l a c t o s e because of i t s fermentative u t i l i z a t i o n . However, w h i l e the lower l e v e l s of l a c t o s e may make these products more compatible to the l a c t o s e i n t o l e r a n t , the p r a c t i c e of f o r t i f y i n g yogurt with l a c t o s e r e s u l t s i n r e s i d u a l l e v e l s as high as 3.3-5.7% (17). Gyuricsek and Thompson (18) reported that among yogurts prepared from l a c t a s e t r e a t e d m i l k i n which more than 90% of set more r a p i d l y than c o n t r o l Advantages claimed f o r the a p p l i c a t i o n of l a c t a s e i n yogurt manuf a c t u r e i n c l u d e a c c e l e r a t e d a c i d development, which reduces the r e q u i r e d set-time, and a r e d u c t i o n of " a c i d " f l a v o r by the glucose and g a l a c t o s e , which made the p l a i n yogurt more acceptable to consumers. Supplying s t a r t e r c u l t u r e organisms with glucose and g a l a c tose, r a t h e r than l a c t o s e , as an energy source can be expected to r e s u l t i n a l t e r e d carbohydrate u t i l i z a t i o n patterns and metabo l i t e production. 0 L e a r y and Woychik (19,20) undertook a study to d e f i n e the m i c r o b i a l and chemical changes that might occur i n c u l t u r e d d a i r y products manufactured from HL milk. They reported that t y p i c a l sugar concentrations i n the HL m i l k f o r yogurt are glucose, 2.6%; g a l a c t o s e , 2.3%; l a c t o s e , 2.0%. Decrease i n pH accompanied fermentation i n the c o n t r o l and HL milk; l e s s time was r e q u i r e d to reach pH 4.6 i n the HL m i l k than i n the c o n t r o l (Figure 2). The f a s t e r a c i d development i n the HL m i l k i s a r e s u l t of a h i g h er i n i t i a l r a t e o f fermentation. These f i n d i n g s support the decreased set-times reported by Gyuricsek and Thompson (18). The growth curves of the mixed s t a r t e r c u l t u r e c o n s i s t i n g of Streptococcus t h e r m o p h i l i s and L a c t o b a c i l l u s b u l g a r i c u s shown i n Figure 3 demonstrate that l a c t o s e h y d r o l y s i s has no e f f e c t on the symbiotic growth r e l a t i o n s h i p s of the s t a r t e r organisms. The c a r bohydrate u t i l i z a t i o n p a t t e r n s by the c u l t u r e s i n the two yogurts were d i f f e r e n t . The values i n Table IV show that almost twice as much galactose was c a t a b o l i z e d i n the c o n t r o l milks as i n HL milk. At the same time, the t o t a l amount of l a c t i c a c i d produced by the s t a r t e r organisms was greater i n the HL m i l k . These f i n d ings r e f l e c t a l t e r e d patterns of metabolite production r e s u l t i n g from the u t i l i z a t i o n of a greater p r o p o r t i o n of the t o t a l a v a i l able sugar i n the form of glucose. f

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

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Figure 2.Change of pH with time during yogurt production

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

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Table IV Galactose U t i l i z a t i o n i n C o n t r o l and HL Yogurts

Control CHO

(%)

HL

Galactose

(%)

CHO

(%)

Galactose

Initial

7.11

3.50

7.01

3.30

Final

5.23

2.73

5.20

2.95

% Utilized

1.88

0.77

1.81

0.35

(%)

The a c c e l e r a t e d a c i d production by yogurt c u l t u r e organisms i n HL milk was a l s o observed by Gyuricsek and Thompson (18) with c u l t u r e s used i n cottage cheese manufacture. Advantages of using HL m i l k reported (18) i n c l u d e reduced s e t t i n g - t i m e , l e s s curd s h a t t e r and reduced l o s s of f i n e s , a more uniform curd, and use of lower cook-out temperatures. With the exception of improved s t a r t e r c u l t u r e s and more automated equipment, few m o d i f i c a t i o n s have been developed f o r improving the manufacture or a c c e l e r a t i n g the r i p e n i n g of Cheddar cheese. Thompson and Brower (11) extended the use of HL milk to the manufacture of Cheddar cheese and found that l a c t o s e h y d r o l y s i s modified the process c o n s i d e r a b l y . The f a s t e r a c i d development reduced renneting times and s i g n i f i c a n t l y reduced curd cooking and cheddaring times. With equivalent times i n cure, the HL Cheddar was s u p e r i o r to c o n t r o l cheeses i n f l a v o r , body, and texture and more r a p i d l y developed these c h a r a c t e r i s t i c s c l o s e r to that of o l d e r c o n t r o l cheeses. The improved q u a l i t i e s were a t t r i b u t e d to the e f f e c t s of l a c t o s e h y d r o l y s i s . The body and t e x t u r e changes may be the r e s u l t of increased osmolarity accompanied by reduced c a s e i n s o l v a t i o n . The a c c e l e r a t e d r i p e n i n g may be a t t r i b u t a b l e to increased l e v e l s of enzymes r e l e a s e d i n t o the cheese by higher b a c t e r i a l populations when glucose i s a v a i l able as a growth s u b s t r a t e . Whey U t i l i z a t i o n The nation's cheese i n d u s t r y annually produces over 32 b i l l i o n pounds of whey, of which l i t t l e more than h a l f i s u t i l i z e d . In the case of l a c t o s e , t h i s excess production over u t i l i z a t i o n represents approximately 600 m i l l i o n pounds which could conceivably be converted i n t o s a l a b l e products. M o d i f i c a t i o n of whey by l a c t a s e h y d r o l y s i s could o f f e r new avenues f o r whey

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

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utilization. Two of the most promising processes appear to be conversion to l a c t o s e - d e r i v e d s i r u p s and fermentative u t i l i z a tion. Sirups. The low sweetness l e v e l of l a c t o s e r e l a t i v e to sucrose does not allow much d i r e c t competition; however, a cons i d e r a b l e increase i n sweetness r e s u l t s f o l l o w i n g h y d r o l y s i s of l a c t o s e . R e l a t i v e sweetness i s dependent on s e v e r a l f a c t o r s , the p r i n c i p a l one being c o n c e n t r a t i o n . Table V l i s t s the sweetness of s e v e r a l sugars at 10% concentrations r e l a t i v e to sucrose.

Table V R e l a t i v e Sweetness of 10% Aqueous Sugar

Sugar

Solutions

Sweetnes

Sucrose

100

Lactose

40

Glucose

75

Galactose

70

Thus, the a v a i l a b i l i t y of HL wheys increases the p o t e n t i a l f o r producing l a c t o s e - d e r i v e d s i r u p s . HL s i r u p s have been prepared by Guy (21) using both hydrolyzed wheys and l a c t o s e s o l u t i o n s . Deproteinized and demineralized s i r u p s concentrated to 65% t o t a l s o l i d s e x h i b i t e d good s o l u b i l i t i e s , showed no mold growth, and had good humectant p r o p e r t i e s . A ready a p p l i c a t i o n f o r these s i r u p s was found i n caramel manufacture where the HL s i r u p showed the same s t a b i l i z i n g e f f e c t against sucrose c r y s t a l l i z a t i o n as d i d " i n v e r t " sugar. Further a p p l i c a t i o n s f o r these s i r u p s await development by food t e c h n o l o g i s t s . Fermentation. U t i l i z a t i o n of whey as a fermentation subs t r a t e , e s p e c i a l l y f o r s i n g l e c e l l p r o t e i n s (22,23,24) and a l c o h o l production (25), has been studied e x t e n s i v e l y . A major o b s t a c l e to broader u t i l i z a t i o n of whey as a fermentation medium has been the f a c t that r e l a t i v e l y few organisms are able to u t i l i z e l a c t o s e . Several yeasts were evaluated f o r a l c o h o l production i n whey (26) and, although Kluyveromyces f r a g i l i s was found to be the most e f f i c i e n t , only 55% of the l a c t o s e was converted to a l c o h o l . The low conversion l e v e l has been a t t r i b u t e d

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

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8.0 h

L. bulgoricus, CONTROL MILK

5.0

S. thermophilic, LH MILK S. thermophillus, CONTROL MILK 1

2 TIME,

3 HOURS

4

Figure 3. Growth of yogurt cultures in control and HL milks

TIME, HOURS Figure 4. Alcohol production by K. fragilis in control and HL acid whey

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

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to a p o s s i b l e i n a b i l i t y o f the yeast to t o l e r a t e high a l c o h o l concentrations p o s s i b l y because of s e n s i t i v i t y of the yeast l a c tase to a l c o h o l . The a v a i l a b i l i t y o f whey having i t s l a c t o s e hydrolyzed to i t s c o n s t i t u e n t monosaccharides permitted an e v a l u a t i o n of nonlactose fermenting organisms such as Saccharomyces c e r e v i s i a e , which t o l e r a t e high a l c o h o l c o n c e n t r a t i o n s , f o r a l c o h o l production. O'Leary et a l . (27) evaluated HL wheys using c e r e v i s i a e and K. f r a g i l i s f o r comparison. Curves i l l u s t r a t ing a l c o h o l production by glucose-pregrown K. f r a g i l i s i n c o n t r o l and HL wheys are shown i n Figure 4. Ethanol production was more r a p i d i n HL whey during the f i r s t 24 h r fermentation, but l a t e r decreased so that the r a t e o f production was l e s s than that i n the c o n t r o l whey. The t o t a l y i e l d o f ethanol was s i m i l a r i n both cases and averaged 1.9%. Reducing sugar l e v e l s decreased at the same r a t e i n the f i r s t 24 hr fermentation, but l e s s u t i l i z a t i o n occured i n HL wheys i n the l a t e r stages of fermentation The p a t t e r n o f sugar u t i l i z a t i o showed that although glucos 24 h r fermentation, there was l i t t l e change i n the galactose conc e n t r a t i o n during t h i s p e r i o d . Galactose u t i l i z a t i o n began only a f t e r 24 hr fermentation and i s t y p i c a l of a d i a u x i c p a t t e r n o f sugar u t i l i z a t i o n . A longer fermentation p e r i o d was r e q u i r e d i n the HL whey due to the d i a u x i c phenomenon. S_. c e r e v i s i a e , a nonlactose fermenting yeast, i s commonly used i n wine and beer production because of i t s a b i l i t y to w i t h stand r e l a t i v e l y high a l c o h o l concentrations. Growth curves of IS. c e r e v i s i a e , pregrown on glucose and on g a l a c t o s e , showed that s i m i l a r c e l l populations e x i s t e d i n HL whey f o r the f i r s t 48 hr growth, but d e c l i n e d t h e r e a f t e r , except that those pregrown on galactose increased s i g n i f i c a n t l y a f t e r the i n t i a l d e c l i n e . A l c o h o l production by these organisms i n the HL wheys showed a s i m i l a r p a t t e r n i n that comparable l e v e l s of a l c o h o l were a t t a i n e d a f t e r 48 h r (Figure 6 ) . A l c o h o l production by the g l u cose pregrown S_. c e r e v i s i a e began to decrease a f t e r 48 h r , whereas the g a l a c t o s e pregrown c e l l s continued t h e i r a l c o h o l production which reached a l e v e l twice that o f the glucosepregrown c e l l s . Sugar u t i l i z a t i o n c l o s e l y p a r a l l e l e d a l c o h o l production. S^. c e r e v i s i a e pregrown on glucose u t i l i z e d glucose r a p i d l y but d i d not u t i l i z e g a l a c t o s e . J3. c e r e v i s i a e pregrown on galactose s i m i l a r l y u t i l i z e d glucose but a l s o u t i l i z e d galactose a f t e r 96 h r . These s t u d i e s i n d i c a t e d that although l a c t o s e h y d r o l y s i s i n whey permits a l c o h o l production by organisms unable to ferment l a c t o s e , the galactose r e l e a s e d by h y d r o l y s i s i s not e f f i c i e n t l y u t i l i z e d . In model system s t u d i e s , the e f f i c i e n c y of converting sugar to a l c o h o l by K. f r a g i l i s was as f o l l o w s : glucose > l a c t o s e > g a l a c t o s e . Conclusions The d a i r y i n d u s t r y today has a v a i l a b l e e x i s t i n g technology f o r the a p p l i c a t i o n o f enzymatic h y d r o l y s i s of l a c t o s e i n m i l k

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

woYCHiK A N D HOLSiNGER

Lactase in Dairy Mdnufdcturing

J 10

*

1 " - - * Γ "

Figure 5. Change in sugar concentrations dur ing fermentation of HL acid whey by K. fragilis

10

50

100

150

200

TIME, HOURS Figure 6. Alcohol production in HL acid whey by glu cose-pregrown and gahctose-pregrown S. cerevisiae

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and m i l k products. U t i l i z a t i o n of the l a c t a s e enzyme, i n e i t h e r " f r e e " o f immobilized systems, can r e s u l t i n q u a l i t y improvements i n a number o f products and y i e l d processing economies i n others. The a p p l i c a t i o n discussed i n t h i s report should be reviewed and assessed f o r p o t e n t i a l b e n e f i t s by d a i r y product manufactur ers i n t h e i r s p e c i f i c operations. Whey, i n p a r t i c u l a r , with i t s ever i n c r e a s i n g production, poses a s p e c i a l problem whose s o l u t i o n can be a t t a i n e d only through a p p l i c a t i o n of unconventional technology. I t i s b e l i e v e d that a p p l i c a t i o n o f l a c t a s e o f f e r s a p o t e n t i a l f o r i n n o v a t i v e whey u t i l i z a t i o n . Literature Cited 1. 2.

3. 4. 5. 6. 7. 8. 9.

10. 11. 12. 13. 14. 15.

16. 17. 18.

Woychik, J . Η., and Wondolowski, M. V. Biochim. Biophys. Acta (1972) 289, 347-351. Woychik, J . Η., Wondolowski, M. V., and Dahl, K. J . In "Immobilized Enzyme A. C. and Cooney, New York, Ν. Y. (1974). Woychik, J . Η., and Wondolowski, M. V. J . M i l k Food Technol. (1973) 36, 31-33. W i e r z b i c k i , L. E., Edwards, V. Η., and Kosikowski, F. V. B i o t e c h n o l . Bioeng. (1974) 16, 397-411. P i t c h e r , W. H. J r . Am. Dairy Rev. (1957) 37, 34B, 34D, 34E. Pastore, Μ., M o r i s i , F., and Viglia, A. J . Dairy Sci. (1974) 57, 269-272. Hustad, G. O., Richardson, T., and Olson, N. F. J . D a i r y Sci. (1973) 56, 1111-1117. Olson, A. C., and Stanley, W. L. J . A g r i . Food Chem. (1973) 21, 440-445. Hasselburger, F. X., A l l e n , B., Paruchuri, Ε. Κ., Charles, Μ., and Coughlin, R. W. Biochem. Biophys. Res. Commun. (1974) 57, 1054-1062. Guy, E. J., Tamsma, Α., Kontson, Α., and H o l s i n g e r , V. H. Food Prod. Develop. (1974) 8, 50-60. Thompson, M. P., and Brower, D. P. Cultured Dairy Prod. J . (1976) 11, 22-23. Paige, D. Μ., Bayless, Τ. Μ., F e r r y , G. C., and Graham, G. C. Johns Hopkins Med. J . (1971) 129, 163-169. Rosensweig, N. S. J. Dairy Sci. (1969) 52, 585-587. Lebenthal, E., Antonowicz, I., and Schwachman, H. Amer. J . Clin. Nutr. (1975) 28, 595-600. Paige, D. Μ., Bayless, Τ. Μ., Huang, S., and Wexler, R. ACS Symposium S e r i e s , " P h y s i o l o g i c a l E f f e c t s of Food Carbohy d r a t e s , " (1974), 191. Tumerman, L., Fram, Η., and C o r n e l y , K. W. J . Dairy Sci. (1954) 37, 830-839. Anonymous. Cultured Dairy Prod. J . (1973) 8, 16-19. Gyuricsek, D. Μ., and Thompson, M. P. Cultured Dairy Prod. J. (1976) 11, 12-13.

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

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19. 20. 21. 22. 23. 24. 25. 26.

27.

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O'Leary, V. S., and Woychik, J. H. J. Food Sci. (1976) 41, 791-793. O'Leary, V. S., and Woychik, J. App. Environmental Micro biol. (1976) 32, 89-94. Guy, E. J. Abstract 71st Annual Meeting, Amer. Dairy Sci. Asso. (1976), D7. Wasserman, A. E., Hopkins, W. J., and Porges, N. Sewage and I n d u s t r i a l Wastes (1958) 30, 913-920. Wasserman, A. E., Hampson, J. W., A l v a r e , N. F., and A l v a r e , N. J. J. Dairy Sci. (1961) 44, 387-392. B e r n s t e i n , S., Tzeng, C. Η., and S i s s o n , D. F i r s t Chem. Congr. N. Amer. Cont. (1975) A b s t r a c t BMPC 68. Rogosa, Μ., Browne, Η. Η., and W h i t t i e r , E. O. J. Dairy Sci. (1947) 30, 263-269. O'Leary, V. S., Green, R., S u l l i v a n , B. C. and H o l s i n g e r , V. H. F i r s t Chem. Congr. N. Amer. Cont. (1975) Abstract BMPC 158. O'Leary, V.S., Sutton H o l s i n g e r , V. H. Unpublished.

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

6 Immunochemical Approach to Questions Related to αand ß-Amylases in Barley and Wheat J. DAUSSANT C.N.R.S., Physiologie des Organes Végétaux, 92190 Meudon, France

I· I n t r o d u c t i o n I n food and beverage processing o( - and £ - amylases have an important f u n c t i o n i n two main areas : beer and bread making. I n these i n d u s t r i e s , there i s s t i l l a need f o r d e f i n i n g the source and the q u a l i t y o f barley, malt, and wheat. Among the biochemical parameters used f o r c h a r a c t e r i z i n g these products, Λ - n d (3 - amylases are of great i n t e r e s t : the a c t i v i t i e s are used f o r d e f i n i n g the seed q u a l i t y and a b e t t e r understanding o f the isoenzymic contents seems promising f o r cha r a c t e r i z i n g seeds. However, Çt- amylase a c t i v i t i e s and Q - amyl a s e zymograms are d i f f i c u l t t o d e f i n e when OK - amylase i s present. The immunochemical c h a r a c t e r i z a t i o n o f oK - and Q- amylases which provided new i n f o r m a t i o n on b a r l e y and wheat amylases i n p h y s i o l o g i c a l s t u d i e s may a l s o be u s e f u l i n the above mentioned characterizations. This a r t i c l e w i l l present r e c e n t l y developed techniques : 1) f o r studying q u a l i t a t i v e l y and q u a n t i t a t i v e l y ^ - amylases i n presence o f θ(- amylases, and 2) f o r i d e n t i f y i n g the o r i g i n o f C ( - amylase sometimes pre sent i n small amounts i n mature and apparently ungerminated wheat seeds. a

I I . Q u a n t i t a t i v e and Q u a l i t a t i v e Techniques f o r @- Amylase Inves t i g a t i o n s i n the Presence o f o Ç - Amylase A. The problem. The c h a r a c t e r i z a t i o n o f amylases i s p a r t i c u l a r l y important f o r e v a l u a t i n g malt used i n brewing. The a c t i v i t i e s o f both C l - and amylases and t h e i r r a t i o c h a r a c t e r i z e malt ; the data are o f major importance f o r the brewing process. E l e c t r o p h o r e t i c a n a l y s i s o f CK- and @- amylase c o n s t i t u e n t s may prov i d e means o f g e t t i n g a deeper i n s i g h t i n t o the q u a l i t y and the o r i g i n o f the malt. However, the measurement and the d e t e c t i o n of these a c t i v i t i e s remain approximate. I n f a c t , OC- amylase may be c h a r a c t e r i z e d i n the presence o f amylase by using s t a r c h

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

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preincubated with β - amylase* T h i s procedure i s used f o r quan t i t a t i v e as w e l l as f o r q u a l i t a t i v e techniques such as e l e c t r o p h o r e s i s . Nevertheless, f o r q u a n t i t a t i n g |3- amylase a c t i v i t y by measurement of reducing sugars that appear upon i n c u b a t i o n of s t a r c h with the enzyme, the e v a l u a t i o n i s approximate when 0C~ amylase i s present. In f a c t , θζ - amylase a l s o produces reducing sugars by i n c u b a t i o n w i t h s t a r c h , and there i s a s y n e r g i s t i c e f f e c t between oC~ and β - amylases. On the other hand, i n e l e c t r o p h o r e t i c a n a l y s i s of β - amylase c o n s t i t u e n t s , some of them may be hidden by OC- amylase components. An i n h i b i t i o n o f OC- amylase and not of (3 - amylase could then be used : 0L- amylase r e q u i r e s Ca f o r i t s a c t i v i t y which i s not the case f o r β - amylase. Thus, the treatment with p o l y phosphates, c i t r a t e or EDTA which i n h i b i t s the OC- amylase a c t i v i t y ( l ) could solve the problem although amylases which may not r e q u i r e C a have bee reported (2) W s h a l l d e s c r i b her techniques i n v o l v i n g th t i t u e n t s by using an immun specifi θ{- amylas techniques permit q u a l i t a t i v e as w e l l as q u a n t i t a t i v e a n a l y s i s of (i - amylases i n presence of 0( - amylases. For q u a n t i t a t i o n of Λ - amylase, we have developed a d i f f u s i o n technique s i m i l a r t o a d i f f u s i o n technique developed f o r OC- amylase determination (3)· Ρ - amylase and, to a smaller extent, OC - amylase are damaged by the r o a s t i n g process of malting ; both enzymes being a l t e r e d in their activities. The r e s u l t s of previous immunochemical s t u d i e s on b a r l e y and malt β- amylases i n d i c a t e d that malting, and i n p a r t i c u l a r r o a s t i n g , do not a f f e c t much the a n t i g e n i c s t r u c t u re of the enzyme. An immunochemical measurement of the enzymatic p r o t e i n contents i n b a r l e y and malt e x t r a c t s could thus be c a r r i e d out (4)· On the b a s i s of immunochemical c h a r a c t e r i z a t i o n of b a r l e y β - amylase, techniques f o r q u a n t i t a t i n g the enzyme are mentioned. Data concerning ^ - amylase a c t i v i t y , even i n presen ce o f θ(- amylase, and {3 - amylase p r o t e i n content provide a means of o b t a i n i n g β - amylase s p e c i f i c a c t i v i t y . T h i s parameter could than serve f o r f u r t h e r c h a r a c t e r i z i n g e f f e c t s of r o a s t i n g i n the malting process. + +

B. Combined immunoabsorption and QÇ - amylase determination i n the same g e l medium (5> 6 ) . amylase s o l u t i o n s were poured i n t o w e l l s cut i n an agarose g e l c o n t a i n i n g 1,5 % s t a r c h p r e i n c u bated with ft- amylase. The enzymes were allowed to d i f f u s e f o r 2.4 h at 20°C and the g e l s were s t a i n e d with an i o d i n e s o l u t i o n . The Q( - amylase a c t i v i t y was r e f l e c t e d by white spots occuring on a red background. There i s a l i n e a r r e l a t i o n s h i p between the diameter of the r e a c t i o n spots and the logarithm o f the OC- amyl a s e c o n c e n t r a t i o n (3)· The immune serum a n t i 0C- amylase of germinated seeds was the one d e s c r i b e d i n s e c t i o n I I I , B. OC- amylase p u r i f i e d by an a f f i n i t y technique (7) from e x t r a c t of germinated barley seeds was used f o r immunization.

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

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For immunoabsorption the w e l l s were f i r s t f i l l e d with the immune serum and when the serum was sucked i n by the g e l they were r e f i l l e d with the malt e x t r a c t . During d i f f u s i o n the c< - amylase meets the a n t i b o d i e s which are a l l around the w e l l s and p r e c i p i t a te, o r are i n h i b i t e d by an excess o f a n t i b o d i e s . F i g . l shows the r e s u l t s o f the immunoabsorption o f - amylase e x t r a c t e d from 7 days germinated b a r l e y seeds. D i f f e r e n t d i l u t i o n s o f the immune serum were t e s t e d f o r the immunoabsorption o f o( - amylase from the malt e x t r a c t . D i l u t i o n 3 o f the immune serum absorbed p r a c t i c a l l y a l l o f the - amylase i n the malt e x t r a c t . This d i l u t i o n o f the immune serum was then used f o r e l i m i n a t i n g amylase from the malt e x t r a c t , i n order t o measure i t s |3 - amyl a s e a c t i v i t y . I t i s noteworthy that, i n order t o e l i m i n a t e the small - amylase-like a c t i v i t y present i n the immune serum, the l a t t e r was heated at 50°C f o r 30 min before use. C. D i f f u s i o n techniqu rose g e l i n the absenc to the one developed by B r i g g s ( 3 ) f o r - amylase determination ; s t a r c h i s used as substrate i n s t e a d of l i m i t d e x t r i n s (Fig.2 upper p a r t ) . D i f f e r e n t d i l u t i o n s o f barley e x t r a c t were poured i n t o

F i g . l - Immunoabsorption o f Q4- amylase from malt e x t r a c t and eval u a t i o n o f the remaining a c t i v i t y a f t e r absorption. This experiment shows that d i l u t i o n 3 o f the a n t i 0^ - amylase immune serum absorbs p r a c t i c a l l y a l l o(,- amylase present i n the malt e x t r a c t . Upper and middle p a r t : w e l l s were c u t i n a 1.2 % agarose g e l prepared i n 0.2 M acetate b u f f e r , pH 5·7> c o n t a i n i n g 1.5 % s t a r c h preincubated with amylase. Upper p a r t : w e l l s were f i l l e d with d i f f e r e n t concentrations o f o(- amylase ( d i l u t i o n s e r i e s ) . I n the i n i t i a l malt e x t r a c t , the oC- amylase concentration was a r b i t r a r i l y designated as 100. Middle p a r t : For OC- amylase immunoabsorption, d i f f e r e n t d i l u t i o n s of the immune serum were f i r s t depos i t e d i n the w e l l s . A f t e r these s o l u t i o n s were sucked i n by the gel the i n i t i a l malt e x t r a c t (cone. 100 o f the enzyme) was deposited i n the w e l l s . A f t e r 24 hr d i f f u s i o n a t 20°C, the g e l was s t a i n e d with i o d i n e . Note that the r e a c t i o n spot corresponding t o the immunoabsorption o f the malt e x t r a c t with d i l u t i o n 3 o f the immune serum (lS/3) i s much smaller than the r e a c t i o n spot corresponding to concentration 0.2 o f the malt Ck- amylase. Lower p a r t o f the f i g u r e : R e l a t i o n s h i p s between diameter o f r e a c t i o n spot and logarithm o f amylase concentration. The p o i n t s represent the mean values o f two measurements made on each o f three r e a c t i o n spots corresponding t o one experiment. Absorption with d i l u t i o n s 27 and 9 o f the immune serum r e s u l t e d i n the absorption o f only p a r t o f the i n i t i a l a c t i v i t y ( r e s p e c t i v e l y about 40 % and 75 °/°) · D i l u t i o n 3 o f the immune serum absorbs more than 99*8 % o f the i n i t i a l activity.

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

DAUSSANT

Amylases in Barley and Wheat

Figure 1

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3 mm diameter w e l l s c u t i n agarose g e l , c o n t a i n i n g 0& % s t a r c h (see legend o f F i g . 2 f o r d e t a i l s ) . The enzyme was allowed t o d i f fuse i n t o the g e l f o r 24 h. at 20°C. The g e l was then s t a i n e d with i o d i n e s o l u t i o n . The amylase a c t i v i t y i n the barley ex t r a c t s o l u t i o n s was r e f l e c t e d by pink spots o f d i f f e r e n t s i z e s , o c c u r r i n g on a blue background. The diameter o f the r e a c t i o n spots and the logarithm o f the corresponding f*> - amylase concen t r a t i o n s c o r r e l a t e i n a l i n e a r r e l a t i o n s h i p ( F i g . 2 , lower p a r t ) . p> - amylase may e x i s t under d i f f e r e n t forms of aggregation which are d i s r u p t e d by reducing agents (see (4) f o r review). I n order t o convert these aggregate forms o f \*> - amylase i n t o t h e i r b a s i c u n i t s , 0.03 % mercapto ethanol was used i n the s o l u t i o n s . When X - amylase i s present, as i s the case i n the malt e x t r a c t s , the r e a c t i o n spots are white and there i s no p o s s i b i l i t y o f measuring - amylase a c t i v i t y without having s p e c i f i c a l l y removed o ( - amy l a s e from the s o l u t i o n ( F i g . 2 upper p a r t M a l t ) D. Combined immuno-absorptio l a s e determination i n the same g e l medium.The w e l l s cut" i n the gel c o n t a i n i n g s t a r c h were f i r s t f i l l e d with d i l u t i o n 3 o f the heated antiCX, - amylase immune serum. When the s o l u t i o n was sucked i n by the g e l , the d i f f e r e n t d i l u t i o n s o f barley e x t r a c t and d i l u t i o n 2 o f the malt e x t r a c t were poured i n t o the w e l l s (Fig.2 mid dle p a r t ) . The a c t i v i t y o f the enzyme was r e f l e c t e d f o r the ex-

F i g . 2 - Π>- amylase a c t i v i t y estimation using a d i f f u s i o n t e c h n i que which a l s o i n v o l v e s the s p e c i f i c e l i m i n a t i o n o f *X - amylase present i n malt e x t r a c t s by immunoabsorption. Upper and middle p a r t s : wells were c u t i n a 1.2 % agarose g e l prepared i n 0.1 M phosphate b u f f e r pH 6 c o n t a i n i n g 0.8 % s t a r c h . Upper part : The w e l l s were f i l l e d with d i f f e r e n t d i l u t i o n s o f the barley e x t r a c t ( P>- amylase concentration i n the i n i t i a l e x t r a c t was a r b i t r a r i l y designated as 100), o r with the malt e x t r a c t d i l u t e d twice. Mid dle p a r t : The w e l l s were f i r s t f i l l e d with d i l u t i o n 3 o f the ant i C X - amylase immune serum. When the s o l u t i o n s were sucked i n by the g e l , the wells were r e f i l l e d with the d i f f e r e n t d i l u t i o n s o f the b a r l e y e x t r a c t * o r with the malt e x t r a c t d i l u t e d twice. A f t e r 24 hr d i f f u s i o n a t 20°C, the g e l was s t a i n e d with i o d i n e . Note that the immune serum has no e f f e c t on the diameter o f the reac t i o n spots corresponding t o the d i l u t i o n s o f the b a r l e y e x t r a c t . With the malt e x t r a c t , the absorption allows one t o get a reac t i o n spot corresponding t o fi - amylase and not t o (A- amylase. Lower p a r t : R e l a t i o n s h i p between diameter o f r e a c t i o n spot and logarithm o f /3- amylase concentration i n barley e x t r a c t s . Each p o i n t represents the mean value o f two measurements made on each of the three r e a c t i o n spots corresponding t o one experiment. fi> amylase a c t i v i t y i n the twice d i l u t e d malt e x t r a c t corresponds t o 60 °/o o f the a c t i v i t y i n the b a r l e y e x t r a c t .

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

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Amylases in Barley and Wheat

Figure 2

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t r a c t s of b a r l e y and malt by pink spots on a blue background. The presence of the immune serum d i d not modify the s i z e of the spot diameters corresponding to the d i l u t i o n s of the b a r l e y ex t r a c t . This i n d i c a t e s that the a c t i v i t y measurement of β» - amyla se was s t i l l p o s s i b l e under these c o n d i t i o n s , β - amylase a c t i v i t y i n the twice d i l u t e d malt e x t r a c t was found to be n e a r l y 60 % of the a c t i v i t y i n barley e x t r a c t . E. Combined immunoelectroabsorption of o< - amylase and e l e c t r o p h o r e s i s of p> - amylase i n the same g e l medium (8). The p r i n c i p l e of t h i s technique i s the same as the preceeding technique, except that enzymatic antigens are brought i n t o contact with the antibodies by means of e l e c t r o p h o r e s i s , i n s t e a d of d i f f u s i o n . The technique aims at p r o v i d i n g from a mixture of <\ - amylase and |°> amylase^ electropherograms of β - amylase alone by e l i m i n a t i n g s p e c i f i c a l l y o{ - amylase c o n s t i t u e n t s : the immune serum a n t i amylase of germinated b a r l e the agarose g e l ; when r e f i l l e d with the e x t r a c t s of e i t h e r b a r l e y or malt. During e l e c t r o p h o r e s i s , o<- amylase meets the a n t i b o d i e s , and when these are i n s u f f i c i e n t amount, the enzyme-antibody complexes form a p r e c i p i t a t e or i n h i b i t the enzyme a c t i v i t y . C h a r a c t e r i z a t i o n r e a c t i o n s were c a r r i e d out on the g e l s a f t e r e l e c t r o p h o r e s i s . D e t a i l s of the technique are described i n Fig.3· On the upper part of the f i g u r e , the immune serum (IS) i s shown to absorb a l l X - amylase present i n the e x t r a c t . The absorption i s s p e c i f i c s i n c e the se rum taken before immunization (s) does not modify the electrophor e t i c p a t t e r n of O^- amylase. On the lower p a r t of f i g u r e 3 i t i s shown that serum and immune serum do not modify the electrophoret i c p a t t e r n of β - amylase i n b a r l e y e x t r a c t . Since a l l amy l a s e c o n s t i t u e n t s can be removed from the malt e x t r a c t by immuno absorption, a comparison between β - amylase c o n s t i t u e n t s in barley and malt can be made · A clearcut d i f f e -

Fig«3 - Electro-immunoabsorption of e x t r a c t s of barley and barley malt using an immune serum s p e c i f i c f o r <X - amylase from germina ted b a r l e y s e e d s . W e l l s were cut i n an 1.2 % agarose g e l prepared i n 0.025 M veronal b u f f e r pH 8.6. Before e l e c t r o p h o r e s i s the wells were f i l l e d with d i f f e r e n t s o l u t i o n s : veronal b u f f e r (V), serum taken before immunization (s), immune serum a n t i t X - amylase (IS). A f t e r the s o l u t i o n s were taken up by the g e l , the w e l l s were r e f i l l e d with e i t h e r the barley or the malt e x t r a c t . A f t e r the s o l u t i o n s were taken up by the g e l , the wells were f i l l e d with hot l i q u i d agarose,and e l e c t r o p h o r e s i s was c a r r i e d out f o r about 5 hr at 3°C, s t a r r i n g with 6 V/cm. A f t e r e l e c t r o p h o r e s i s , charac t e r i z a t i o n r e a c t i o n s were c a r r i e d out on the g e l s using l i m i t dex t r i n e f o r <X- amylase c h a r a c t e r i z a t i o n and s t a r c h f o r c< - and p>- amylase c h a r a c t e r i z a t i o 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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Figure 3

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rence i n the e l e c t r o p h o r e t i c m o b i l i t y o f b a r l e y and barley malt |3 " amylase c o n s t i t u e n t s was thus evinced. F. Remarks. l ) the non d e t e c t a b l e r e a c t i v i t y o f the a n t i <X - amylase immune serum with β - amylase o f b a r l e y e x t r a c t i n d i c a t e s that with t h i s immune serum, c< - and β - amylases d i s p l a y no a n t i g e n i c s i m i l a r i t i e s . T h i s confirms that they correspond t o q u i t e d i f f e r e n t molecular s p e c i e s . However, i f i n the malt ex t r a c t , o< - and β - amylases form h y b r i d molecules, the immuno absorption o f - amylase could a c t on the β - amylase analyses. The complexes formed by '\ - amylases and the corresponding a n t i bodies would thus c o n t a i n those β - amylase molecules which are h y b r i d i z e d with :χ^- amylases. I f the complexes p r e c i p i t a t e , i n both techniques the β - amylase molecules would escape the analy s i s . I f the complexes s t i l l remain s o l u b l e , because o f a l a r g e excess o f a n t i b o d i e s ^ the β - amylase contained i n the complexes may be i n h i b i t e d . I n an of the complexes are q u i t In p a r t i c u l a r , the complexes are o f much l a r g e r s i z e than β - amy l a s e and must t h e r e f o r e d i f f u s e with a much slower r a t e i n the g e l than β - amylase. The β - amylase molecules would then escape the /'- amylase determination i n the d i f f u s i o n q u a n t i t a t i v e tech nique. The p o s s i b i l i t y o f h y b r i d i z a t i o n between X - and β - amy lases must be s t u d i e d f u r t h e r . Immunoabsorption o f o( - amylase using an a n t i β - amylase immune serum could be c a r r i e d out ; a d i minution o f 0^ - amylase a c t i v i t y would evidence t h a t 'X- amylase i s enclosed i n β - amylase-antibody complexes. 2) The d i f f e r e n c e i n electrophoretic m o b i l i t i e s between β amylases from b a r l e y and malt shown by t h i s technique confirms the preceding r e s u l t s obtained using immunoelectrophoretic a n a l y s i s . - amylases from b a r l e y and malt appeared a n t i g e n i c a l l y i d e n t i c a l using v a r i o u s immune sera, whereas one immune serum allowed the observance o f a n t i g e n i c s i m i l a r i t i e s as w e l l as a n t i g e n i c d i f f e rences (see (4) f o r review). The same observations were reported f o r wheat β - amylases. The causes of the β - amylase m o d i f i c a t i o n s were i n v e s t i g a t e d by experiments combining i n v i v o l a b e l l e d amino acids i n c o r p o r a t i o n i n t o p r o t e i n s and immunochemical i d e n t i f i c a t i o n o f β - amylase. I t was found that the d i f f e r e n c e s were due t o a process modifying the enzymes e x i s t i n g i n mature wheat upon germination r a t h e r than t o a disappearance of these enzymes and synthesis o f new β - amylase molecules during germination (9)· This phenomenon i s probably a l s o the one i n v o l v e d i n barley β amylase m o d i f i c a t i o n upon germination. I t remains t o be seen whe ther each c o n s t i t u e n t of mature seed β- amylase i s converted i n t o a s p e c i f i c c o n s t i t u e n t i n germinated seed β- amylase. 3) We described a technique f o r e v a l u a t i n g β- amylase a c t i v i t y i n the presence o f <X- amylase by s p e c i f i c a l l y removing 'X - amylase from the mixture. Immunochemical s t u d i e s on b a r l e 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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and malt β- amylases allowed one to envisage immunochemical quan t i t a t i o n of p - amylase p r o t e i n s i n p r o t e i n mixtures, such as bar l e y and malt e x t r a c t s . Both e v a l u a t i o n s provide means f o r estima t i n g s p e c i f i c β - amylase a c t i v i t y i n the e x t r a c t s (see (4) f o r review) : β - amylase from b a r l e y and malt appeared to be a n t i g e n i c a l l y i d e n t i c a l using v a r i o u s immune sera, whereas with other immune s e r a , a n t i g e n i c d i f f e r e n c e s between these enzymes were de t e c t e d . Only the f i r s t s o r t of immune serum could be used f o r comparing amounts of β - amylase p r o t e i n s i n b a r l e y and malt ex t r a c t s . With the immunoabsorption technique, a l l /3- amylase c o n s t i t u e n t s of barley and malt were shown t o r e a c t with the immu ne serum. Thus, a l l /3- amylase c o n s t i t u e n t s could a c t u a l l y be q u a n t i t a t e d at once. Techniques i n v o l v i n g a migration of the en zyme i n a g e l c o n t a i n i n g the immune serum could be a p p l i e d . The migration may be promoted by d i f f u s i o n (lO) or by e l e c t r o p h o r e s i s ( l l ) , the l a t t e r technique f o r q u a n t i t a t i o n of one - amylase constituent i s describe The study o f s p e c i f i and e s p e c i a l l y during k i l n i n g i s thus p o s s i b l e f o r f u r t h e r cha r a c t e r i z a t i o n o f malt. I I I . Immunochemical I d e n t i f i c a t i o n of X- Amylase i n Wheat Seeds A. The problem. For breadmaking, too high a l e v e l o f amylase a c t i v i t y i n wheat c o n s t i t u t e s a major concern i n Europe (12, 13)· °( - amylase a c t i v i t y i n mature wheat o f t e n occurs i n northern and western Europe as a consequence of the moist and tem perate c l i m a t e p r e v a i l i n g during the r i p e n i n g p e r i o d o f the g r a i n (14)· This a c t i v i t y may r e s u l t from the presence of sprouted g r a i n s i n the crops, but not n e c e s s a r i l y so ; the development of "X - amylase need not be a s s o c i a t e d with sprouting (12). In fact, the c o r r e l a t i o n between v i s i b l e sprouted g r a i n s and o( - amylase a c t i v i t y i s poor (15)· I t was observed that the enzyme l e v e l i n absence of v i s i b l e s p r o u t i n g may already be much higher i n sprou ted seeds than i n sound g r a i n s (l6). An i n c r e a s e i n 0^ - amylase a c t i v i t y i n wheat seeds during storage and without apparent s p r o u t i n g was a l s o reported (17)· Several sorts of amylases may be accounted f o r by the ac t i v i t y i n sound and apparently ungerminated seeds : enzymes exoge nous t o the seeds ( f u n g i , b a c t e r i a ) or enzymes endogenous to the seeds. I n the l a t t e r case, s e v e r a l isoenzymes may be i n v o l v e d and i t i s worthy t o r e c a l l b r i e f l y the f e a t u r e of 0^- amylase i n de v e l o p i n g , maturing and germinating wheat seeds.c<- amylase a c t i v i t y present i n developing seeds vanishes during maturation and reoccurs d r a m a t i c a l l y during germination (l8, 19> 20). A s e t of - amylases present i n developing seeds appears to be e l e c t r o p h o r e t i c a l l y and a n t i g e n i c a l l y i d e n t i c a l to O^- amylases o f germina ted seeds (21, 22, 23, 24)· Another s e t o f oC- amylases i n deve l o p i n g seeds appears t o be s p e c i f i c f o r the developing seeds (2l) whereas the major p a r t o f the a c t i v i t y i n germinating seeds seems

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to be due to enzymes not present i n developing seeds (21, 22, 23> 24> 25)· X - amylases o c c u r r i n g i n c e r e a l s during germination are known t o be synthesized i n the aleurone l a y e r , the enzymes found i n developing seeds, however, have been l o c a l i z e d i n the p e r i c a r p (13, 19, 20, 26, 27, 28). I t i s worth n o t i n g that - amylase found i n endosperm and aleurone during l a t e stages of development was a s s o c i a t e d with damaged areas of the k e r n e l (26). One may speculate t h a t amylase secreted i n the aleurone l a y e r may, i n v i v o s l i g h t l y d i f f u s e i n t o the starchy endosperm. Thus, i t could mo d i f y p a r t of the s t a r c h , whereas the (χ- amylase l o c a t e d i n the p e r i c a r p would have l e s s opportunity to attack i n v i v o the s t a r chy endosperm. The d i f f e r e n c e i n the l o c a l i z a t i o n of the two t y pes of X- amylases and the d i f f e r e n c e s i n the s p e c i f i c a c t i v i t y of these enzymes may e x p l a i n some cases concerning t r i t i c a l e , whe re both f a l l i n g number and a c t i v i t y l e v e l s were found to be high (29)· The disappearance of χ amylase during maturation seems more probably due to a d e s t r u c t i o t i v a t i o n or to an i n s o l u b i l i z a t i o Further evidence was provided, i n d i c a t i n g that the g r e a t e s t p r o p o r t i o n of (X - amylase which appears during germination and i s i d e n t i c a l to c<- amylase o f developing seeds, r e s u l t s more probably from i n v i v o s y n t h e s i s than from a regeneration of pre e x i s t i n g enzymes (21, 30)· The problem concerning o^- amylase a c t i v i t y i n wheat i s q u i t e d i f f e r e n t i n c o u n t r i e s with a more a r i d c l i m a t e compared to north western Europe, and f l o u r must o f t e n be complemented with amylases of exogenous o r i g i n (14)· Nevertheless, the s o r t of o<^- amylase added to f l o u r (enz/mes from germinated wheat or b a r l e y seeds, from f u n g i , or from b a c t e r i a ) i s not i n d i f f e r e n t to the process of breadmaking and to the q u a l i t y of the end product, due p a r t i c u l a r l y to the d i f f e r e n t thermostability of the enzymes (17)· The c h a r a c t e r i z a t i o n of O^- amylase present i n wheat seeds and f l o u r would thus c o n s t i t u t e an i n t e r e s t i n g complementary means of e v a l u a t i n g these products. I w i l l now show how an immunoche m i c a l approach to the problem could be attempted on the b a s i s of

F i g . 4 - E l e c t r o p h o r e t i c and immunoelectrophoretic analyses of ex t r a c t s of germinated wheat seeds (upper w e l l s ) and developing wheat seeds (lower w e l l s ) . E l e c t r o p h o r e s i s was performed i n 1.2 % agarose g e l prepared i n 0.025 M veronal b u f f e r pH 8.6 at 4°C un der 6 V/cm f o r 50 min. Q{ - amylase c h a r a c t e r i z a t i o n r e a c t i o n was performed a f t e r e l e c t r o p h o r e s i s or Immunoelectrophoresis. Upper p a r t : electropherogram. Middle p a r t : immunoelectropherogram i n v o l v i n g a serum s p e c i f i c f o r X- amylases of developing seeds. Lower p a r t : immunoelectropherogram i n v o l v i n g a serum s p e c i f i c f o r o<, - amylases of germinated seeds. U2> G2 designate amylase groups detected i n e x t r a c t s of developing and of germina t i n g seeds, r e s p e c t i v e l 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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Figure 4

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immunochemical data concerning wheat p a r t i c u l a r technique t o be described.

- amylases and using a

B. Immunochemical c h a r a c t e r i z a t i o n o f wheat Ok- amylases. The c h a r a c t e r i z a t i o n was c a r r i e d out using an immune serum s p e c i f i c for - amylases o f developing seeds and another immune serum specific for amylases o f germinating seeds (4> 21, 30). The r e s u l t s may be summarized as f o l l o w s : 1. The immune sera were prepared by i n j e c t i n g r a b b i t s with 'X - amylase p u r i f i e d from seed e x t r a c t s according t o Loyter and Schramm (?)· Freezedried wheat seeds taken 15 days a f t e r anthesis and a mixture o f f r e e z e d r i e d b a r l e y seeds germinated f o r 3>5 and 7 days were used f o r p r o t e i n e x t r a c t i o n . Previous studies showing i d e n t i t y r e a c t i o n s between oC~ amylasesof germinated seeds, from b a r l e y and wheat using the a n t i - b a r l e y o{- amylase immune serum i n d i c a t e d the p o s s i b i l i t amylase s t u d i e s (32). Later with the p r o t e i n s bearing most o f the 0(- amylase a c t i v i t y i n wheat germinated seeds (25)· 2. Absorption techniques showed that the immune serum s p e c i f i c f o r θ( - amylase o f developing seeds reacted with a l l ^ - amy l a s e enzymes e x t r a c t e d from the developing seeds. As j u s t repor ted, i t a l s o confirmed that the immune serum s p e c i f i c f o r <X - amy l a s e o f germinated barley seeds reacted with a l l cX,- amylase enzy mes extracted from germinated wheat seeds. Thus, the reagents permitted the i d e n t i f i c a t i o n o f a l l c<- amylase types endogenous to developing and germinating wheat seeds ( 21 ) . 3· I d e n t i f i c a t i o n with the immune serum s p e c i f i c f o r X amylases o f developing seeds i s shown on Fig.4· By using immunoe l e c t r o p h o r e t i c a n a l y s i s , two a n t i g e n i c θ{- amylases ( and D2) were i d e n t i f i e d i n e x t r a c t s o f developing seeds. They correspond to the two 0 ^ - amylase groups, one anodic, the other cathodic, de t e c t e d by agarose g e l e l e c t r o p h o r e s i s a t pH 8.6. The two c<- amy l a s e s were shown t o be a n t i g e n i c a l l y q u i t e d i f f e r e n t . I n e x t r a c t s of germinating seeds, two a n t i g e n i c C<- amylases (G^ and G2) were detected, which corresponded t o the two Ok- amylase groups e v i n ced by agarose g e l e l e c t r o p h o r e s i s a t pH 8.6. A partial identity r e a c t i o n was suggested between these two c o n s t i t u e n t s using d i f f e r e n t techniques. The p a r t i a l i d e n t i t y r e a c t i o n i n d i c a t e d that, while antibodies r e a c t with both G^ and G2 c o n s t i t u e n t s , other antibodies r e a c t only with G^. An i d e n t i t y r e a c t i o n was observed between and G^. This means that a l l antibodies which r e a c t with D i r e a c t a l s o with G^. (By using long d u r a t i o n agarose g e l e l e c t r o p h o r e s i s a t pH 8.6, i t was shown that and G^ each con t a i n e d three c o n s t i t u e n t s d i f f e r i n g by t h e i r e l e c t r o p h o r e t i c mobi l i t y but a l l a n t i g e n i c a l l y i d e n t i c a l ) ^ ( 2 1 , 30) . To summ up, the immune serum permitted the i d e n t i f i c a t i o n o f

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one c o n s t i t u e n t s p e c i f i c f o r developing seeds (D^) and another with e l e c t r o p h o r e t i c a l l y and a n t i g e n i c a l l y i d e n t i c a l p r o p e r t i e s i n developing and germinating seeds. Furthermore, c e r t a i n a n t i genic r e l a t i o n s h i p s w e r e suggested and a n t i g e n i c d i f f e r e n c e s were shown between and G^, consequently between and G^» 4· I d e n t i f i c a t i o n with the immune serum s p e c i f i c f o r ok - amy l a s e s o f germinating seeds i s shown on Fig.4· Using immunoelect r o p h o r e t i c a n a l y s i s , two groups of a n t i g e n i c - amylase (G^ and G^) were detected i n e x t r a c t s of germinating seeds. The antigens corresponded t o the enzymatic groups detected by agarose e l e c t r o p h o r e s i s . A p a r t i a l i d e n t i t y r e a c t i o n between the c o n s t i t u e n t s was suggested : t h i s i n d i c a t e d that, while a n t i b o d i e s reacted with G-. and G£9 f u r t h e r a n t i b o d i e s reacted only with G^. The p a r t i a l i d e n t i t y r e a c t i o n i m p l i e s , i n p a r t i c u l a r , that more a n t i b o d i e s r e a c t with G^ than with G^ The immune serum r e a c t e d with only one o f the a n t i g e n i c ok A r e a c t i o n of i d e n t i t y wa s u l t s i n d i c a t e , i n p a r t i c u l a r , that the enzymic group which bears most o f the ok - amylase a c t i v i t y i n germinating seeds (G2) has an a n t i g e n i c s t r u c t u r e which does not e x i s t i n o^- amylase of develo ping seeds (31>30). C. P r i n c i p l e of i d e n t i f i c a t i o n of ok - amylases p a r t i c u l a r t o germinating seeds ( G p J . T h e above mentioned r e s u l t s i n d i c a t e that the immune serum a n t i «χ- amylase from germinated seeds, con t a i n s a n t i b o d i e s s p e c i f i c f o r enzymes of the germinated seeds, G-L and G2> but some of these a n t i b o d i e s a l s o r e a c t with one o
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amylase c h a r a c t e r i z a t i o n r e a c t i o n was performed on the g e l a f t e r electrophoresis. In these p r e l i m i n a r y experiments designated f o r i n v e s t i g a t i n g seeds with low Ck- amylase a c t i v i t y , d i l u t i o n s s e r i e s of a d i l u t e d e x t r a c t of 7 days germinated seeds was used. The highest a c t i v i ty o f the d i l u t i o n s e r i e s evaluated according to the d i f f u s i o n technique (3) described e a r l i e r f o r determination of malt ok - amy l a s e was 14·75 mm/24 h. This a c t i v i t y was designated as 100. (Such an a c t i v i t y was found f o r seeds d i s p l a y i n g 4·5 u n i t s accor ding the S Κ Β technique). The concentration of the absorbed im mune serum i n the g e l was 0.03 %· Under these c o n d i t i o n s one p r e c i p i t i n peak was detected f o r the s o l u t i o n s with a c t i v i t y 100, 30, 10 r e s p e c t i v e l y ( F i g . 5A, g ) . I t i s worthy to note that the θ( amylase c h a r a c t e r i z a t i o n on the p r e c i p i t i n band increases the sen s i t i v i t y of the technique ; the immunopeaks are not v i s i b l e a f t e r p r o t e i n amidoblack s t a i n i n g In order to i n c r e a s more, we combined rocke e l e c t r o p h o r e s i s (34) with the enzymatic c h a r a c t e r i z a t i o n r e a c t i o n ( F i g . 5 B). A g e l s t r i p c o n t a i n i n g a d i l u t i o n of the germinated seed e x t r a c t was i n s e r t e d between the g e l c o n t a i n i n g the immune serum and the g e l where the w e l l s were c u t . During electrophore s i s , the ok - amylase of the g e l s t r i p migrates as a continuous.

F i g - 5 - Immunochemical technique f o r i d e n t i f y i n g the ok- amylase antigen c h a r a c t e r i s t i c f o r germinated seeds i n e x t r a c t s of appa r e n t l y ungerminated seeds. The technique was performed i n 1.2 °/o agarose prepared i n 0.025 M veronal b u f f e r pH 8.6. Electropho r e s i s was c a r r i e d out at 4°C f o r 5 hrs s t a r t i n g with 6 V/cm. ok amylase c h a r a c t e r i z a t i o n r e a c t i o n was performed on the g e l a f t e r electrophoresis. A : Rocket Immunoelectrophoresis (see t e x t ) . Β and C : Combination of rocket and l i n e Immunoelectrophoresis (see t e x t ) . g : i n c r e a s i n g d i l u t i o n s e r i e s of e x t r a c t of 7 days germinated wheat seeds. d : d i l u t i o n s of e x t r a c t s of wheat seeds taken 15 days a f t e r anthesis. b : d i l u t i o n s of b a c t e r i a l ok- amylase s o l u t i o n . C l , Ca, Ma : e x t r a c t s of three apparently ungerminated seed sam ples. A c t i v i t i e s : a c t i v i t y i n each s o l u t i o n was evaluated by a d i f f u s i o n technique (3)· A c t i v i t i e s are expressed i n percent of the a c t i v i t y i n the f i r s t d i l u t i o n of the germinated seed e x t r a c t (see text)· Note that the shape of the w e l l s i n C permits t o pour more s o l u t i o n than i n the w e l l s shown i n Β ; t h i s r e s u l t s i n a b e t t e r v i z u a l i z a t i o n o f the peaks.

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

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Amylases in Barley and Wheat

Figure 5

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band i n the g e l c o n t a i n i n g a n t i b o d i e s , i t forms complexes with the a n t i b o d i e s and p r e c i p i t a t e s as a l i n e when a c e r t a i n r a t i o between antigen and a n t i b o d i e s i s reached. Nevertheless, i n the zones where there are a d d i t i o n a l amounts of enzyme moved from the w e l l s by e l e c t r o p h o r e s i s , the l i n e i s broken and forms peaks, the s i z e of which i s i n d i r e c t r e l a t i o n to the amount of a d d i t i o n a l antigen ( F i g . 5 B). In order t o o b t a i n the highest s e n s i t i v i t y , a maximum d i l u t i o n of the wheat e x t r a c t was used ; here the 5 broad g e l s t r i p contained 12 °/o of the d i l u t e d germinated seed e x t r a c t ( s o l u t i o n with a c t i v i t y designated as 100). The ok - amyl a s e G2 was then detected with t h i s technique f o r the s o l u t i o n s with a c t i v i t y 100, 30, 10, 3 r e s p e c t i v e l y (Fig.5 B g ) . Thus, the l a t t e r technique appears three times more s e n s i t i v e than the preceeding one. The combination of rocket and l i n e Immunoelectrophoresis was then t e s t e d f o r i t s s p e c i f i c i t y and f o r i t s c a p a c i t y to evaluate the c o n s t i t u e n t c h a r a c t e r i s t i p a r e n t l y not germinated amylase a c t i v i t y . The a c t i v i t y of a l l the p r e p a r a t i o n s t e s t e d was evaluated by the d i f f u s i o n technique (3)· The s c a l e was e s t a b l i s h e d using the d i l u t i o n s e r i e s of the d i l u t e d germinated wheat seed e x t r a c t . The a c t i v i t y i n the f i r s t d i l u t i o n was designated as 100. The a c t i v i t y i n the d i f f e r e n t p r e p a r a t i o n s i s then expressed as per cent of the a c t i v i t y i n t h i s germinated seed extract dilution. A small peak on the p r e c i p i t i n band was s t i l l detected f o r the d i l u t e d germinating seed e x t r a c t with a c t i v i t y 3 (Fig.50> g,3)» Such a peak was not detected on the l i n e above the w e l l s c o n t a i ning the s o l u t i o n s of b a c t e r i a l <X - amylase with a c t i v i t y 70 and 210 (Fig.5C, b ) . T h i s was a l s o true f o r much higher b a c t e r i a l o< amylase a c t i v i t y so that the enzyme d i d not at a l l r e a c t with antibodies s p e c i f i c f o r the G2 c o n s t i t u e n t . Concerning o(- amylase of developing seeds no peak was detected on the l i n e above the w e l l s c o n t a i n i n g d i l u t i o n s of the e x t r a c t with a c t i v i t y 23 and 70 (Fig.5C, d, 23, d, 70). However f o r a l e s s d i l u t e d e x t r a c t , with a c t i v i t y 210, a small peak was detected (compare Fig.5C, d, 210 and Fig.5C, g, 3)· I t i s not yet known whether t h i s i s due t o the presence i n developing seeds of very small amounts of a n t i g e n i c a l c o n s t i t u e n t G2 or to an e f f e c t which i s due to the a n t i g e n i c r e semblance between G^ and G2 and which i s not e l i m i n a t e d by the absorption as performed under our c o n d i t i o n s ; t h i s p o i n t remains to be f u r t h e r i n v e s t i g a t e d . Nevertheless,the technique makes i t p o s s i b l e t o compare the t o t a l <X - amylase a c t i v i t y of the seed and the corresponding amount of c o n s t i t u e n t G2 and to i n d i c a t e whether the a c t i v i t y i s mainly due to t h i s c o n s t i t u e n t . For example, e x t r a c t s of apparently not germinated seeds which presented a small ^ - amylase a c t i v i t y were submitted to the a n a l y s i s ( F i g . 50, C l , Ca, Ma). The a c t i v i t y i n these ext r a c t s were estimated by the d i f f u s i o n technique (3) and approximated r e s p e c t i v e l y a c t i v i t i e s 10, 30 and 120 i n our s c a l e . In m m

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these e x t r a c t s , the amounts o f the a n t i g e n i c c o n s t i t u e n t G 2 r e f l e c t e d by the s i z e o f the peaks were s i m i l a r t o the amounts o f t h i s c o n s t i t u e n t i n the d i l u t i o n s o f the germinated seed e x t r a c t d i s p l a y i n g a c t i v i t y 10, 30 and 100 r e s p e c t i v e l y . P r a c t i c a l l y the r e s u l t s i n d i c a t e that i n these apparently ungerminated samples, the small amylase a c t i v i t y was mainly due t o the c o n s t i t u e n t c h a r a c t e r i s t i c f o r germination. E. Remarks. The immunochemical c h a r a c t e r i z a t i o n o f X - amy l a s e s from germinated seeds served t o s e t up q u a l i t a t i v e and quan t i t a t i v e techniques f o r d i s t i n g u i s h i n g between λ - amylases from germinated seeds and Λ - amylases exogenous t o the seeds ( f u n g i , bactéries), (6, 8 ) . We have d e s c r i b e d here a s e n s i t i v e technique f o r i d e n t i f y i n g the a n t i g e n i c X - amylase s p e c i f i c f o r germination i n seeds showing a small X. - amylase a c t i v i t y The q u a n t i t a t i v e p o s s i b i l i t i e s o f the techniqu f i e d p r e p a r a t i o n s o f th t i n g seeds ( G 2 ) , i n order t o e s t a b l i s h a r e f e r e n c e s c a l e . The same technique could be developed f o r d e t e c t i n g and quan t i t a t i n g the two other a n t i g e n i c Λ - amylases evinced i n germi n a t i n g and developing wheat seeds. For t h i s purpose, the immune serum s p e c i f i c f o r X - amylases from developing seeds must be used. An immune serum s p e c i f i c f o r the amylase c o n s t i t u e n t common to developing and germinating seeds (D^ and G^) could be obtained by absorbing the immune serum with the p u r i f i e d antigen s p e c i f i c f o r germinated seeds ( G 2 ) · As f a r as D 2 i s concerned, s i n c e the enzyme migrates towards the cathode by e l e c t r o p h o r e s i s a t pH 8 . 6 , the w e l l s must be c u t i n the anodic s i d e o f the g e l , the g e l s t r i p c o n t a i n i n g the e x t r a c t o f developing seeds being i n s e r t e d between the g e l with the w e l l s and the g e l c o n t a i n i n g the immune serum. A n t i g e n i c a l r e l a t i o n s h i p s have been reported between X - amy l a s e s o f c e r t a i n c e r e a l s (32, 35)· Thus, some o f the immune s e r a could be used f o r d e t e c t i o n i n s e v e r a l c e r e a l s . IV. A b s t r a c t s . Two types of application of the immunochemical characteriza tion of α- amylases i n barley and wheat are presented. The f i r s t type of application allows the elimination of αamylase from seed extracts i n a specific and easy way without any interference with β- amylase i n electrophoretic or diffusion techniques i n g e l . Thus, electrophorograms of β- amylase from seed extracts also containing α- amylase can be obtained, and quantitation of β- amylase a c t i v i t y i n extracts containing αamylase can be carried out using a gel diffusion technique. The second type of application aims at identifying i n sound

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seeds, the origin of the very low α- amylase activity present : whether it i s due to the seed enzymes or to enzymes exogenous to the seeds (bacteria,fungi). I t also tends at identifying whether the enzymes are characteristic of the developmental or of the ger minating part of the l i f e of the seed. V. L i t e r a t u r e c i t e d . 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.

SCRIBAN, R. - B r a s s e r i e (1965), 221, 3-15. JACOBSEN, J.V., SCANDALIOS, J.C. and VARNER, J.E. - P l a n t . P h y s i o l . (1970), 45, 367-371. BRIGGS, D.E. - J. I n s t . Brew. (1962), 68, 27-33. DAUSSANT, J. - in "Immunological aspects o f food", N. CATSIMPOOLAS ( E d i t o r ) AVI pub. Co. (in p r e s s ) . DAUSSANT, J., SKAKOUN, A. and NIKU-PAAVOLA, M.L. - J. I n s t . Brew. (1974) 80 55-58 DAUSSANT, J. and SKAKOUN 4, 127-133. LOYTER, A. and SCHRAMM, M. - Biochem. Biophys. Acta (1962), 65, 200-206. DAUSSANT, J. and CARFANTAN, N. - J. of Immunol. Methods (1975) 8, 373-382. DAUSSANT, J. and CORVAZIER, P., FEBS l e t t e r s (1970), 7, 191194. MANCINI, G., CARBONARA, A.O. and HEREMANS, J.F., in Immunochemistry, Pergamon Press (1965), 235-254. LAURELL, G.B., Annal. Biochem. (1966), 15, 45-52. LACROIX, L.J., WAIKAKUL, P. and YOUNG, G.M., C e r e a l . Res. Comm. (1976), 4, 139-146. KRUGER, J.E., C e r e a l Res. Comm. (1976), 4, 187-194· OLERED, R., P u b l i c a t i o n o f the department of Plant Husbandry, Upsala, Almqvist and W i k s e l l s (1967), 23, 1-106. BELDEROCK, Β., Field Crop Abstr. (1968), 21, 203-211. GALE, M.D., C e r e a l Res. Comm. (1976), 4, 231-243. MERCIER, C. and COLAS, Α., Ann. Nutr. Alim. (1967), 21, 299340. KNEEN, E., C e r e a l Chem. (1944), 21, 304-314. OLERED, R., A r k i v . Kemi. (1964), 22, 175-183. SANDSTEDT, R.M. and BECKORD, O.C., Cereal Chem., (1946), 23, 548-559. DAUSSANT, J. and RENARD, M., C e r e a l Res. Comm. (1976), 4, 201212. KRUGER,J.E.,C e r e a l Chem. (1972 a), 49, 391-398. OLERED, R. and JÖNSSON, G., J. Sci. Fd. A g r i c . (1970), 21, 385-392. OLERED, R., C e r e a l Res. Comm., (1976), 4, 195-199· DAUSSANT, J. and RENARD, M., FEBS l e t t e r s , (1972), 22, 301304. DEDIO, W., SIMMONDS, D.H., HILL, R.D. and SHEALY, Η., Can J. P l a n t . Sci., (1975), 55, 29-36.

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6.

27. 28.

DAUSSANT

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KRUGER, J.E., C e r e a l Chem. (1972 b ) , 49, 379-390. MacGREGOR, A.W., GORDON, A.G., MEREDITH, W.O.S. and LACROIX,L.

J. I n s t . Brew., (1972), 78, 174-179. 29. CHOJNACKI, G., BRYKCZYINSKI, J. and TYMIENIECKA, Ε., C e r e a l

Res. Comm., (1976), 4, 111-114.

30.

DAUSSANT, J., LAURI ERE, C., CARFANTAN, N. and SKAKOUN, A., i n Phytochemical S o c i e t y Symposia, Academic Press ( i n Press). 31. GUILBOT, A. and DRAPRON, R., Ann. P h y s i o l . Veg. (1963), 5,

5-18.

32. DAUSSANT, J. and GRABAR, P., Ann. I n s t . Pasteur, (1966),

110,

79-83. 33. CLARKE, H.G.M. and FREEMAN, T., in "Prot. Biol. F l u i d s " , H. Peeters ( E d i t o r ) , E l s e v i e r Amsterdam (1967), 503-

509. 34. KRØLL, J., Scand. J. o f Immunology (1973), 2,

( S u p p l . l ) , 83-

87. 35.

ALEXANDRESCU, V. an

mie, (1973), 10, 89-94. VI. Acknowledgements. The author thanks Miss C. M AYER f o r s k i l f u l t e c h n i c a l a s s i s tance, Mrs R. DAUSSANT f o r her help i n c o r r e c t i n g the e n g l i s h text and Dr R.L. ORY f o r having reviewed the a r t i c l e . T h i s study was supported i n p a r t by the "Centre Technique de la B r a s s e r i e e t M a l t e r i e françaises", Nancy, France.

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

7 Immobilized Enzymes A L F R E D C. OLSON and ROGER A. KORUS Western Regional Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Berkeley, Calif. 94710

An immobilized enzym i s c o n s t r a i n e d one way o r a n o t h e r w i t h i n the l i m i t e d c o n f i n e s of a s o l i d support. T h e subject of i m m o b i l i z e d e n z y m e s has r e c e i v e d a s i g n i f i c a n t a m o u n t of attention i n r e c e n t y e a r s b e c a u s e of a d v a n t a g e s that h a v e b e e n and m i g h t be a c h i e v e d through s u c c e s s f u l a p p l i c a t i o n s i n a n u m b e r of f i e l d s i n c l u d i n g food p r o c e s s i n g . T h i s c h a p t e r on i m m o b i l i z e d e n z y m e s h a s b e e n divided into f i v e p a r t s b e g i n n i n g with an i n t r o d u c t i o n in w h i c h we h a v e t r i e d to p o s i t i o n i m m o b i l i z e d e n z y m e s w i t h i n the larger context of the use of e n z y m e s i n food p r o c e s s i n g , p o i n t i n g out a d v a n t a g e s and l i m i t a t i o n s to the method. T h i s i s f o l l o w e d by a s e c t i o n on s u p p o r t s that h a v e b e e n u s e d to i m m o b i l i z e e n z y m e s p a r t i c u l a r l y f o r food a p p l i c a t i o n s . A t h i r d s e c t i o n c o v e r s s o m e e x a m p l e s of i m m o b i l i z e d e n z y m e s c u r r e n t l y i n u s e i n food p r o c e s s i n g f o l l o w e d by a s e c t i o n on p r o p o s e d a p p l i c a t i o n s of i m m o b i l i z e d e n z y m e s . S i n c e m u c h of the l i t e r a t u r e on the s u b j e c t e x p r e s s e s r e s u l t s i n t e r m s that m a k e c o m p a r i s o n between d i f f e r e n t m e t h o d s of i m m o b i l i z a t i o n d i f f i c u l t , the f i n a l s e c t i o n i s a b r i e f d i s c u s s i o n of s o m e of the c o m m o n p a r a m e t e r s u s e d to d e s c r i b e i m m o b i l i z e d e n z y m e s y s t e m s . The c h a p t e r i s a s e l e c t e d s u r v e y of the r e c e n t l i t e r a t u r e with s o m e a p p l i c a t i o n s c o v e r e d i n m o r e d e t a i l i n o t h e r c h a p t e r s i n this book. T h e i n genuity, c o m p l e x i t y and d i v e r s i t y of m e t h o d s f o r i m m o b i l i z i n g e n z y m e s has b e e n l o o k e d at from the point of v i e w of t h e i r app l i c a t i o n to food p r o c e s s i n g , with s o m e s u g g e s t i o n s as to the f u t u r e u s e s of i m m o b i l i z e d e n z y m e s i n food p r o c e s s i n g . A s l i t t l e as 20-30 y e a r s ago a g r e a t d e a l of a r t and t r a d i t i o n w e r e still i n v o l v e d i n the use of e n z y m e s to p r o c e s s foods. M u c h of the t e c h n i c a l knowledge we now h a v e r e g a r d i n g e n z y m e s and t h e i r a p p l i c a t i o n to food p r o c e s s i n g has b e e n d i s c o v e r e d and d e v e l o p e d i n the i n t e r v e n i n g y e a r s . B a s i c i n f o r m a t i o n on e n z y m e s r e l a t e d to food c a n be found i n b o o k s b y R e e d (1) and W h i t a k e r (2,_3). M o r e s p e c i a l i z e d b o o k s by Z a b o r s k y (4), P y e and W i n g a r d (5), D u n l a p (6), O l s o n and C o o n e y (7) and S a l m o n a , S a r o n i o and G a r a t t i n i (8) d i s c u s s the u s e of i m m o b i l i z e d

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

7.

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AND

KORUS

101

Immobilized Enzymes

e n z y m e s and r e l a t e d s u b j e c t s . A n u m b e r of r e v i e w s on i m m o b i l i z e d e n z y m e s h a v e a l s o a p p e a r e d (9-15). W i t h the s i t u a t i o n c h a n g i n g so r a p i d l y the p r o b l e m of des c r i b i n g new d e v e l o p m e n t s i n a p p l i e d food e n z y m o l o g y b e c o m e s v e r y d i f f i c u l t . T h e r e i s often a d i c h o t o m y of i n t e r e s t between b a s i c s c i e n t i f i c studies and c o m m e r c i a l a p p l i c a t i o n s . Ideas, e x p e r i m e n t s , p r o c e s s e s and p r o d u c t s that l o o k good i n the l a b o r a t o r y m a y not m e e t m a r k e t i n g r e q u i r e m e n t s , c u s t o m e r d e s i r e s or the i n t e r e s t of a p r o m o t e r . A l a r g e i n v e s t m e n t by a c o m p a n y in a new p r o c e s s i n v o l v i n g e n z y m e s w i l l p r o b a b l y r e s u l t i n the c o m p a n y k e e p i n g that i n f o r m a t i o n p r o p r i e t a r y a s l o n g a s p o s s i ble. T o p r o j e c t what r o l e e n z y m e s , i n c l u d i n g i m m o b i l i z e d enz y m e s , m a y h a v e i n food p r o c e s s i n g i n the f u t u r e i s even m o r e d i f f i c u l t . T h e e x e r c i s e i s not without m e r i t , h o w e v e r , f o r an o v e r v i e w of the s i t u a t i o n often c l a r i f i e s o b j e c t i v e s and b r i n g s into f o c u s a r e a s w h e r e p r o g r e s s can be m a d e A study of r e c e n t d e v e l o p m e n t p r o c e s s i n g can be d i v i d e e n z y m e p r o d u c t i o n , e n z y m e p u r i f i c a t i o n and e n z y m e a p p l i c a t i o n . A l l t h r e e a r e a s a r e of i m p o r t a n c e and p r o m i s e and a r e c u r r e n t l y r e c e i v i n g a g r e a t d e a l of attention. F o r e x a m p l e , by a p p l y i n g knowledge f r o m studies m a d e i n m i c r o b i a l g e n e t i c s and i n d u c e d e n z y m e s y n t h e s i s it m a y be p o s s i b l e to p r e p a r e m u c h l a r g e r a m o u n t s of i n t r a - and e x t r a - c e l l u l a r food grade e n z y m e s than was p o s s i b l e b e f o r e . T h e s e e n z y m e s can be u s e d as h e a t s t a b i l i z e d whole c e l l s o r p u r i f i e d b y a v a r i e t y of p r o c e d u r e s . A s u m m a r y of the steps i n v o l v e d i n the c o m m e r c i a l p r o d u c t i o n and p u r i f i c a t i o n of e n z y m e s f o r food use i s shown i n F i g u r e 1.

ENZYME

SOURCES

ENZYME

PURIFICATION

ANIMAL GLANDS EVAPORATOR

CENTRIFUGE OR FILTER PRESS

PLANT TISSUE

MEMBRANE FILTRATION

REVERSE OSMOSIS

CHROMATOGRAPHY

ACETONE PRECIPITATION

ELECTROPHORESIS SPRAY DRIER FRACTIONAL PRECIPITATION

FREEZE DRYER

~ T ~ MICROORGANISMS r

I

DRIER

I

DRY CRUDE PRODUCT FERMENTOR

CONCENTRATED LIQUID PRODUCT

FRACTIONATED PRODUCT

STANDARDIZED DRY PRODUCT

DILUTE LIQUID PRODUCT

Figure 1. Enzyme production and purification

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

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PROCESSING

W h i l e s o m e e n z y m e s s u c h a s r e n n i n and p a p a i n a r e obtained f r o m a n i m a l and plant s o u r c e s , the m a j o r i t y a r e f r o m m i c r o o r g a n i s m s . P u r i f i c a t i o n i n v o l v e s s o m e k i n d of c o l l e c t i o n f o l l o w e d by a c o n c e n t r a t i o n step. E n z y m e p r e p a r a t i o n and purification that i s done i n b a t c h e s m a k e s r e p r o d u c i b i l i t y d i f f i c u l t and c o s t s h i g h . E f f o r t s a r e u n d e r w a y i n a n u m b e r of l a b o r a t o r i e s to be a b l e to c o n t i n u o u s l y p r o d u c e , p u r i f y , and u s e e n z y m e s . In the c o m m e r c i a l a p p l i c a t i o n of e n z y m e s it s h o u l d be advantageous to c o m b i n e continuous p r o d u c t i o n , p u r i f i c a t i o n and u s e of e n z y m e s , i n c l u d i n g i m m o b i l i z e d e n z y m e s , at a s i n g l e l o c a t i o n . T h e a b i l i t y to i m m o b i l i z e e n z y m e s , then, r e p r e s e n t s only one p a r t of a l a r g e r r e s e a r c h effort that i s c h a n g i n g food p r o c e s s i n g . What a r e s o m e of the advantages to i m m o b i l i z e an enzyme? B r i e f l y , they i n c l u d e (1) the a b i l i t y to u s e the e n z y m e m a n y t i m e s m o r e than the s o l u b l e e n z y m e can be u s e d , (2) l o w e r e n z y m e c o s t s (because of 1), (3) no e n z y m e left i n the p r o d u c t , (4) i n c r e a s e d s t a b i l i t b e e n shown to s t a b i l i z on e x p o s u r e to s u b s t r a t e ) , (5) i m p r o v e d e n z y m e b e h a v i o r ( p H o p t i m a shifted to m o r e advantageous p H on i m m o b i l i z a t i o n to c e r t a i n s u p p o r t s ) , (6) continuous p r o c e s s p o s s i b l e , (7) b e t t e r q u a l i t y c o n t r o l of the p r o d u c t (because of 6), (8) l e s s a d d i t i o n a l p r o c e s s i n g (because of 3), (9) l o w e r l a b o r c o s t s , and (10) a d vantageous u s e of m u l t i p l e e n z y m e s y s t e m s . There are also p o t e n t i a l d i s a d v a n t a g e s w h i c h i n c l u d e (1) the cost of the i m m o b i l i z a t i o n , (2) l o s s of e n z y m e a c t i v i t y on i m m o b i l i z a t i o n , (3) g r e a t e r i n i t i a l plant i n v e s t m e n t , (4) m o r e t e c h n i c a l l y c o m p l e x p r o c e s s , (5) m o r e s k i l l e d s u p e r v i s i o n of p r o c e s s r e q u i r e d (because of 4), (6) h i g h e r l a b o r c o s t s (because of 5), (7) unique s a n i t a t i o n a n d t o x i c o l o g y p r o b l e m s , and (8) a p p l i c a b l e m a i n l y to soluble substrates. W h i l e these g e n e r a l i z a t i o n s c a n p r o v i d e s o m e g u i d e l i n e s , f i n a l c o n s i d e r a t i o n s that d e t e r m i n e w h e t h e r it is advantageous to i m m o b i l i z e an e n z y m e depend on the p a r t i c u l a r p r o c e s s one i s c o n s i d e r i n g . E n z y m e Supports In this s e c t i o n we h a v e c o v e r e d i n f o r m a t i o n on e n z y m e i m m o b i l i z a t i o n i n t e r m s of s u p p o r t s that h a v e b e e n u s e d f o r this purpose including polysaccharides, inorganic supports, fibrous p r o t e i n s , s y n t h e t i c p o l y m e r s , h y d r o g e l s and h o l l o w f i b e r s . E n z y m e s c a n be c o n s t r a i n e d o r h e l d to these s u p p o r t s b y a d sorption, covalent attachment, c r o s s l i n k i n g , entrapment, m i c r o e n c a p s u l a t i o n o r b y v a r i o u s c o m b i n a t i o n s of the f o r e g o i n g m e t h o d s . E x a m p l e s of these m e t h o d s of c o n s t r a i n t w i l l be d i s c u s s e d w h e r e they h a v e b e e n u s e d w i t h p a r t i c u l a r s u p p o r t m a t e rials. T h e v a r i e t y of s u p p o r t m a t e r i a l s that h a v e b e e n p r o p o s e d with s o m e s u i t a b i l i t y f o r a food p r o c e s s i n g a p p l i c a t i o n i s worth noting.

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

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103

P o l y s a c c h a r i d e S u p p o r t s . A l a r g e n u m b e r of p o l y s a c c h a r i d e and substituted p o l y s a c c h a r i d e m a t e r i a l s have b e e n u s e d as s u p p o r t s for e n z y m e i m m o b i l i z a t i o n . T h e s e s u p p o r t s h a v e b e e n u s e d f o r a d s o r p t i o n of e n z y m e s i n a n u m b e r of food p r o c e s s i n g a p p l i c a t i o n s s u c h as p r o d u c t i o n of L - a m i n o a c i d s i n J a p a n w h i c h u s e s D E A E - S e p h a d e x (16, 17) and the C l i n t o n C o r n P r o c e s s i n g C o m p a n y p r o c e s s for h i g h f r u c t o s e c o r n s y r u p i n w h i c h g l u c o s e i s o m e r a s e a d s o r b e d on D E A E - c e l l u l o s e i s u s e d i n r e a c t o r s c o n s i s t i n g of a n a s s e m b l y of s h a l l o w beds of the i m m o b i l i z e d e n z y m e (18). C e l l u l o s e i s the m a j o r constituent of f i b r o u s plants and the m o s t abundant o r g a n i c m a t e r i a l i n n a t u r e . T h e m o n o m e r units of c e l l u l o s e a r e D - g l u c o s e . A l a r g e n u m b e r of substituted c e l l u l o s e s have b e e n p r e p a r e d by r e p l a c i n g the h y d r o x y l g r o u p s b y v a r i o u s substituents. R e p l a c e m e n t by d i e t h y l a m i n o e t h y l ( D E A E ) substituents i n t r o d u c e s a n a m i n o g r o u p w h i c h p r o v i d e s a polycationic, weakly c e l l u l o s e h a s b e e n one z y m e i m m o b i l i z a t i o n . O n the other h a n d , r e p l a c e m e n t of h y d r o x y l g r o u p s by c a r b o x y m e t h y l ( C M ) substituents i n t r o d u c e s c a r b o x y l g r o u p s w h i c h p r o v i d e a w e a k l y a c i d i c c a t i o n exchange material. O t h e r p o p u l a r s u p p o r t s for e n z y m e i m m o b i l i z a t i o n a r e Sephadex and substituted Sephadex. Sephadex i s a b e a d e d , c r o s s l i n k e d d e x t r a n . D e x t r a n s a r e p o l y s a c c h a r i d e s of g l u c o s e units that a r e p r o d u c e d by s t r a i n s of L e u c o n o s t o c mesenteroides. Substituted Sephadex c a n be p r e p a r e d i n m u c h the s a m e way a s substituted c e l l u l o s e and shows a s i m i l a r ability for enzyme a d s o r p t i o n . E n z y m e s h a v i n g a r e l a t i v e l y h i g h content of a c i d i c a m i n o a c i d s r e m a i n f i r m l y bound to D E A E - c e l l u l o s e o r D E A E Sephadex even at h i g h s u b s t r a t e c o n c e n t r a t i o n a s l o n g as c e r t a i n p h y s i c a l c o n d i t i o n s s u c h as i o n i c s t r e n g t h and p H a r e m a i n t a i n e d . T h e a m i n o a c y l a s e f r o m A s p e r g i l l u s o r y z a e a d s o r b e d on D E A E - S e p h a d e x l o s e s 40% of its a c t i v i t y o v e r a 3 2 - d a y p e r i o d when u s e d at 5 0 ° C f o r continuous h y d r o l y s i s of a c e t y l a t e d L m e t h i o n i n e ( 19). D e t e r i o r a t e d c o l u m n s c o u l d be r e a c t i v a t e d b y d i r e c t a d d i t i o n of a m i n o a c y l a s e . H i g h e r e n z y m e a c t i v i t i e s w e r e obtained with D E A E - S e p h a d e x than with D E A E - c e l l u l o s e . U p w a r d flow i n a p a c k e d c o l u m n was u s e d to a v o i d c o m p r e s s i o n of the p a c k e d bed and e x c e s s i v e p r e s s u r e b u i l d - u p . T h e o p t i m u m c o n d i t i o n s of p H , i o n i c s t r e n g t h and t e m p e r a t u r e m u s t be d e t e r m i n e d to a c h i e v e and m a i n t a i n good a d s o r p t i o n and good a c t i v i t y . If a s t r o n g a d s o r p t i o n does not o c c u r , the e n z y m e i s r e a d i l y d e s o r b e d with subsequent l o s s of a c t i v i t y and c o n t a m i n a t i o n of the product. T h e s e l e c t i o n of o p t i m u m c o n d i t i o n s h a s m o s t l y b e e n a m a t t e r of t r i a l and e r r o r . M i c r o c r y s t a l l i n e D E A E - c e l l u l o s e powder is easily c o m p r e s s e d . O n l y s h a l l o w p a c k e d beds c a n be u s e d i n a l a r g e s c a l e c o m m e r c i a l o p e r a t i o n to a v o i d e x c e s s i v e p r e s s u r e b u i l d - u p .

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

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E N Z Y M E S IN FOOD AND

B E V E R A G E PROCESSING

T h i s f l o w b e h a v i o r can be i m p r o v e d by u s i n g p o r o u s D E A E c e l l u l o s e beads w h i c h show a h i g h e n z y m e l o a d i n g c a p a c i t y f o r i m m o b i l i z a t i o n of g l u c o s e i s o m e r a s e (20). G l u c o s e i s o m e r a s e i m m o b i l i z e d by a d s o r p t i o n on D E A E - c e l l u l o s e was found to be s o m e w h a t l e s s stable than the f r e e e n z y m e a t 6 0 C . At50°C the i m m o b i l i z e d g l u c o s e i s o m e r a s e has a h a l f - l i f e of 10-11 d a y s u n d e r continuous o p e r a t i o n (21). A n o t h e r p r o m i s i n g p o l y s a c c h a r i d e support i s chitin. Chitin f o r m s the s t r u c t u r a l m a t e r i a l of the s k e l e t o n s of m a r i n e c r u s t a c e a n s and b e a r s the s a m e r e l a t i o n to i n v e r t e b r a t e s as c e l l u l o s e d o e s to p l a n t s . T h e m o n o m e r unit i s g l u c o s a m i n e . On about f i v e out of e v e r y s i x g l u c o s a m i n e u n i t s the a m i n o n i t r o g e n i s b l o c k e d with an a c e t y l group. N a t i v e c h i t i n contains a p p r o x i m a t e l y equal a m o u n t s of c a l c i u m c a r b o n a t e and c h i t i n . The c a l c i u m c a r b o n a t e can be r e m o v e d by a c i d g i v i n g a r i g i d p o r o u s m a t e r i a l that i s r e l a t i v e l y i n e r t to both c h e m i c a l and m i c r o b i a l attack. G l u c o s e i s o m e r a s b e e n i m m o b i l i z e d on c h i t i e n z y m e s w e r e i m m o b i l i z e d on c h i t i n with g l u t a r a l d e h y d e : l a c t a s e ( a c i d t o l e r a n t ) , a c i d p h o s p h a t a s e , α-chymotrypsin (24) and g l u c o a m y l a s e (23). W h e n c h i t i n i s heated u n d e r p r e s s u r e with s t r o n g a l k a l i , i t is d e a c e t y l a t e d to p r o d u c e c h i t o s a n . T y p i c a l l y 8 0 % of the a c e t y l groups in chitin a r e r e m o v e d l e a v i n g f r e e amino groups in c h i t o s a n . C h i t o s a n has a l s o b e e n u s e d with g l u t a r a l d e h y d e to p r e p a r e i m m o b i l i z e d a c i d - t o l e r a n t l a c t a s e (25). T h e s e p o l y s a c c h a r i d e s a r e r e l a t i v e l y cheap, food g r a d e ma t e r i a l s and s o m e h a v e a h i g h c a p a c i t y f o r p r o t e i n a d s o r p t i o n . A s a g r o u p they a r e c u r r e n t l y a m o n g the m o s t w i d e l y u s e d sup p o r t s f o r e n z y m e i m m o b i l i z a t i o n i n the food i n d u s t r y . e

I n o r g a n i c Supports. Porous glass, alumina, hydroxya p a t i t e , n i c k e l oxide on n i c k e l s c r e e n , s i l i c a a l u m i n a i m p r e g nated with n i c k e l oxide, p o r o u s c e r a m i c s of v a r i o u s types, s t a i n l e s s s t e e l and sand a r e s o m e of the i n o r g a n i c m a t e r i a l s to w h i c h e n z y m e s h a v e b e e n attached. A p p a r e n t a d v a n t a g e s of i n o r g a n i c s u p p o r t s f o r e n z y m e s i n c l u d e : (1) s t r u c t u r a l s t a b i l i t y o v e r a wide r a n g e of pH, p r e s s u r e , t e m p e r a t u r e , and s o l v e n t c o m p o s i t i o n , (2) e x c e l l e n t f l o w p r o p e r t i e s i n r e a c t o r s , (3) i n e r t n e s s to m i c r o b i a l a t t a c k or a t t a c k by e n z y m e s , (4) e a s e of a d a p t a b i l i t y to v a r i o u s p a r t i c l e shapes and s i z e s , and (5) good regeneration capability. A t t a c h m e n t of e n z y m e to these s u p p o r t s has b e e n by a d s o r p tion and by c o v a l e n t a t t a c h m e n t to the i n o r g a n i c s u r f a c e . S i n c e W e e t a l l (26, 27) f i r s t r e p o r t e d the u s e of p o r o u s g l a s s to i m m o b i l i z e e n z y m e s , m a n y e n z y m e s h a v e b e e n c o v a l e n t l y a t t a c h e d to m o d i f i e d p o r o u s g l a s s and r e l a t e d m a t e r i a l s (4, 28, 29). P o r o u s g l a s s c o m m o n l y u s e d f o r this p u r p o s e has p o r e d i a m e t e r s i n the range of 200-1500 A and p a r t i c l e s i z e i n the range of 20-80 m e s h . It i s 9 6 % s i l i c a and can be r e a c t e d with an a l k y l a m i n o

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E n z y m e s c a n then be c o u p l e d to the a m i n e d e r i v a t i v e by the c a r bodiimide, thiourea or a z o linkage methods which probably i n v o l v e a t t a c h m e n t to c a r b o x y l , a m i n e o r a r o m a t i c r e s i d u e s , r e s p e c t i v e l y , on the e n z y m e . A n a l t e r n a t i v e p r o c e d u r e i s to t r e a t the a l k y l a m i n e g l a s s w i t h g l u t a r a l d e h y d e f o l l o w e d b y the e n z y m e to effect a s t a b l e c o u p l i n g . P o r o u s g l a s s h a s b e e n r e p o r t e d to be p a r t l y s o l u b l e a n d b r e a k s down at p H v a l u e s n e a r 7 o r above. T h i s p r o b l e m c a n be o v e r c o m e by c o a t i n g the g l a s s w i t h z i r c o n i u m oxide, a v e r y i n s o l u b l e m a t e r i a l , p r i o r to u s e (30). Sand i s a n o t h e r s i l i c a s u p p o r t that h a s r e c e n t l y b e e n u s e d s u c c e s s f u l l y to i m m o b i l i z e β- gala c to s i d a se (31), a l c o h o l dehy d r o g e n a s e a n d u r e a s e (32). W h i l e sand h a s a l o w e r e f f e c t i v e s u r f a c e a r e a a n d e n z y m e l o a d i n g c a p a c i t y than p o r o u s g l a s s , i t i s l e s s e x p e n s i v e and thus m a y be s u i t a b l e f o r l a r g e v o l u m e a p p l i c a t i o n s w i t h l o w c o n c e n t r a t i o n s of s u b s t r a t e . A p p l i c a t i o n of the e n z y m e to sand c a n be a c c o m p l i s h e d b y p r e p a r i n g a l k y l a m i n e sand, t r e a t i n g this with g l u t a r a l d e h y d e f o l l o w e d b y the enzyme. P o r o u s c e r a m i c c a r r i e r s h a v e a l s o b e e n d e v e l o p e d and u s e d s u c c e s s f u l l y to b i n d e n z y m e s . T h e s e s u p p o r t s a r e l e s s e x p e n s i v e than p o r o u s g l a s s and m o r e i n s o l u b l e . A m i n o a c y l a s e h a s b e e n i m m o b i l i z e d on c e r a m i c c a r r i e r s c o m p o s e d of S i O , A l ^ O ^ , and T i O (33). C o u p l i n g was a g a i n a c h i e v e d by f i r s t p r e p a r i n g the s i i a n i z e d c e r a m i c s f o l l o w e d by t r e a t i n g w i t h g l u t a r a l d e h y d e and f i n a l l y a d d i n g e n z y m e . S o m e a p p l i c a t i o n s of i m m o b i l i z e d e n z y m e s i n v o l v e the s i m ple a d s o r p t i o n of e n z y m e s . F o r e x a m p l e , a p o r o u s c e r a m i c i s u s e d a s a n e n z y m e s u p p o r t i n the p r o d u c t i o n of 5'-mononucleo tide f l a v o r e n h a n c e r s (34). G l u c o s e i s o m e r a s e h a s b e e n s u c c e s s f u l l y a d s o r b e d on the i n t e r n a l s u r f a c e of c o n t r o l l e d p o r e a l u m i n a (35). T h e a l u m i n a was p r e t r e a t e d w i t h a d i l u t e s o l u t i o n of m a g n e s i u m a c e t a t e a n d cobalt a c e t a t e p r i o r to a d d i n g the

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enzyme. The resulting adsorbed glucose i s o m e r a s e exhibited c o n v e r s i o n s i n the r a n g e of 85% w i t h a h a l f l i f e i n c o l u m n o p e r a t i o n of 40 d a y s . G l u c o s e o x i d a s e has b e e n c o v a l e n t l y c o u p l e d to n i c k e l oxide on n i c k e l s c r e e n s (36) and to n i c k e l oxide i m p r e g n a t e d s i l i c a a l u m i n a p e l l e t s (37). M o r e r e c e n t l y M a r k e y , G r e e n f i e l d and K i t t r e l l (38) studied the i m m o b i l i z a t i o n of g l u c o s e o x i d a s e a n d c a t a l a s e on a n u m b e r of s i l a n i z e d i n o r g a n i c s u p p o r t s u s i n g g l u t a r a l d e h y d e as a c r o s s l i n k i n g agent. A d e c r e a s e i n a c t i v i t y of both e n z y m e s was o b s e r v e d as p a r t i c l e s i z e of the s u p p o r t s i n c r e a s e d up to 1000 m i c r o n s . T h i s d e c r e a s e was a t t r i b u t e d to both e x t e r n a l f i l m and i n t e r n a l d i f f u s i o n a l r e s i s t a n c e s . Particle c o m p o s i t i o n , s i z e , shape and p o r e v o l u m e a r e i m p o r t a n t f a c t o r s in d e t e r m i n i n g the e f f e c t i v e n e s s of these k i n d s of s u p p o r t s f o r e n z y m e s (38, 39). G r e e n f i e l d and L a u r e n c e (39) and B o u i n et a l . (40) point out that g l u c o s e o x i d a s e i m m o b i l i z e d s i m u l t a n e o u s l y with c a t a l a s e s i g n i f i c a n t l o x i d a s e a c t i v i t y by d e c o m p o s i n in the g l u c o s e o x i d a s e r e a c t i o n . I m m o b i l i z e d c a t a l a s e and g l u c o s e o x i d a s e u n d e r g o s u b s t r a t e and p r o d u c t i n a c t i v a t i o n , r e s p e c t i v e l y , b y h y d r o g e n p e r o x i d e (41). A s a c o n s e q u e n c e the i n d u s t r i a l p e r f o r m a n c e of these e n z y m e s w i l l p r o b a b l y be d e t e r m i n e d by h o w w e l l this i n a c t i v a t i o n c a n be r e d u c e d and controlled. Fibrous Proteins. T h e f i b r o u s p r o t e i n s offer a n u m b e r of p o t e n t i a l advantages as e n z y m e s u p p o r t s for food a p p l i c a t i o n s . T h e y a r e n a t u r a l m a t e r i a l s , with good m e c h a n i c a l p r o p e r t i e s , offer a l o w r e s i s t a n c e to s u b s t r a t e d i f f u s i o n , and h a v e a n open i n t e r n a l s t r u c t u r e with m a n y p o t e n t i a l b i n d i n g s i t e s f o r e n z y m e a t t a c h m e n t and s t a b i l i z a t i o n . T h e y a r e abundant and i n e x p e n s i v e and can be p r o c e s s e d into f i l m s , m e m b r a n e s and other f o r m s that r e t a i n t h e i r s t r u c t u r a l i d e n t i t y . O f the f i b r o u s p r o t e i n s the c o l l a g e n s h a v e b e e n the m o s t w i d e l y studied as e n z y m e s u p p o r t s (42, 43). Collagen proteins in c o m b i n a t i o n w i t h e l a s t i n , m u c o p o l y s a c c h a r i d e s and m i n e r a l s a l t s f o r m the c o n n e c t i v e t i s s u e r e s p o n s i b l e f o r the s t r u c t u r a l i n t e g r i t y of the a n i m a l b o d y . T h e d i s t i n c t i v e c h a r a c t e r i s t i c of the a m i n o a c i d c o m p o s i t i o n of a v a r i e t y of v e r t e b r a t e c o l l a g e n s i s a h i g h g l y c i n e content w h i c h i s c l o s e to o n e - t h i r d of the total n u m b e r of r e s i d u e s . C o l l a g e n s a r e a l s o r i c h i n the i m i n o a c i d s p r o l i n e and h y d r o x y p r o l i n e w h i c h c a n a m o u n t to 20-25% of the total n u m b e r of r e s i d u e s . T h e unique t r i p l e s t r a n d e d h e l i c a l s t r u c t u r e of c o l l a g e n is c l o s e l y r e l a t e d to the h i g h content of g l y c i n e and i m i n o a c i d r e s i d u e s . T h e r e i s a c o m p l e x r e g u l a r i t y to the o r g a n i z a t i o n of the c o l l a g e n m o l e c u l e s into m i c r o f i b r i l s that r e s u l t s i n the p r e s e n c e of h o l e s s p a c e d at r e p e a t i n g i n t e r v a l s a l o n g the l e n g t h of the m i c r o f i b r i l . It i s b e l i e v e d that these r e g i o n s a r e the p r i m a r y s i t e s f o r e n z y m e b i n d i n g (44). C o l l a g e n s w e l l s i n the p r e s e n c e of

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w a t e r , i s h y d r o p h i l i c , and at n e u t r a l p H takes up m o r e than i t s own weight of w a t e r . I m m o b i l i z a t i o n of e n z y m e s on c o l l a g e n i n v o l v e s the f o r m a t i o n of a n e t w o r k of n o n - c o v a l e n t bonds s u c h a s s a l t l i n k a g e s ( i o n i c i n t e r a c t i o n s ) , h y d r o g e n bonds and V a n d e r W a a l s i n t e r a c t i o n s a c t i n g together between c o l l a g e n and the e n z y m e (43). W h i l e i n d i v i d u a l l y these bonds a r e weak, taken together they f o r m a v e r y stable n e t w o r k between e n z y m e and collagen. C o l l a g e n - e n z y m e c o m p l e x e s h a v e b e e n p r e p a r e d by t h r e e d i f f e r e n t p r o c e d u r e s : (1) i m p r e g n a t i n g a p r e s w o l l e n m e m b r a n e w i t h e n z y m e , (2) m a c r o m o l e c u l a r c o m p l e x a t i o n i n w h i c h a n e n z y m e - c o l l a g e n d i s p e r s i o n at the d e s i r e d p H i s c a s t and d r i e d and then r e h y d r a t e d for u s e , and (3) e l e c t r o c o d e p o s i t i o n i n w h i c h e n z y m e i s d e p o s i t e d i n a c o l l a g e n m e m b r a n e u n d e r the i n f l u e n c e of an e l e c t r i c f i e l d . The complexes can subsequently be t r e a t e d w i t h g l u t a r a l d e h y d e to i n d u c e c r o s s l i n k s to s t r e n g t h en the m e m b r a n e and th C o l l a g e n - e n z y m e m e m b r a n e s have b e e n c o i l e d and p l a c e d i n a c y l i n d e r with s u i t a b l e s p a c e r s to m a k e efficient continuous r e a c t o r s f o r e n z y m e c o n v e r s i o n . A l a r g e n u m b e r of e n z y m e s , i n c l u d i n g whole c e l l s , h a v e b e e n i m m o b i l i z e d on c o l l a g e n m e m branes. T h i s technique s e e m s a p p l i c a b l e i n g e n e r a l to e n z y m e immobilization. K e r a t i n i s a n o t h e r f i b r o u s p r o t e i n that h a s b e e n e x a m i n e d a s an e n z y m e s u p p o r t . K e r a t i n i s found i n the outer l a y e r s of v e r t e b r a t e s , is un r e a c t i v e t o w a r d the e n v i r o n m e n t and i s m e c h a n i c a l l y s t r o n g and d u r a b l e . L i k e the c o l l a g e n s , k e r a t i n s v a r y d e p e n d i n g on t h e i r s o u r c e . T h e y h a v e a h i g h content of the s u l f u r c o n t a i n i n g a m i n o a c i d s and c r o s s l i n k i n g p r o v i d e d b y the S-S bond of c y s t i n e w h i c h m a i n t a i n s s t r u c t u r a l i n t e g r i t y . Stanley and c o - w o r k e r s (45) h a v e r e c e n t l y p r e p a r e d a g r a n u l a r k e r a t i n f r o m f e a t h e r m e a l , a b y p r o d u c t of the p o u l t r y i n d u s t r y , and h a v e s u c c e s s f u l l y bound l a c t a s e to the g r a n u l e s w i t h g l u t a r a l d e h y d e . F e a t h e r k e r a t i n i n r e d u c e d f o r m h a s s o m e unique a t t r i butes w o r t h e x p l o i t i n g . T h e h i g h c o n c e n t r a t i o n of f r e e s u l f h y d r y l g r o u p s c o u l d be u s e d i n p u r i f y i n g s u l f u r - c o n t a i n i n g e n z y m e s t h r o u g h the technique of t h i o l - d i s u l f i d e i n t e r c h a n g e (46) o r s i m p l y by a d s o r p t i o n i n s o m e c a s e s . U r e a s e h a s b e e n i m m o b i l i z e d on f e a t h e r k e r a t i n b y a d s o r p t i o n (45). P h e n o l i c R e s i n s . A r e s i n made f r o m phenol and f o r m a l d e hyde h a s b e e n found to be a n e x c e l l e n t s u p p o r t f o r s o m e e n z y m e s by s e v e r a l i n v e s t i g a t o r s (47, 48, 49). A r e s i n of this type i s c o m m e r c i a l l y a v a i l a b l e f r o m D i a m o n d S h a m r o c k C h e m i cal C o m p a n y , Redwood C i t y , C a l i f o r n i a as Duolite E S - 7 6 2 . T h i s p o r o u s r e s i n i s r e l a t i v e l y stable to a c i d s , b a s e s a n d m o s t organic solvents. T h e 10-50 m e s h p a r t i c l e s a r e r i g i d , i r r e g u l a r and g r a n u l a r , and r e s i s t a n t attrition. They have a pore v o l u m e of a b o u £ 0. 6^cm p e r c m of r e s i n and a s u r f a c e a r e a of about 100 m / c m . T h i s type of r e s i n i s u s e d a s a n a d s o r b a n t

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f o r p r o t e i n s , a r o m a t i c s and other c o m p o u n d s a n d a s s u c h it w i l l b i n d e n z y m e s without f u r t h e r t r e a t m e n t . H o w e v e r , the a d d i t i o n of g l u t a r a l d e h y d e to the r e s i n e i t h e r b e f o r e o r a f t e r a p p l i c a t i o n of the e n z y m e c a n i n c r e a s e the r e t e n t i o n of e n z y m e a c t i v i t y s i g nificantly* I m m o b i l i z a t i o n is r a p i d at r o o m t e m p e r a t u r e and can be done i n a b a t c h p r o c e s s i n a n open v e s s e l o r with the r e s i n p a c k e d i n a c o l u m n . In m o s t c a s e s the e n z y m e can be r e m o v e d f r o m the r e s i n b y t r e a t m e n t w i t h d i l u t e a c i d a n d / o r b a s e and new e n z y m e a d s o r b e d to the s u p p o r t . T h i s r e s i n has been s u c c e s s f u l l y u s e d to i m m o b i l i z e l a c t a s e (47, 48), α-amylase (49), t r y p s i n (50) and other e n z y m e s . M o d i f i c a t i o n s of the p h e n o l f o r m a l d e h y d e r e s i n a r e a l s o p o s s i b l e . R e s i n s substituted with v a r i o u s p r i m a r y , s e c o n d a r y and t e r t i a r y m e t h y l a m i n o groups a r e a l s o available. T h e s e a n i o n exchange r e s i n s h a v e b e e n u s e d to i m m o b i l i z e i n v e r t a s e (48), g l u c o s e i s o m e r a s e and a s p a r t a s e (51). T h e a d s o r p t i o n p r o c e s s with the u n s u b s t i t u t e d resin most probably involve aromatic amino acids i of p r o t e i n s with p h e n o l i c c o m p o u n d s i s a l s o o b s e r v e d i n t h e i r r e a c t i o n w i t h tannins and i n p a r t i c u l a r tannic a c i d to f o r m i n s o l u b l e c o m p l e x e s . S u c h c o m p l e x e s w i t h i n v e r t a s e and l a c t a s e a r e e n z y m a t i c a l l y a c t i v e a n d r e m a i n i n s o l u b l e i f the c o m p l e x i s s t a b i l i z e d with g l u t a r a l d e h y d e (48). It is a l s o n e c e s s a r y to d e p o s i t this c o m p l e x on a s u p p o r t s u c h a s C e l i t e f o r c o l u m n o p e r a t i o n . W h i l e these l i m i t a t i o n s on the u s e of tannins to p r e c i p i tate e n z y m e s a p p e a r s g r e a t , the c o m b i n a t i o n s of different t a n n i n s and s u p p o r t s that c o u l d be t r i e d s u g g e s t s that for s o m e a p p l i c a t i o n s this a p p r o a c h c o u l d be u s e f u l . H y d r o g e l s . T h e m e t h o d of g e l e n t r a p m e n t i n v o l v e s the f o r m a t i o n of a c r o s s l i n k e d p o l y m e r n e t w o r k i n the p r e s e n c e of an e n z y m e o r m i c r o b i a l c e l l . T h i s technique s e e m s to be p a r t i c u l a r l y a t t r a c t i v e f o r the i m m o b i l i z a t i o n of whole c e l l s . With whole c e l l i m m o b i l i z a t i o n , the c o s t l y e n z y m e p u r i f i c a t i o n p r o c e s s e s c a n be a v o i d e d . S e q u e n t i a l r e a c t i o n s c a n be c a r r i e d out u t i l i z i n g the v a r i o u s e n z y m e s and c o f a c t o r s w h i c h a r e left intact i n the m i c r o b i a l c e l l s . W i t h i n the m i c r o b i a l c e l l s m a n y e n z y m e s a r e a l r e a d y i m m o b i l i z e d and c o m p a r t m e n t a l i z e d i n o r d e r to c a r r y out c o m p l e x m e t a b o l i c and synthetic r e a c t i o n s . By entrapping cells in a hydrophilic p o l y m e r network, a sup p o r t i n g s t r u c t u r e can be p r o v i d e d that h a s l i t t l e effect on c e l l properties. T h e p o l y m e r m o s t often u s e d f o r e n t r a p m e n t i s p o l y a c r y l amide. T h e c r o s s l i n k i n g agent u s e d to f o r m a t h r e e d i m e n s i o n al network is usually Ν , N - m e t h y l e n e b i s ( a c r y l a m i d e ) . Poly m e r i z a t i o n i s c a r r i e d out i n a n aqueous s o l u t i o n c o n t a i n i n g e n z y m e o r m i c r o b i a l c e l l , m o n o m e r , c r o s s l i n k i n g agent, and free r a d i c a l initiator. A three d i m e n s i o n a l p o l y m e r network is f o r m e d entrapping enzyme or m i c r o b i a l c e l l . T a n a b e S e i y a k u C o m p a n y i n J a p a n h a s d e v e l o p e d two 1

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p r o c e s s e s f o r a m i n o a c i d p r o d u c t i o n b a s e d on m i c r o b i a l c e l l e n t r a p m e n t i n p o l y a c r y l a m i d e g e l s . In one p r o c e s s , L - a s p a r t i c a c i d i s p r o d u c e d f r o m f u m a r i c a c i d u s i n g the a s p a r t a s e a c t i v i t y of e n t r a p p e d E . c o l i b a c t e r i a l c e l l s . In the other p r o c e s s , L m a l i c a c i d i s p r o d u c e d f r o m f u m a r i c a c i d u s i n g the f u m a r a s e a c t i v i t y of B r e v i b a c t e r i u m a m m o n i a gene s (52). Β . a m m o n i a genes c e l l s e n t r a p p e d i n p o l y a c r y l a m i d e gels h a v e a l s o b e e n studied f o r the s y n t h e s i s of c o e n z y m e A f r o m pantothenic a c i d , c y s t e i n e , and A T P . This synthesis uses five sequential enzymic steps and i l l u s t r a t e s the advantage of u s i n g i m m o b i l i z e d whole c e l l s f o r m u l t i - s t e p s y s t e m s (53). A l t h o u g h i t i s u s u a l l y a s s u m e d that e n z y m e s e n t r a p p e d i n p o l y a c r y l a m i d e gels r e m a i n unattached to the g e l m a t r i x and r e t a i n t h e i r n a t i v e c o n f o r m a t i o n , this m a y not a l w a y s be t r u e . H a r r i s o n has shown that the e n z y m e g l u c o s e 6-phosphate dehy d r o g e n a s e i s bound c o v a l e n t l d r y l or amino groups durin E n z y m e i m m o b i l i z a t i o n by g e l e n t r a p m e n t h a s b e e n r e c e n t l y r e v i e w e d (55). A l t h o u g h m o s t e n z y m e e n t r a p m e n t has been i n p o l y a c r y l a m i d e , o t h e r h y d r o g e l s m a y be s u p e r i o r with r e g a r d to m e c h a n i c a l s t r e n g t h and e l i m i n a t i o n of e n z y m e l e a k a g e . A l a r g e n u m b e r of e n z y m e s h a v e b e e n e n t r a p p e d i n poly( 2 - h y d r o x y e t h y l m e t h y l a c r y l a t e ) g e l s , (5_6). T h i s g e l m a y be p r e f e r r e d f o r s o m e a p p l i c a t i o n s b e c a u s e of i t s s u p e r i o r m e c h a n i c a l s t r e n g t h . E n z y m e s h a v e a l s o b e e n i m m o b i l i z e d u s i n g a l a r g e n u m b e r of synthetic m o n o m e r s and p o l y m e r s s u c h a s Ν-vinyl p y r r o l i d one, p o l y ( v i n y l a l c o h o l ) , and p o l y ( v i n y l p y r r o l i d o n e ) u n d e r r a d i a n t r a y i r r a d i a t i o n (57). Whole y e a s t c e l l s h a v e b e e n e n t r a p p e d i n s p h e r i c a l a g a r p e l l e t s and u s e d f o r s u c r o s e h y d r o l y s i s (58). H o l l o w F i b e r s . A n i m m o b i l i z a t i o n technique w h i c h o f f e r s s e v e r a l a d v a n t a g e s f o r food s y s t e m s i s the u s e of h o l l o w f i b e r s . Two types of h o l l o w f i b e r d e v i c e s that can be u s e d f o r e n z y m e i m m o b i l i z a t i o n a r e the i s o t r o p i c , c e l l u l o s i c f i b e r s m a d e by Dow C h e m i c a l C o m p a n y and the a n i s o t r o p i c , n o n c e l l u l o s i c f i b e r s m a d e by A m i c o n C o r p o r a t i o n and R o m i c o n C o r p o r a t i o n . The Dow s e m i p e r m e a b l e h o l l o w f i b e r s a r e m a d e of c e l l u l o s e a c e t a t e o r other c e l l u l o s i c m a t e r i a l s . T h e y a r e h o m o g e n e o u s ( i s o t r o p i c ) and p e r m i t p e r m e a t i o n of s o l u t e o r s o l v e n t f r o m e i t h e r s i d e of the f i b e r . A m i c o n and R o m i c o n f i b e r s a r e a n i s o t r o p i c and c o m p o s e d of an a p p r o x i m a t e l y 0. 5 μιη t h i c k s e m i p e r m e a b l e m e m b r a n e s u p p o r t e d by a t h i c k o p e n - c e l l e d sponge ( F i g u r e 2). T h e X50 h o l l o w f i b e r s a r e c o p o l y m e r s of p o l y ( v i n y l c h l o r i d e ) and p o l y a c r y l o n i t r i l e and h a v e a n o m i n a l m o l e c u l a r weight c u t - o f f of 50,000. The P10 f i b e r s a r e p o l y s u l f o n e p o l y m e r s with a n o m i n a l m o l e c u l a r weight c u t - o f f of 10, 000. T h e i m m o b i l i z a t i o n technique often u s e d with the A m i c o n and R o m i c o n h o l l o w f i b e r s c o n s i s t s of p h y s i c a l l y e n t r a i n i n g an aque ous p r e p a r a t i o n of e n z y m e or whole c e l l s w i t h i n the p o r o u s sponge r e g i o n of the f i b e r s . S u b s t r a t e s o l u t i o n i s then p a s s e d

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AXIAL CROSS SECTION Figure 2.

RADIAL CROSS SECTION

Structure of assymmetric hollow fiber

t h r o u g h the f i b e r l u m e n g i v i n g a continuous flow e n z y m e r e a c tor. T h e e n z y m e - s u b s t r a t e s y s t e m m u s t be s e l e c t e d so that the e n z y m e i s too l a r g e to p a s s t h r o u g h the f i b e r m e m b r a n e w h i l e the s u b s t r a t e i s s u f f i c i e n t l y s m a l l so a s to p a s s t h r o u g h the membrane. A theoretical model describing enzymic catalysis using hollow fiber m e m b r a n e s has been developed by Waterland et a l . (59). R e a c t o r c o n v e r s i o n data f o r β - gala c to s i d a se a g r e e w e l l w i t h p r e d i c t i o n s obtained f r o m the t h e o r e t i c a l m o d e l (60). H o l l o w fiber enzyme r e a c t o r s have also been used for i m m o b i l i z a t i o n of g l u c o s e i s o m e r a s e (61), cz-galactosidase a n d i n v e r t a s e (62). T h e s t a b i l i t y of e n z y m e s i m m o b i l i z e d i n h o l l o w f i b e r r e a c t o r s i s a p p r o x i m a t e l y the s a m e as f o r the c o r r e s p o n d i n g e n z y m e s i n f r e e s o l u t i o n . H o w e v e r , the R o m i c o n and A m i c o n P 1 0 h o l l o w f i b e r s r e q u i r e a p r e c o n d i t i o n i n g with a n i n e r t p r o t e i n b e f o r e u s e i n o r d e r to a v o i d e n z y m e i n a c t i v a t i o n on contact w i t h the f i b e r s (61, 62). S o m e of the advantages of u s i n g h o l l o w f i b e r s f o r e n z y m e i m m o b i l i z a t i o n a r e : h i g h throughput with a l o w p r e s s u r e d r o p can be a c h i e v e d , n o e n z y m e m o d i f i c a t i o n i s r e q u i r e d and t h e r e i s n o p r o d u c t c o n t a m i n a t i o n f r o m c h e m i c a l s u s e d i n the i m m o b i l i z a t i o n p r o c e s s , h o l l o w f i b e r c a r t r i d g e s c a n be e a s i l y c l e a n e d and r e l o a d e d w i t h e n z y m e , a n d the f i b e r s have a l a r g e s u r f a c e t o - v o l u m e r a t i o a n d a thin m e m b r a n e w h i c h offers a l o w r e s i s t a n c e to s u b s t r a t e t r a n s p o r t . Microbial Pellets. In a s u b m e r g e d c u l t u r e of f i l a m e n t o u s m i c r o o r g a n i s m s , the m y c e l i a a r e l i k e l y to a g g r e g a t e to f o r m pellets. M e d i a c o m p o s i t i o n and p H of the f e r m e n t a t i o n m e d i a can d e t e r m i n e whether pulpy m y c e l i a o r pellets a r e f o r m e d (63). M i c r o b i a l p e l l e t s c a n be v i e w e d a s i m m o b i l i z e d whole c e l l s w h e r e the i n t r a c e l l u l a r e n z y m e s a r e a v a i l a b l e f o r u t i l i z a tion. T h e s e u n t r e a t e d c e l l s c a n be d i r e c t l y u s e d a s b i o c a t a l y s t s

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for i n d u s t r i a l p r o c e s s e s . T h i s e l i m i n a t e s the n e e d f o r e n z y m e p u r i f i c a t i o n and g r e a t l y s i m p l i f i e s the i m m o b i l i z a t i o n p r o c e s s . P e l l e t s p r o d u c e d by f e r m e n t a t i o n of the fungus M o r t i e r e l l a v i n a c e a e have b e e n s u c c e s s f u l l y u s e d i n the beet s u g a r i n d u s t r y a s a n e n z y m e s o u r c e of ar-galactosidase. This enzyme hydrol y z e s the i n d i g e s t i b l e s u g a r r a f f i n o s e to s u c r o s e . Raffinose r e d u c e s s u g a r y i e l d by r e t a r d i n g the r a t e of s u c r o s e p r e c i p i t a tion f r o m beet s u g a r m o l a s s e s . K i n e t i c studies of or-galactosidase c o n t a i n i n g p e l l e t s of M . v i n a c e a e show that the b e h a v i o r of p e l l e t s is s i m i l a r to that of gel e n t r a p p e d e n z y m e s (64). T h e r e is a decrease in enzyme a c t i v i t y w i t h i n c r e a s i n g p e l l e t s i z e above 0. 25 m m b e c a u s e of d i f f u s i o n a l effects. F o r p e l l e t s s m a l l e r than about 0. 25 m m i n d i a m e t e r , d i f f u s i o n a l effects a r e a b s e n t . The effectiveness f a c t o r s found e x p e r i m e n t a l l y f o r p e l l e t s of M . v i n a c e a e with ag a l a c t o s i d a s e a c t i v i t y w e r e found to a g r e e f a i r l y w e l l w i t h a theoretical model describin particles. C o m m e r c i a l A p p l i c a t i o n of I m m o b i l i z e d E n z y m e s D e t a i l e d i n f o r m a t i o n on c o m m e r c i a l p r o c e s s e s u s i n g i m m o b i l i z e d e n z y m e s i n food p r o c e s s i n g i s g e n e r a l l y p r o p r i e t a r y . F o r s o m e p r o c e s s e s the only a v a i l a b l e i n f o r m a t i o n i s the m i c r o o r g a n i s m f r o m w h i c h the e n z y m e u s e d was obtained and a g e n e r a l d e s c r i p t i o n of the i m m o b i l i z a t i o n m e t h o d . T h e c o m m e r c i a l a p p l i c a t i o n s of i m m o b i l i z e d e n z y m e s p r e s e n t e d i n this s e c t i o n a r e w e l l d o c u m e n t e d and r e p r e s e n t s i g n i f i c a n t a d v a n c e s in p r o c e s s d e v e l o p m e n t i n the food i n d u s t r y . Aminoacylase. T h e f i r s t l a r g e s c a l e use of i m m o b i l i z e d e n z y m e s was the T a n a b e S e i y a k u C o m p a n y p r o c e s s f o r L - a m i n o a c i d production in Japan. This p r o c e s s uses aminoacylase a d s o r b e d on D E A E - S e p h a d e x f o r the c o n t i n u o u s , a u t o m a t i c a l l y c o n t r o l l e d p r e p a r a t i o n of L - m e t h i o n i n e , L - p h e n y l a l a n i n e , L tryptophane and L - v a l i n e f r o m r a c e m i c m i x t u r e s of those a m i n o acids. T h e r e d u c e d c o s t s of e n z y m e , s u b s t r a t e and l a b o r h a v e cut p r o d u c t i o n c o s t s of the i m m o b i l i z e d e n z y m e p r o c e s s to 60% of that f o r the c o n v e n t i o n a l b a t c h p r o c e s s u s i n g s o l u b l e e n z y m e s (65, 66). A p r o c e s s flow sheet f o r the continuous p r o d u c t i o n of L a m i n o a c i d f r o m r a c e m i c a c e t y l - D , L - a m i n o a c i d i s shown i n F i g u r e 3. T h e a c y l a t e d a m i n o a c i d m i x t u r e i s fed to a c o l u m n c o n t a i n i n g a m i n o a c y l a s e i m m o b i l i z e d b y a d s o r p t i o n onto D E A E Sephadex. T h i s e n z y m e h y d r o l y z e s only the a c e t y l - L - a m i n o acid. T h e r e s u l t i n g m i x t u r e of a c e t y l - D - a m i n o a c i d and L a m i n o a c i d c a n be s e p a r a t e d b y c r y s t a l l i z a t i o n b e c a u s e of solubility differences. L - m e t h i o n i n e h a s been p r o d u c e d w i t h a r e p o r t e d output of 20 m e t r i c tons p e r m o n t h (67).

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ACETYLDL-AMINOACID

CRYSTALLIZER

RACEMIZATION TANK

EVAPORATOR IMMOBILIZED HEAT EXCHANGER

AMINOACYLASE COLUMN

CRYSTALLINE L-AMINO

ACID

Figure 3.Process flow sheet

Glucose Isomerase. U n t i l 1938 c o m m e r c i a l c o r n s y r u p s w e r e p r o d u c e d by a c i d h y d r o l y s i s of c o r n s t a r c h . This process p r o d u c e d a s y r u p with a d e x t r o s e - e q u i v a l e n t ( D . E . ) of 5 6 - 5 8 . A s w e e t e r s y r u p was not p r o d u c e d b e c a u s e h i g h e r c o n v e r s i o n s by a c i d h y d r o l y s i s gave b i t t e r f l a v o r s . W i t h the i n t r o d u c t i o n of s a c c h a r i f y i n g e n z y m e s it b e c a m e p o s s i b l e to i n c r e a s e the D . E . without p r o d u c i n g b i t t e r f l a v o r s . The glucoamylase currently u s e d f o r s t a r c h s a c c h a r i f i c a t i o n p r o d u c e c o r n s y r u p s with a D . E . of 9 5 - 9 7 . T h i s h i g h d e x t r o s e c o r n s y r u p , w h i c h i s 70-75% as sweet as s u c r o s e , c a n be t r a n s f o r m e d to a p r o d u c t w h i c h i s e q u i v a l e n t i n s w e e t n e s s to s u c r o s e by a p a r t i a l i s o m e r i z a t i o n of g l u c o s e ( d e x t r o s e ) to f r u c t o s e . T h i s i s o m e r i z a t i o n has b e e n c a r r i e d out on a c o m m e r c i a l s c a l e s i n c e 1968. The c o m m e r c i a l a p p l i c a t i o n of i m m o b i l i z e d g l u c o s e i s o m e r a s e to p r o d u c e h i g h f r u c t o s e c o r n s y r u p i s c u r r e n t l y the l a r g e s t v o l u m e u s e of a n i m m o b i l i z e d e n z y m e i n the w o r l d . A p p r o x i m a t e l y one b i l l i o n pounds of h i g h f r u c t o s e s y r u p w e r e p r o d u c e d i n 1974 (9). The c o m m e r c i a l p r o c e s s for high fructose c o r n s y r u p p r o d u c t i o n i s shown i n F i g u r e 4. The process involves liquefying r a w c o r n s t a r c h , s a c c h a r i f y i n g the s t a r c h to a h i g h d e x t r o s e s y r u p u s i n g a c i d a n d e n z y m i c h y d r o l y s i s , and r e f i n i n g the syrup. T h e c a r b o n and i o n exchange r e f i n e d g l u c o s e s y r u p c o n tains about 93% D - g l u c o s e on a d r y b a s i s . T h e r e m a i n i n g 7% i s m a d e up of o t h e r c o r n s y r u p s a c c h a r i d e s . T h i s glucose syrup i s p r e p a r e d for i s o m e r i z a t i o n by the a d d i t i o n of r e q u i r e d s a l t s (Co and Mg) and a d j u s t m e n t of p H . T h e g l u c o s e s y r u p i s p u m p e d t h r o u g h a s e r i e s of s h a l l o w bed r e a c t o r s w h i c h c o n t a i n g l u c o s e i s o m e r a s e a d s o r b e d onto D E A E - c e l l u l o s e . T h e flow r a t e i s c o n t r o l l e d so that the p r o d u c t c o n t a i n s about 42% f r u c tose. T h e flow r a t e c a n be r e d u c e d as i s o m e r a s e 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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Immobilized Enzymes

LIGHT EVAPORATION

HEAVY EVAPORATION

SALTS pH

COOLING & STORAGE

ADJUSTMENT CARBON REFINING

Figure 4.

CD

High-fructose

d i m i n i s h e s i n o r d e r to m a i n t a i n this c o n v e r s i o n . T h e f r u c t o s e s y r u p i s then r e f i n e d and c o n c e n t r a t e d by e v a p o r a t i o n . T h e r e fined h i g h f r u c t o s e c o r n s y r u p i s s u b s t a n t i a l l y c o l o r l e s s , a s h f r e e and h a s l i t t l e tendency to f o r m c o l o r on s t o r a g e (68). A h i g h e r f r u c t o s e content can be obtained by s e p a r a t i n g g l u c o s e and f r u c t o s e i n i o n exchange c o l u m n s , t h e r e b y p r o d u c i n g 90% f r u c t o s e s y r u p s on a l a r g e s c a l e w i t h h i g h y i e l d s (69). These s y r u p s a r e now c o m m e r c i a l l y a v a i l a b l e . cy-Galactosidase. T h e beet s u g a r i n d u s t r y u s e s i m m o b i l i z e d a- gala c to s i d a se to h y d r o l y z e the r a f f i n o s e i n s u g a r b e e t s . This e n z y m e c a n be h e l d w i t h i n p e l l e t s of the fungus M . v i n a c e a e which are formed under specific fermentation conditions. D i l u t e d s u g a r beet m o l a s s e s i s p a s s e d t h r o u g h a r e a c t o r c o n t a i n i n g the p e l l e t s . T h e p e l l e t s a r e v e r y c o m p r e s s i b l e and c a n not be r e a d i l y p a c k e d i n a c o l u m n without an e x c e s s i v e p r e s s u r e b u i l d - u p d u r i n g continuous o p e r a t i o n . A n e n z y m e r e a c t o r that has b e e n d e v e l o p e d f o r the c o m m e r c i a l p r o c e s s (70) c o n s i s t s of a h o r i z o n t a l vat d i v i d e d into s e v e r a l c h a m b e r s , e a c h b e i n g a g i tated, a n d e a c h s e p a r a t e d by a s c r e e n to p r e v e n t the p a s s a g e of p e l l e t s f r o m one c h a m b e r to a n o t h e r . T h e m o l a s s e s i s fed into one end of the r e a c t o r and c o n t a c t s the s u s p e n d e d p e l l e t s as it p a s s e s f r o m c h a m b e r to c h a m b e r . T y p i c a l l y , a s u g a r beet p r o c e s s i n g plant m i g h t p r o c e s s 3000 tons of beets a d a y and obtain w a t e r - s o l u b l e e x t r a c t s r e p r e s e n t ing 600 tons of s u c r o s e and as m u c h as 4 o r 5 tons of raffinose (71). A b o u t 80% of the r a f f i n o s e c a n be h y d r o l y z e d as the m o l a s s e s i s p a s s e d t h r o u g h the a g i t a t e d c h a m b e r s . S i n c e r a f f i n o s e i n t e r f e r e s with the c r y s t a l l i z a t i o n of s u c r o s e , s u c r o s e r e c o v e r y i s i n c r e a s e d by r a f f i n o s e h y d r o l y s i s to a g r e a t e r extent than the a m o u n t of r a f f i n o s e h y d r o l y z e d . E x p e r i e n c e i n J a p a n i n d i c a t e d

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ENZYMES

IN FOOD AND B E V E R A G E

PROCESSING

that p l a n t throughput was i n c r e a s e d by at l e a s t 5 % and r e c o v e r i e s of s u c r o s e i n c r e a s e d (71). T h e r e i s a l s o i n t e r e s t i n or-ga l a c to s i d a se f o r h y d r o l y s i s of the or-galactosides, r a f f i n o s e , s t a c h y o s e and v e r b a s c o s e , that a r e c o n s i d e r e d to be p a r t i a l l y r e s p o n s i b l e f o r the f l a t u l e n c e a s s o c i a t e d w i t h d r y b e a n p r o d u c t s s u c h as s o y b e a n m i l k (62, 72). H o w e v e r , t h e r e h a v e b e e n no r e p o r t s of a c o m m e r c i a l application. R i b o n u c l e a s e . A r e c e n t a p p l i c a t i o n of i m m o b i l i z e d e n z y m e s i n J a p a n i s the p r o d u c t i o n of 5'-mononucleotide f l a v o r e n h a n c e r s (34). I n o s i n e - 5 - m o n o p h o s p h a t e (IMP) and guanosine-5'-mono phosphate ( G M P ) a c t s y n e r g i s t i c a l l y w i t h m o n o s o d i u m g l u t a m a t e to e n h a n c e food f l a v o r s . T h e 5'- r i b o n u c l e o t i d e s , a d e n o s i n e - 5 ' monophosphate (AMP), cytidine-5'-monophosphate ( C M P ) , u r i d i n e - 5 ' - m o n o p h o s p h a t e ( U M P ) and G M P are produced f r o m ribonucleic acids usin i m m o b i l i z e d on a p o r o u the r e p l a c e m e n t of the a m i n o g r o u p of A M P with a h y d r o x y l group. T h i s r e a c t i o n i s c a t a l y z e d by 5'-adenylate d e a m i n a s e i m m o b i l i z e d on a p o r o u s c e r a m i c s u p p o r t . f

RNA—=τ—r r—τ: : 5 -phosphodiesterase

AMP

• 5'-ribonucleotides Μ Ρ , GMP, CMP,

. -ζ : • u 5 -adenylate d e a m i n a s e

Γ

Ί

(

Α

UMP)

IMP

F r o m these o b s e r v a t i o n s on what h a s s u c c e e d e d i n the f i e l d of i m m o b i l i z e d e n z y m e s a p p l i e d to food p r o c e s s i n g we w o u l d suggest that f o r l a r g e v o l u m e , l o w unit p r i c e i t e m s , the m o r e s i m p l e i m m o b i l i z a t i o n s c h e m e s on i n e x p e n s i v e food grade sup p o r t s a r e n e c e s s a r y . A d s o r p t i o n on D E A E c e l l u l o s e and s t a b i l i z e d m i c r o b i a l p e l l e t s h a v e s u c c e e d e d . O f c o u r s e , a s the v a l u e of the p r o d u c t goes up a m o r e c o m p l e x s c h e m e m a y be p o s s i b l e . P o t e n t i a l New

A p p l i c a t i o n s of I m m o b i l i z e d

Enzymes

S o m e e x a m p l e s of the m a n y p r o p o s e d u s e s of i m m o b i l i z e d e n z y m e s to food p r o c e s s i n g a r e l i s t e d i n T a b l e I. E a c h of these a p p l i c a t i o n s i s a s i g n i f i c a n t s u b j e c t i n and of i t s e l f . We h a v e s e l e c t e d a few p r o p o s a l s to d i s c u s s i n m o r e d e t a i l as i l l u s t r a t i v e of what m i g h t be done w i t h i m m o b i l i z e d e n z y m e s i n food p r o c e s s i n g i n the f u t u r e . Immobilized Starch H y d r o l y z i n g E n z y m e Systems. Sugar syrups prepared f r o m enzymatically hydrolyzed starch are i m p o r t a n t i n g r e d i e n t s i n m a n y m o d e r n food p r o d u c t s . S y r u p s a r e a v a i l a b l e c o n t a i n i n g a wide range of s u g a r s w i t h v a r y i n g sweet-

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

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Immobilized Enzymes

P r o p o s e d U s e s of I m m o b i l i z e d Processing

Application

E n z y m e s to F o o d

Examples

Sugar h y d r o l y s i s

F o r fermentation processes l a c t a s e on whey, p r i o r to wine fermentation

Clarification or haze removal

In b e e r and wine i n d u s t r i e s u s e papain, b r o m e l a i n , and o t h e r proteases

Sterilization

L y s o z y m e to r e m o v e m i c o o r g a n i s m s from air C a t a l a s e to r e m o v e e x c e s s H^O^

Food stabilization catalase system Prevent gelling

In f r o z e n o r a n g e j u i c e c o n c e n t r a t e s use pectic enzymes

Flavor modification

D e b i t t e r i n g p r o t e i n s with p r o t e a s e s Naringinase for grapefruit juice Limonate dehydrogenase f o r citrus juices

Nutritional improvement

P l a s t e i n r e a c t i o n to add a m i n o a c i d to protein Cellulases, hemicellulases, pectina s e s , etc, , to i m p r o v e food d i g e s t i bility

Removing undesirable factors

Decaffeinating coffee R e m o v i n g tannins f r o m tea

n e s s and f u n c t i o n a l p r o p e r t i e s . T h e p r o c e s s e s i n v o l v e d i n p r e p a r i n g these s y r u p s constitute one of the l a r g e s t c o m m e r c i a l u s e s of e n z y m e s . C h a n g e s and i m p r o v e m e n t s i n e n z y m e s o u r c e s and m e a n s of u t i l i z a t i o n c a n thus be of c o n s i d e r a b l e i m p o r t a n c e to the i n d u s t r i e s i n v o l v e d , the q u a l i t y and p r o p e r t i e s of the p r o d u c t s p r o d u c e d and the c o n s u m e r . P o t e n t i a l a d v a n tages f o r u s i n g i m m o b i l i z e d e n z y m e s i n these p r o c e s s e s i n c l u d e l o w e r e n z y m e c o s t s , continuous p r o c e s s e s , l o w e r l a b o r c o s t s , c l e a n e r p r o d u c t s , b e t t e r q u a l i t y c o n t r o l and l e s s a d d i t i o n a l p r o c e s s i n g . W i t h these p o t e n t i a l advantages i t i s not s u r p r i s i n g that w e l l o v e r 100 p a p e r s h a v e b e e n p u b l i s h e d i n the l a s t few y e a r s d e a l i n g w i t h the i m m o b i l i z a t i o n of one o r m o r e of the

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e n z y m e s i n v o l v e d i n s t a r c h h y d r o l y s i s . S o m e of this w o r k h a s b e e n r e v i e w e d b y Z a b o r s k y (4) a n d O l s o n and R i c h a r d s o n ( 11). T h e p r o c e s s of p r e p a r i n g s u g a r s y r u p s f r o m s t a r c h c a n be d i v i d e d into s e v e r a l steps ( F i g u r e 5). T h e g e l a t i n i z a t i o n a n d Starch Gelatinization pH 2 Swollen Starch L i q u e f a c t i o n a-Amylase,

p H 6. 5,

85°C

Thinned Starch Sac cha r if i c a ti on G l u c oa m y l a s e p H 3. 5-6, 5, 3 5 - 6 5

β-Amylase <

Glucose Syrups

.

pH 6.0,

45°C

Maltose Syrups

Isomerization Glucose Isomerase p H 7, 5 0 - 6 0 ° C Glucose F r u c t o s e Syrups Figure 5. A summary of the steps involved in preparing sugar syrups from starch

l i q u e f a c t i o n steps c a n be c o m b i n e d i n a s i n g l e a c i d - e n z y m e p r o c e s s i n w h i c h a n α - a m y l a s e i s added to a 30-40% (w/w) s t a r c h s l u r r y at p H 6, 5. T h e m i x t u r e i s s u b s e q u e n t l y h e a t e d to 1051 1 0 ° C f o r 5-15 m i n u t e s f o r g e l a t i n i z a t i o n and then c o o l e d a n d h e l d at 95 ° C f o r 1-20 h o u r s f o r l i q u e f a c t i o n by the or-amylase. While a-amylase preparations f r o m B a c i l l u s subtilis have been u s e d to thin s t a r c h , a r e c e n t l y c o m m e r c i a l l y i n t r o d u c e d , m o r e heat stable or-amylase f r o m B a c i l l u s l i c h e n i f o r m i s jWorks e v e n b e t t e r i n a s i n g l e step p r o c e s s with l o w e r total C a require m e n t s f o r e n z y m e t e m p e r a t u r e s t a b i l i t y (73). I m m o b i l i z e d α - a m y l a s e m i g h t a l s o be u s e d i n d u s t r i a l l y . T h e e n z y m e h a s b e e n e n t r a p p e d i n gels (74), c o v a l e n t l y bound to c r o s s l i n k e d p o l y a c r y l a m i d e d e r i v a t i v e s (75), a n d i m m o b i l i z e d on c o l l a g e n m e m b r a n e s (76). a-Amylase attached to p o l y s t y r e n e p a r t i c l e s i n a continuous s t i r r e d r e a c t o r of-amylase showed a g r e a t e r extent of m u l t i p l e a t t a c k on s t a r c h than the s o l u b l e e n z y m e with a n i n c r e a s e i n the p r o d u c t i o n of g l u c o s e (77). When α - a m y l a s e was i m m o b i l i z e d i n a n u l t r a f i l t r a t i o n r e a c t o r s o m e of the e n z y m e p a s s e d t h r o u g h the m e m b r a n e (78). T o o v e r c o m e this d i f f i c u l t y W y k e s et a l . (79) i n c r e a s e d the s i z e of

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the s o l u b l e e n z y m e by c o u p l i n g i t to s o l u b l e s - t r i a z i n e d e r i v a t i v e s of d e x t r a n , D E A E - d e x t r a n and c a r b o x y m e t h y l c e l l u l o s e . T h e l a r g e r s o l u b l e e n z y m e d e r i v a t i v e s d i d not p a s s through the p o r e s of the u l t r a f i l t r a t i o n m e m b r a n e s as r e a d i l y a s the nond e r i v a t i z e d e n z y m e but w e r e e f f e c t i v e i n h y d r o l y z i n g s t a r c h solutions. a - A m y l a s e h y d r o l y z e s bonds i n the i n t e r i o r of the s t a r c h m o l e c u l e to p r o d u c e o l i g o s a c c h a r i d e s and v a r i o u s a m o u n t of g l u cose, m a l t o s e and m a l t o t r i o s e d e p e n d i n g on the s o u r c e of the e n z y m e . It i s u s e d c o m m e r c i a l l y f o r the l i q u e f a c t i o n of g e l a t i n i z e d s t a r c h b e f o r e s a c c h a r i f i c a t i o n by a d d i n g the e n z y m e to a s l u r r y and h e a t i n g to 8 0 - 9 0 ° C . U n d e r these c o n d i t i o n s the enz y m e h y d r o l y z e s enough of the s t a r c h to effect l i q u e f a c t i o n but i n the p r o c e s s i t can be i n a c t i v a t e d by the h i g h t e m p e r a t u r e . Imm o b i l i z e d ûf-amylase should p r o b a b l y be u s e d f o r the l i q u e f a c t i o n p r o c e d u r e u n d e r s u c h c o n d i t i o n s that i t i s not i n a c t i v a t e d Wheat s t a r c h has b e e n and p a r t i a l l y h y d r o l y z e m o b i l i z e d on c y a n o g e n b r o m i d e a c t i v a t e d c a r b o x y m e t h y l c e l l u l o s e i n a s t i r r e d tank r e a c t o r (80). T h e i m m o b i l i z e d α - a m y l a s e p r o d u c e d r e l a t i v e l y m o r e g l u c o s e and m a l t o s e than the s o l u b l e enzyme. β - A m y l a s e h y d r o l y s e s α-1, 4 - g l y c o s i d i c l i n k a g e s f r o m the n o n - r e d u c i n g ends of s t a r c h c h a i n s to give m a l t o s e . The en z y m e i s b e i n g u s e d c o m m e r c i a l l y i n the b r e w i n g , d i s t i l l i n g and b a k i n g i n d u s t r i e s to c o n v e r t s t a r c h to f e r m e n t a b l e s u g a r . High m a l t o s e s y r u p s , p r o d u c e d with a m i x t u r e of α - a m y l a s e and βa m y l a s e , a r e u s e d i n the c o n f e c t i o n a r y i n d u s t r y b e c a u s e they a r e n o n h y d r o s c o p i e and do not c r y s t a l l i z e as r e a d i l y a s h i g h g l u c o s e s y r u p s . β - A m y l a s e h a s b e e n i m m o b i l i z e d to c r o s s l i n k e d p o l y a c r y l a m i d e s (75), entrapped i n g e l l a t t i c e (81), a t t a c h e d to a l k y l a m i n e p o r o u s g l a s s with g l u t a r a l d e h y d e (82), and c h e m i c a l l y a t t a c h e d to a c r o s s l i n k e d c o p o l y m e r of a c r y l a m i d e - a c r y l i c acid using a water soluble carbodiimide (83). β - A m y l a s e a c t i n g a l o n e on s t a r c h g i v e s m a l t o s e and a l a r g e p o r t i o n of a b r a n c h e d u n d e g r a d e d a m y l o p e c t i n β-limit dex t r i n . In o r d e r to i n c r e a s e the y i e l d of m a l t o s e f r o m s t a r c h M a r t e n s son (84, 85) has i m m o b i l i z e d β-amylase together w i t h p u l l u l a n a s e , a d e b r a n c h i n g e n z y m e . β - A m y l a s e was a t t a c h e d to the support f i r s t b e c a u s e i t was m o r e stable to the c a r b o d i i m i d e c o u p l i n g reagent. C o u p l i n g y i e l d s of 4 0 % β-amylase p r o t e i n and 3 8 % p u l l u l a n a s e p r o t e i n w e r e obtained with r e s i d u a l e n z y m e a c t i v i t i e s of 2 2 % and 3 2 % r e s p e c t i v e l y . T h i s two e n z y m e s y s t e m was c o n s i d e r e d advantageous as a s t e r i c opening of the sub s t r a t e i s p e r f o r m e d by one e n z y m e f a c i l i t a t i n g the a c t i o n of the other e n z y m e . M a r k e d i n c r e a s e d o p e r a t i o n a l s t a b i l i t y was ob s e r v e d f o r the i m m o b i l i z e d two e n z y m e s y s t e m c o m p a r e d to the f r e e e n z y m e s i n s o l u t i o n . P u l l u l a n a s e c o u l d a l s o be u s e d a l o n e to p r o d u c e l o w D. E . s y r u p s of c o m m e r c i a l v a l u e . Glucoamylase hydrolyzes a-l,4-glucan links removing

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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

g l u c o s e units c o n s e c u t i v e l y f r o m the n o n r e d u c i n g ends of s t a r c h molecules. T h e s o l u b l e e n z y m e is u s e d i n the e n z y m a t i c c o n v e r s i o n of s t a r c h to g l u c o s e . T h e p o t e n t i a l advantages of u s i n g i m m o b i l i z e d glucoamylase have been well recognized. The e n z y m e i o n i c a l l y bound to D E A E - c e l l u l o s e (86) w i l l r e m a i n so a s l o n g a s c e r t a i n p h y s i c a l c o n d i t i o n s s u c h as l o w i o n i c s t r e n g t h and p H (about 4) a r e m a i n t a i n e d . C o l u m n s of D E A E - c e l l u l o s e bound e n z y m e m u s t be l o n g to a l l o w f o r sufficient r e s i d e n c e t i m e f o r p r a c t i c a l c o n v e r s i o n s . A s a r e s u l t the c e l l u l o s e e n z y m e c o m p l e x p a c k s v e r y tight r e s t r i c t i n g the flow. It i s , h o w e v e r , p o s s i b l e to u s e g l u c o a m y l a s e on D E A E - c e l l u l o s e i n a continuous s t i r r e d tank r e a c t o r ( C S T R ) at 55 ° C f o r 3-4 w e e k s without l o s s of e n z y m e (87). G l u c o a m y l a s e can be c o v a l e n t l y bound to D E A E - c e l l u l o s e b y f i r s t a c t i v a t i n g the s u p p o r t w i t h 2 - a m i n o - 4 , 6 - d i c h l o r o - s t r i a z i n e and then c o u p l i n g the e n z y m e to the a c t i v a t e d s u p p o r t (88, 89, 90). A s w i t h m o s c a t a l y t i c b e h a v i o r of the bound e n z y m e was d i f f e r e n t f r o m that of the f r e e e n z y m e . O n i m m o b i l i z a t i o n the p H o p t i m u m shifted f r o m 5 to 4. 3, Κ d e c r e a s e d f r o m 6. 1 m M to 1.4 m M , and t e m p e r a t u r e s t a b i l i t y at 50 ° C i n c r e a s e d . P a r t of the g l u c o a m y l a s e m o l e c u l e i s a c a r b o h y d r a t e w h i c h offers an u n u s u a l m e a n s f o r a t t a c h i n g the e n z y m e to a c e l l u l o s e s u p p o r t . C h r i s t i s o n (9!) h a s o x i d i z e d the c a r b o h y d r a t e w i t h s o d i u m m e t a p e r i o d a t e and c o u p l e d the r e s u l t i n g o x y g l u c o a m y l a s e to c a r b o x y m e t h y l c e l l u l o s e h y d r a z i d e . A m b e r l i t e I R - 4 5 ( O H ) , a s t r o n g a n i o n exchange r e s i n , w i l l b i n d s i g n i f i c a n t a m o u n t s of g l u c o a m y l a s e (92). T h e bead f o r m of this s u p p o r t could m a k e it s u p e r i o r to c e l l u l o s e s u p p o r t s f o r l o n g c o l u m n o p e r a t i o n s s i n c e c o m p a c t i o n of the s u p p o r t s h o u l d be l e s s . G l u c o a m y l a s e h a s a l s o b e e n c o v a l e n t l y bound to v a r i ous d e r i v a t i v e s of m a c r o r e t i c u l a r p o l y s t y r e n e b e a d s (93). A d e r i v a t i v e p r e p a r e d by n i t r a t i o n of the beads f o l l o w e d by r e d u c tion to a p o l y ( p - a m i n o s t y r e n e ) was a good s u p p o r t to w h i c h g l u c o a m y l a s e c o u l d be bound with g l u t a r a l d e h y d e . Stable gels c a n be p r e p a r e d c o n t a i n i n g g l u c o a m y l a s e w h i c h can h y d r o l y z e d e x t r i n s to g l u c o s e and f r o m w h i c h t h e r e i s n o l e a k a g e of e n z y m e . F o r e x a m p l e , g l u c o a m y l a s e h a s b e e n i m m o b i l i z e d i n thin p o l y a c r y l a m i d e gels b y p h o t o p o l y m e r i z i n g aqueous s o l u t i o n s of a c r y l a m i d e , a c r o s s l i n k i n g m o n o m e r ( a l k y l g l y c i d y l e t h e r ) , an e n z y m e r e a c t i v e m o n o m e r ( g l y c i d y l a c r y l a t e ) and g l u c o a m y l a s e (94). M a e d a and c o - w o r k e r s (95) h a v e p r e p a r e d i m m o b i l i z e d g l u c o a m y l a s e i n p o l y a c r y l a m i d e gels by 7 - r a y i r r a d i a t i o n but without the u s e of a n y c r o s s l i n k i n g agent. T h e e n z y m e has a l s o b e e n e n t r a p p e d i n p o l y ( v i n y l a l c o hol) (95) and p o l y ( v i n y l p y r r o l i d o n e ) gels (96) b y i n i t i a t i n g p o l y m e r i z a t i o n of s o l u t i o n s of m o n o m e r s and the e n z y m e with 7 rays. P a c k e d beds of m a t r i x bound g l u c o a m y l a s e i n p o l y a c r y l a m i d e gels h a v e b e e n u s e d to c o n v e r t 28 D . E . s t a r c h h y d r o l y zate s y r u p s to s y r u p s c o n t a i n i n g 90% o r m o r e d e x t r o s e (94).

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C o l u m n s of g l u c o a m y l a s e i m m o b l i z e d on c h i t i n with g l u t a r a l d e h y d e i n b e n c h s c a l e o p e r a t i o n s gave r e m a r k a b l y good o p e r ational characteristics. U s i n g a feed of a - a m y l a s e t r e a t e d s t a r c h c o n t a i n i n g 30% s o l i d s a p r o d u c t was obtained i n w h i c h the d e x t r o s e content was 95. 6%. A n a l y s e s by h i g h p r e s s u r e l i q u i d c h r o m a t o g r a p h y i n d i c a t e d that u n h y d r o l y z e d r e s i d u e was e s s e n t i a l l y r e s t r i c t e d to d i s a c c h a r i d e s (23). Collagen m e m b r a n e s containing glucoamylase have been p r e p a r e d by a d d i n g e n z y m e to c o l l a g e n h o m o g e n a t e s p r i o r to c a s t i n g the m e m b r a n e ( c o d i s p e r s i o n ) and b y s o a k i n g a p r e f o r m e d m e m b r a n e i n a s o l u t i o n of the e n z y m e ( i m p r e g n a t i o n ) (97). In both c a s e s the e n z y m e w a s s u b s e q u e n t l y fixed to the m e m b r a n e b y c r o s s l i n k i n g with glutaraldehyde. The impregnated prepared m e m b r a n e bound l e s s e n z y m e than the c o d i s p e r s e d m e m b r a n e but r e t a i n e d h i g h e r a c t i v i t y f o r a l o n g e r p e r i o d of t i m e . T h e d u r a t i o n of e x p o s u r e a n d l e v e l of g l u t a r a l d e h y d e u s e d to fix the e n z y m e to the m e m b r a n e i m m o b i l i z a t i o n of a c t i v e e n z y m e . G l u c o a m y l a s e h a s b e e n i m m o b i l i z e d to a n u m b e r of i n o r g a n i c c a r r i e r s i n c l u d i n g c o n t r o l l e d p o r e g l a s s (_30, 98., 99), a c i d a c t i v a t e d m o l e c u l a r s i e v e and a l u m i n a (100). E n z y m e l o a d i n g s of 100 m g / g c a r r i e r with f u l l r e t e n t i o n of e n z y m e a c t i v i t y c o u l d be obtained on a m o l e c u l a r s i e v e that h a d b e e n p r e v i o u s l y a c t i vated w i t h 2 N H C 1 . T h e g e l - l i k e n a t u r e of the p r e p a r a t i o n , h o w e v e r , m a k e s it u n s u i t a b l e f o r c o l u m n w o r k . A l u m i n a s u p p o r t e d g l u c o a m y l a s e p r e p a r e d by s e v e r a l different p r o c e d u r e s showed l o w e r a c t i v i t y than the m o l e c u l a r s i e v e p r e p a r a t i o n s . T h e y d i d have good m e c h a n i c a l a n d flow c h a r a c t e r i s t i c s s u i t a b l e for column operation. W h e n g l u c o a m y l a s e c o u p l e d to p o r o u s g l a s s was u s e d i n c o l u m n o p e r a t i o n , s o m e d e c l i n e i n e n z y m e a c t i v i t y was o b s e r v e d that w a s a t t r i b u t e d to d i s s o l u t i o n of the g l a s s c a r r i e r . T h i s p r o b l e m w a s s o l v e d b y c o a t i n g the g l a s s w i t h Z r O - (30). When g l u c o a m y l a s e was c o u p l e d to a r y l a m i n e - g l a s s b y the a z o l i n k a g e p r o c e d u r e , a n u m b e r of c o v a l e n t l i n k s a r e f o r m e d b e tween g l a s s a n d p r o t e i n m o l e c u l e s a n d the bond e n e r g i e s of these l i n k s a r e sufficient to p r e v e n t s h e a r i n g of the e n z y m e f r o m the support u n d e r s t r e s s e s that c o u l d be e x p e c t e d i n c o l u m n o r p a c k e d b e d r e a c t o r s (101). L o s s e s i n e n z y m e a c t i v i t y f r o m r e a c t o r s u s i n g s u c h bound g l u c o a m y l a s e w o u l d h a v e to be due to s o m e other c a u s e s s u c h a s e n z y m e d e n a t u r a t i o n o r p o i s o n i n g of the e n z y m e b y a n i n h i b i t o r . S e v e r a l studies have b e e n m a d e on the p i l o t plant p r o d u c t i o n of g l u c o s e w i t h g l u c o a m y l a s e i m m o b i l i z e d to p o r o u s s i l i c a (102, column was p r e p a r e d by f i l l i n g the c o l u m n w i t h a l k y l a m i n e s i l i c a b e a d s i n w a t e r . A f t e r the c o l u m n w a s f u l l y p a c k e d a g l u t a r a l d e h y d e s o l u t i o n was p u m p e d into it to r e p l a c e the w a t e r . F o l l o w i n g t h i s , the c o l u m n was w a s h e d w i t h p H 7 phosphate buffer a n d a 20% g l u c o a m y l a s e s o l u t i o n i n the s a m e buffer w a s r e c i r c u l a t e d t h r o u g h the c o l u m n

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at r o o m t e m p e r a t u r e f o r 2 h o u r s . T h e c o l u m n was s t e r i l i z e d with a s a t u r a t e d aqueous s o l u t i o n of c h l o r o f o r m . B o u n d i n this way g l u c o a m y l a s e was found to be v e r y s t a b l e i n p i l o t plant o p e r a t i o n . A c t i v i t y l o s s e s at 4 0 ° C w e r e p r o b a b l y due to p l u g ging of c a r r i e r p o r e s with i n c o m p l e t e l y p r o c e s s e d s u b s t r a t e . M i c r o b i a l c o n t a m i n a t i o n was m i n i m a l and c o u l d be kept u n d e r c o n t r o l by w a s h i n g the c o l u m n w i t h a s a t u r a t e d s o l u t i o n of c h l o r o f o r m i n w a t e r . T h e r e a c t o r was c a p a b l e of p r o d u c i n g 1000 l b s / d a y of g l u c o s e at 40 °C and pH 4. 5. R e s i d e n c e t i m e of the f e e d i n the c o l u m n was i m p o r t a n t i n o b t a i n i n g h i g h D. E . and g l u c o s e c o n c e n t r a t i o n i n the p r o d u c t . A t l o n g r e s i d e n c e t i m e s c o n v e r s i o n of g l u c o s e to i s o m a l t o s e b e c a m e s i g n i f i c a n t , aA m y l a s e t r e a t e d d e x t r i n a s feed gave the h i g h e s t v a l u e of D . E . and g l u c o s e c o n c e n t r a t i o n . M a n u f a c t u r e of C h e e s e I th c o m m e r c i a l p r o d u c t i o f c h e e s e the c o n t r o l l e d c u r d l i n g a c c o m p l i s h e d with proteolyti to d e v e l o p a continuous, a u t o m a t e d p r o c e s s f o r c h e e s e p r o d u c tion i t would be advantageous to use i m m o b i l i z e d r e n n e t s . In this p r o c e s s c o o l e d m i l k (about 1 5 ° C ) would p a s s o v e r the i m m o b i l i z e d e n z y m e c o l u m n w h e r e p r o t e o l y s i s of the m i l k p r o tein, c a s e i n , would o c c u r . N o c o a g u l a t i o n a c t u a l l y o c c u r s u n t i l the m i l k i s heated. H e a t i n g of the e n z y m e - t r e a t e d m i l k would take p l a c e i n a s e p a r a t e s e c o n d a r y step to i n d u c e c u r d l i n g . The m a j o r d i f f i c u l t i e s e n c o u n t e r e d i n u s i n g i m m o b i l i z e d rennet a r e l o w a c t i v i t y and p o o r s t a b i l i t y of the i m m o b i l i z e d e n z y m e s , r e l e a s e of e n z y m e d u r i n g continuous o p e r a t i o n , c o l u m n plugging, and m i c r o b i a l c o n t a m i n a t i o n (105). Cofactor Regeneration. C o m m e r c i a l enzymic processes in the food i n d u s t r y u s e d e g r a d a t i v e h y d r o l y t i c e n z y m e s . I m m o b i l i z e d e n z y m e s m a y be a p p l i e d to s y n t h e s i z e o r a s s e m b l e r e l a t i v e l y c o m p l e x m o l e c u l e s s u c h as e x p e n s i v e p h a r m a c e u t i c a l c h e m i c a l s . A b i g d i f f i c u l t y i n s u c h an a p p l i c a t i o n i s the r e q u i r e m e n t f o r e x p e n s i v e c o f a c t o r s that w i l l p r o b a b l y h a v e to be r e g e n e r a t e d by m e a n s of a n i m m o b i l i z e d e n z y m e s y s t e m . It i s u n l i k e l y that s u c h an a p p l i c a t i o n w i l l s o o n be u s e d by the food i n d u s t r y . P r e l i m i n a r y c o s t e s t i m a t e s i n d i c a t e that, e v e n w i t h e f f i c i e n t c o f a c t o r r e c y c l i n g , o n l y p r o d u c t i o n of a v e r y h i g h c o s t p r o d u c t i s e c o n o m i c a l l y f e a s i b l e (9., 106). A n a l y s i s . E n z y m e s i n c l u d i n g i m m o b i l i z e d e n z y m e s (107, 108) a r e b e i n g u s e d m o r e r o u t i n e l y i n a n a l y t i c a l s y s t e m s . G l u c o s e a n a l y z e r s a r e a l r e a d y b e i n g m a r k e t e d by Y e l l o w S p r i n g s I n s t r u m e n t C o m p a n y ( Y e l l o w S p r i n g s , Ohio) and L e e d s & N o r t h rup Company (North Wales, Pa.). U s i n g different systems, these a n a l y z e r s e m p l o y a m p e r o m e t r i c d e t e c t o r s and i m m o b i l i z e d g l u c o s e o x i d a s e . In the Y e l l o w S p r i n g s i n s t r u m e n t an e n z y m e - c o n t a i n i n g m e m b r a n e i s f i x e d d i r e c t l y to a n e l e c t r o d e

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

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s e n s o r w h i c h r e s p o n d s to the H^O^ p r o d u c e d d u r i n g g l u c o s e o x i d a t i o n . T h e L e e d s & N o r t h r u p i n s t r u m e n t c o n s i s t s of a c o l u m n of i m m o b i l i z e d e n z y m e f o l l o w e d by a d e t e c t o r . C e l l u l o s e , H e m i c e l l u l o s e , and L i g n i n D e g r a d a t i o n . One of the b i g g e s t p o t e n t i a l a p p l i c a t i o n s of e n z y m e s to food s y s t e m s i s the e n z y m i c c o n v e r s i o n of c e l l u l o s e to g l u c o s e . W h i l e not i n v o l v i n g i m m o b i l i z e d e n z y m e s as we h a v e b e e n c o n s i d e r i n g t h e m the p r o b l e m s i n v o l v e d i n this s y s t e m a r e r e l a t e d . In woody p l a n t s c e l l u l o s e i s p h y s i c a l l y p r o t e c t e d f r o m enz y m e h y d r o l y s i s by the interpénétration of l i g n i n into a c r y s t a l l i n e c e l l u l o s i c n e t w o r k . T h i s s t r u c t u r e m u s t be d i s r u p t e d by c h e m i c a l or e n z y m i c t r e a t m e n t s or by m i l l i n g b e f o r e e n z y m e s c a n a t t a c k the c e l l u l o s e . T o h e l p d e t e r m i n e the p r o b l e m s i n v o l v e d and the e c o n o m i c v i a b i l i t y of c e l l u l o s e d e g r a d a t i o n a highly instrumented prepilot plant containing f e r m e n t o r s en z y m e r e a c t o r s , and a u x i l l i a r U. S. A r m y N a t i c k D e v e l o p m e n One of the p r o c e s s i n g s c h e m e s p r o p o s e d f o r c e l l u l o s e h y d r o l y s i s i s shown i n F i g u r e 6 (110). C e l l u l a s e i s p r o d u c e d by

»

SOLIDS (LIGNIN) TO FURNACE

Applied Polymer Symposium No. 2

Figure 6*

Berkeley process for the enzymatic conversion of cellulose to glucose

(110) T r i c h o d e r m a v i r i d e i n a two-stage f e r m e n t a t i o n s y s t e m w i t h m i c r o b i a l c e l l growth with s o l u b l e s u g a r s i n a g r o w t h f e r m e n t o r f o l l o w e d by c e l l u l a s e i n d u c t i o n on s o l i d c e l l u l o s e i n i n d u c t i o n f e r m e n t o r s . W h i l e this p r o c e s s was d e s i g n e d to handle w a s t e n e w s p r i n t , once the f e a s i b i l i t y of c e l l u l o s e h y d r o l y s i s i s e s t a b l i s h e d , i t m a y a l s o be a d v a n t a g e o u s to c o n v e r t h e m i c e l l u l o s e and l i g n i n (111) into s i m p l e , s o l u b l e s u b s t a n c e s . It w o u l d then be p o s s i b l e to t r e a t c r o p r e s i d u e s , g a r b a g e and food p r o c e s s i n g plant w a s t e s and p r o d u c e i m p o r t a n t r a w m a t e r i a l s and energy. T h e s u g a r s p r o d u c e d i n this p r o c e s s c o u l d be r e c o v e r e d o r c o n v e r t e d into ethyl a l c o h o l , m e t h a n e o r s i n g l e c e l l p r o t e i n by

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

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continuous f e r m e n t a t i o n . T h e f o r m of h e m i c e l l u l o s e that i s found i n g r e a t e s t abund a n c e i n food p r o d u c t s s u c h as c o r n i s x y l a n . X y l a n c a n be h y d r o l y z e d by x y l a n a s e and β-xylosidase e n z y m e s to give a h i g h y i e l d of the m o n o s a c c h a r i d e x y l o s e (82). A l t h o u g h x y l o s e h a s l i t t l e v a l u e a s a foodstuff, i t can be c o n v e r t e d to x y l i t o l by c a t a l y t i c hydrogénation o r u s e d a s a c a r b o n s o u r c e f o r f e r m e n t a tion. X y l i t o l h a s a s w e e t n e s s e q u i v a l e n t to s u c r o s e and h a s b e e n s u g g e s t e d as a r e p l a c e m e n t f o r s u c r o s e i n food f o r m u l a tions (112). Appendix: Enzymes

Terminology

and T h e o r e t i c a l A s p e c t s of I m m o b i l i z e d

M o s t i m m o b i l i z e d e n z y m e s exhibit a l t e r e d k i n e t i c b e h a v i o r when c o m p a r e d w i t h the c o r r e s p o n d i n g s o l u b l e e n z y m e s (113) A c t i v i t y of e n z y m e s a l m o s m o b i l i z a t i o n . T h i s ca change i n the e n z y m e s t r u c t u r e d u r i n g i m m o b i l i z a t i o n o r bec a u s e the e n z y m e i s i n a d i f f e r e n t e n v i r o n m e n t a f t e r i m m o b i l i z a t i o n . A c o n f o r m a t i o n a l change i s l i k e l y to o c c u r i f the e n z y m e i s c o v a l e n t l y bound to a support. E n v i r o n m e n t a l e f f e c t s a r e g r e a t e s t when the e n z y m e e n v i r o n m e n t i s m o s t u n l i k e a f r e e aqueous e n v i r o m e n t . F o r example, a c i d i c functional groups i n the v i c i n i t y of an e n z y m e w i l l c a u s e a shift i n m a x i m u m a c t i v i t y to a h i g h e r pH. Reduction i n i m m o b i l i z e d enzyme activity can a l s o o c c u r as a r e s u l t of d i f f u s i o n a l e f f e c t s . In the i m m o b i l i z a t i o n p r o c e s s the e n z y m e can be bound to a s u r f a c e o r h e l d w i t h i n a m a t r i x F i g u r e 7). In o r d e r f o r an e n z y m e c a t a l y z e d r e a c t i o n to o c c u r the s u b s t r a t e m u s t f i r s t d i f f u s e f r o m s o l u t i o n to the s u p p o r t s u r f a c e and, i n the c a s e of an e n t r a p p e d e n z y m e , d i f f u s e w i t h i n the s u p p o r t to r e a c h the e n z y m e . A f t e r r e a c t i o n the p r o d u c t s m u s t then d i f f u s e b a c k into the s o l u t i o n . The r e m o v a l of p r o d uct i s e s p e c i a l l y i m p o r t a n t i f the r e a c t i o n i s r e v e r s i b l e o r i n h i b i t e d by p r o d u c t . In both c a s e s e n z y m e a c t i v i t y i s r e d u c e d by increased diffusional resistance. In o r d e r to c o m p a r e d i f f e r e n t i m m o b i l i z e d e n z y m e p r e p a r a tions, it i s i m p o r t a n t to h a v e a s t a n d a r d i z e d n o m e n c l a t u r e . R e c o m m e n d a t i o n s f o r n o m e n c l a t u r e s t a n d a r d i z a t i o n ( 114) s u g gest that the a c t i v i t y of an i m m o b i l i z e d e n z y m e should be exp r e s s e d a s an i n i t i a l r e a c t i o n r a t e i n t e r m s of m i c r o m o l e s of s u b s t r a t e c o n v e r t e d p e r s e c o n d p e r m g of d r y i m m o b i l i z e d enzyme preparation. A v e r y u s e f u l concept i n d i s c u s s i n g m a s s t r a n s f e r e f f e c t s i s the e f f e c t i v e n e s s f a c t o r , η, w h i c h i s d e f i n e d as the r a t i o of the r a t e of r e a c t i o n f o r an i m m o b i l i z e d e n z y m e to the r a t e of r e a c tion f o r the s a m e a m o u n t of e n z y m e i n s o l u t i o n ( i . e. i n the ab sence of d i f f u s i o n a l e f f e c t s ) . T h e e f f e c t i v e n e s s f a c t o r can be r e a d i l y d e t e r m i n e d e x p e r i m e n t a l l y and g i v e s a good i n d i c a t i o 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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Figure 7.

Immobilized Enzymes

123

Schematic of surface-bound and entrapped enzymes illustrating the important diffusional effects for each

of the extent of d i f f u s i o n a l e f f e c t s . T h e s e e f f e c t s a r i s e f r o m m a s s t r a n s f e r c o n s i d e r a t i o n s e i t h e r e x t e r n a l o r i n t e r n a l to the e n z y m e s u p p o r t s u r f a c e . F o r e n z y m e attached to a s u r f a c e only e x t e r n a l m a s s t r a n s f e r e f f e c t s a r e i n v o l v e d a n d the r a t e of r e a c t i o n w i l l be d e t e r m i n e d b y the r a t e at w h i c h s u b s t r a t e c a n be b r o u g h t to the s u r f a c e . T h i s f l u x of s u b s t r a t e to the s u r f a c e can be c h a r a c t e r i z e d by a m a s s t r a n s f e r c o e f f i c i e n t , k w h e r e c F l u x = k (c - c ) c s' 7

v

w h e r e c = c o n c e n t r a t i o n of s u b s t r a t e i n s o l u t i o n and c = c o n c e n t r a t i o n of s u b s t r a t e a t the s u p p o r t s u r f a c e . T h i s f l u x of s u b s t r a t e i s g r e a t l y a f f e c t e d by the m o v e m e n t of f l u i d p a s t the s u p p o r t s u r f a c e . T h e v a l u e of k i n c r e a s e s a s the f l o w i n c r e a s e s . A n e s t i m a t e of the f l u x f o r s p h e r i c a l p a r t i c l e s can be m a d e b y s e t t i n g c = 0 and l e t t i n g k = 2D/d w h e r e D i s the s u b s t r a t e d i f f u s i v i t y and d i s the p a r t i c l e diarrreter. This i s the v a l u e of k f o r a stagnant f l u i d and r e p r e s e n t s a l o w e r limit for k F o r a m o r e exact e s t i m a t e of k , c o r r e l a tions a r e a v a i l a b l e f o r f l o w through a p a c k e d b e d QjL6). There w i l l be no d i f f u s i o n a l r e d u c t i o n i n the r e a c t i o n r a t e i f the c a l c u l a t e d o v e r a l l f l u x of s u b s t r a t e i s g r e a t e r than the total f r e e enzyme activity. E x t e r n a l d i f f u s i o n a l r a t e l i m i t a t i o n w i l l be a p p a r e n t e x p e r i m e n t a l l y when a c t i v i t y i s m e a s u r e d a s a f u n c t i o n of f l o w r a t e . In the a b s e n c e of e x t e r n a l d i f f u s i o n a l e f f e c t s no change i n

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a c t i v i t y w i l l o c c u r when the f l o w r a t e i s changed. A c t i v i t y w i l l i n c r e a s e w i t h i n c r e a s i n g f l o w r a t e when e x t e r n a l d i f f u s i o n i s l i m i t i n g the r a t e of r e a c t i o n . T h i s i s b e c a u s e i n c r e a s e d f l o w w i l l f a c i l i t a t e t r a n s p o r t of s u b s t r a t e f r o m s o l u t i o n to the support surface. F o r enzymes held within a m a t r i x internal m a s s t r a n s f e r e f f e c t s m u s t be c o n s i d e r e d . P a r t i t i o n i n g of s u b s t r a t e w i l l o c c u r at the s u p p o r t s u r f a c e b e c a u s e of the s o l u b i l i t y d i f f e r e n c e between f r e e s o l u t i o n and w i t h i n the support. E x t e r n a l d i f f u s i o n a l e f f e c t s can be e s t i m a t e d and e x p e r i m e n t a l l y e x a m i n e d as d e s c r i b e d a b o v e . In o r d e r to r i g o r o u s l y c a l c u l a t e the extent of i n t e r n a l d i f f u s i o n a l e f f e c t s on the r a t e of r e a c t i o n , it i s n e c e s s a r y to s o l v e the d i f f e r e n t i a l equations equating the r a t e of s u b s t r a t e d i f f u s i o n w i t h i n the s u p p o r t to the r a t e of r e a c t i o n of s u b s t r a t e . In g e n e r a l n u m e r i c a l s o l u t i o n s a r e r e q u i r e d by these n o n - l i n e a r p a r t i a l d i f f e r e n t i a l equations ( 117 118) The results are usually presente

The T h i e l e m o d u l u s i s a m e a s u r e of the r a t i o of the r a t e of r e a c t i o n to the r a t e of s u b s t r a t e d i f f u s i o n w i t h i n the m a t r i x . It can be e x p r e s s e d i n a n u m b e r of d i f f e r e n t f o r m s , but i n g e n e r a 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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T h i e l e modulus = (support dimension) / i m m o b i l i z e d e n z y m e activity. * Κ ' χ substrate diffusivity m

1/2

7

T h e s u p p o r t d i m e n s i o n i s the r a d i u s f o r s p h e r i c a l p a r t i c l e s o r the t h i c k n e s s f o r m e m b r a n e s and Κ ' i s the M i c h a e l i s constant for the i m m o b i l i z e d e n z y m e . T h e e f f e c t i v e n e s s f a c t o r h a s a v a l u e of one when t h e r e i s no d i f f u s i o n a l l i m i t a t i o n , a s s u m i n g that e n z y m e a c t i v i t y h a s not b e e n a l t e r e d as a r e s u l t of i m m o b i l i z a t i o n . A s can be s e e n f r o m F i g u r e 8 the e f f e c t i v e n e s s f a c t o r a p p r o a c h e s one f o r s m a l l v a l u e s of the T h i e l e m o d u l u s . A l s o , the e f f e c t i v e n e s s f a c t o r i s i n c r e a s e d a s c b e c o m e s l a r g e r than Κ ' s i n c e h i g h s u b s t r a t e c o n c e n t r a t i o n s s a t u r a t e the i m m o b i l i z e c P e n z y m e s u p p o r t . This i n c r e a s e s s u b s t r a t e c o n c e n t r a t i o n i n the i n t e r i o r of the s u p p o r t making m o r e substrat The effectiveness factor is i n c r e a s e d by s m a l l support d i m e n sions, low i m m o b i l i z e d enzyme activity, high substrate diffus i v i t y , and a h i g h s u b s t r a t e c o n c e n t r a t i o n . A l l of these f a c t o r s r e d u c e d i f f u s i o n a l effects. A s i m m o b i l i z e d e n z y m e a c t i v i t y is i n c r e a s e d b y a d d i n g m o r e e n z y m e p e r unit v o l u m e of s u p p o r t , the e f f e c t i v e n e s s f a c t o r d e c r e a s e s s i n c e h i g h e n z y m i c a c t i v i t y c a u s e s the r e a c t i o n to b e c o m e d i f f u s i o n c o n t r o l l e d . In the r e g i o n of d i f f u s i o n c o n t r o l i n F i g u r e 8 the r a t e of the i m m o b i l i z e d e n z y m e r e a c t i o n i s p r o p o r t i o n a l to the s q u a r e root of the e n z y m e c o n c e n t r a t i o n . A p r i m a r y c o n s i d e r a t i o n i n i m m o b i l i z e d e n z y m e u s e i s the a b i l i t y to r e t a i n a c t i v i t y u n d e r c o n d i t i o n s of s t o r a g e and u s a g e . When the r a t e of r e a c t i o n i s l i m i t e d by d i f f u s i o n a l effects, t h e r e i s a n a p p a r e n t i n c r e a s e i n the h a l f - l i f e of the i m m o b i l i z e d e n z y m e ( 119). F i g u r e 9 shows the a p p a r e n t i n c r e a s e i n s t a b i l i t y that i s p o s s i b l e f o r a n e n z y m e u n i f o r m l y d i s t r i b u t e d w i t h i n a s u p p o r t i n g m a t r i x . In the a b s e n c e of d i f f u s i o n a l effects this i m m o b i l i z e d e n z y m e h a s a t r u e h a l f - l i f e of one t i m e u n i t . T h e a p p a r e n t h a l f - l i f e i s i n c r e a s e d at l o w s u b s t r a t e c o n c e n t r a t i o n s , h i g h e n z y m e a c t i v i t y , l a r g e s u p p o r t d i m e n s i o n s , and l o w s u b s t r a t e d i f f u s i v i t y w i t h i n the s u p p o r t o r , i n other w o r d s , by those f a c t o r s that c a u s e a r e a c t i o n to be d i f f u s i o n c o n t r o l l e d . When the r e a c t i o n is c o m p l e t e l y c o n t r o l l e d b y d i f f u s i o n , the r a t e w i l l be p r o p o r t i o n a l to the s q u a r e r o o t of the e n z y m e c o n c e n t r a t i o n and the a p p a r e n t h a l f - l i f e w i l l be twice the true h a l f - l i f e of the e n z y m e . A true m e a s u r e of i m m o b i l i z e d e n z y m e s t a b i l i t y i s obtained only u n d e r c o n d i t i o n s w h e r e d i f f u s i o n a l effects a r e absent. In r e v i e w i n g the l i t e r a t u r e on the k i n e t i c s of i m m o b i l i z e d e n z y m e s , m a n y t h e o r e t i c a l a r t i c l e s c a n be found w h i c h m a k e s u c h r e s t r i c t i v e a s s u m p t i o n s that they a r e of v i r t u a l l y no u s e in m o s t p r a c t i c a l s y s t e m s . F o r i n s t a n c e , i n o r d e r to obtain c l o s e d f o r m a n a l y t i c a l s o l u t i o n s for i m m o b i l i z e d e n z y m e S

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

126

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

LOG Figure 9.

(Cs/K' ) m

Apparent half-life of an enzyme which is uniformly distributed within a spherical particle (119)

r e a c t o r p e r f o r m a n c e , m a n y a u t h o r s a s s u m e that the e n z y m e r e a c t i o n i s f i r s t o r d e r i n s u b s t r a t e c o n c e n t r a t i o n ( c « K ' ). T h i s i s c e r t a i n l y not the c a s e for m o s t r e a c t i o n s of i n t e r e s t i n food s y s t e m s . Literature Cited 1. 2.

3.

4. 5.

Reed, Gerald, "Enzymes in Food Processing," second e d i t i o n , 573 p a g e s , A c a d e m i c Press, N e w Y o r k , 1975. W h i t a k e r , J. R., " P r i n c i p l e s of E n z y m o l o g y f o r the F o o d S c i e n c e s ," 636 p a g e s , M a r c e l D e k k e r , I n c . , N e w Y o r k , 1972. W h i t a k e r , J. R., E d i t o r , " F o o d R e l a t e d E n z y m e s ," 365 pages, American C h e m i c a l Society Advances in C h e m i s t r y S e r i e s N o . 136, 1976. Z a b o r s k y , Ο . Α., " I m m o b i l i z e d Enzymes," 175 p a g e s , CRC Press, Cleveland, Ohio, 1973. P y e , E. K e n d a l l and W i n g a r d , L e m u e l B . Jr., E d i t o r s , " E n z y m e E n g i n e e r i n g , " V o l u m e 2, 470 p a g e s , P l e n u m Press, New Y o r k , 1974.

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

7.

OLSON

6.

7.

8.

9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.

23. 24. 25. 26. 27. 28. 29. 30.

AND

KORUS

Immobilized Enzymes

127

D u n l a p , R . Β., E d i t o r , " I m m o b i l i z e d B i o c h e m i c a l s a n d A f f i n i t y C h r o m a t o g r a p h y , " 377 p a g e s , P l e n u m Press, N e w Y o r k , 1974. O l s o n , A. C. a n d C o o n e y , C . L., E d i t o r s , " I m m o b i l i z e d E n z y m e s i n F o o d a n d Microbial P r o c e s s e s ," 268 p a g e s , P l e n u m Press, N e w Y o r k , 1974. S a l m o n a , M., S a r o n i o , C . a n d G a r a t t i n i , S., E d i t o r s , " I n s o l u b i l i z e d Enzymes," 226 p a g e s , R a v e n Press, N e w Y o r k , 1974. S k i n n e r , K . J., C h e m . a n d Eng. N e w s (1975) 53(33), 2241. R i c h a r d s o n , T., J . Food S c i . (1974) 39, 6 4 5 - 6 4 6 . O l s o n , N. F. a n d R i c h a r d s o n , T., J . F o o d S c i . (1974) 39, 6 5 3 - 6 5 9 . Stanley, W. L. a n d O l s o n , A. C., J . F o o d S c i . (1974) 39, 660-666. H u l t i n , H. O., J L e e , Y. Y. a n d T s a o , G . T., F o o d S c i . (1974) 39, 667-672. S m i l e y , K . L. a n d S t r a n d b e r g , G . W., A d v a n c e s i n A p p l i e d M i c r o b i o l o g y (1972) 15, 13-38. T o s a , T., M o r i , T., F u s e , Ν., a n d C h i b a t a , I., B i o t e c h n o l . B i o e n g . (1967) 9, 603-615. T o s a , T., Mori, T., a n d C h i b a t a , I., Agr. Biol. C h e m . (1969) 33, 1053-1059. S c h n y d e r , B . J., Die S t a r k e (1974) 26, 409-412. T o s a , T., Mori, T. and C h i b a t a , I., Agr. Biol. C h e m . (1969) 33, 1047-1052. C h e n , L. F. a n d T s a o , G . T. (1976), s u b m i t t e d f o r p u b lication. P a r k , Y. K . a n d T o m a , M., J . F o o d S c i . (1975) 40, 1112-1114. Stanley, W . L., W a t t e r s , G . G., K e l l y , S. Η., C h a n , B . G., G a r i b a l d i , J . A., a n d S c h a d e , J . Ε., B i o t e c h n o l . B i o e n g . (1976) 18, 4 3 9 - 4 4 3 . Stanley, W . L., W a t t e r s , G . G., K e l l y , S. Η., a n d O l s o n , A. C . (1976), s u b m i t t e d f o r p u b l i c a t i o n . Stanley, W . L., W a t t e r s , G . G., C h a n , Β., a n d Mercer, J . Μ., B i o t e c h n o l . B i o e n g . (1975) 17, 315-326. M a s r i , M. S., R a n d a l l , V. G. a n d S t a n l e y , W . L., P o l y m e r P r e p r i n t s (1975) 16(2), 7 0 - 7 5 . W e e t a l l , Η . Η., N a t u r e (1969) 223, 9 5 9 - 9 6 0 . W e e t a l l , Η . Η., S c i e n c e (1969) 166, 615-617. M e s s i n g , R . Α., P r o c e s s B i o c h e m i s t r y (1974) 9 (9), 2628. W e e t a l l , Η . Η., F o o d P r o d u c t D e v e l o p m e n t (1973) 7 (3), 46-52. W e e t a l l , Η . H. a n d H a v e w a l a , Ν . Β., B i o t e c h n o l . B i o e n g . S y m p . (1972) 3, 241-266.

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

128 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44.

45. 46. 47. 48. 49. 50. 51.

52. 53. 54.

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

B y r n e , M. J. a n d J o h n s o n , D. Β., B i o c h e m . S o c . T r a n s . (1974) 2, 4 9 6 - 4 9 7 . B r o t h e r t o n , J. Ε., E m e r y , A. a n d R o d w e l l , V . W., B i o t e c h n o l . B i o e n g . (1976) 18, 5 2 7 - 5 4 3 . W e e t a l l , H. H. a n d D e t a r , C. C., B i o t e c h n o l . B i o e n g . (1974) 16, 1537-1544. S a m e j i m a , Η., " E n z y m e E n g i n e e r i n g , " V o l u m e 3, P l e n u m Press, to be p u b l i s h e d . M e s s i n g , R . A. a n d Filbert, A. M., J. Agr. F o o d C h e m . (1975) 23, 9 2 0 - 9 2 3 . W e e t a l l , H. H. a n d Hersh, L. S., B i o c h i m . B i o p h y s . A c t a (1970) 206, 5 4 - 6 0 . H e r r i n g , W . Μ., L a u r e n c e , R . L. a n d Kittrell, J. R., B i o t e c h n o l . B i o e n g . (1972) 14, 975-984. M a r k e y , P . Ε., G r e e n f i e l d , P . F. a n d Kittrell, J. R., B i o t e c h n o l . B i o e n g . (1975) 17, 2 8 5 - 2 8 9 . G r e e n f i e l d , P . F. (1975) 40, 906-910. B o u i n , J. C., D u d g e o n , P . H. a n d H u l t i n , H. O., J. F o o d S c i . (1976) 41, 886-890. A l t o m a r e , R., G r e e n f i e l d , P . F. and Kittrell, J. R., B i o t e c h n o l . B i o e n g . (1974) 16, 1675-1680. B a r n d t , R . L., W a n g , S. S. a n d Leeder, J. G., J. F o o d S c i . (1976) 4 1 , 4 9 4 - 4 9 7 . V i e t h , W . R . a n d V e n k a t a s u b r a m a n i a n , Κ., C h e m . T e c h . (1974) 4, 4 7 - 5 5 . B e r n a t h , F. R . a n d Vieth, W . R., " I m m o b i l i z e d E n z y m e s i n F o o d a n d Microbial P r o c e s s e s ," A. C. O l s o n and C. L. C o o n e y , E d i t o r s , p . 157-185, P l e n u m Press, N e w Y o r k , 1974. Stanley, W. L., W a t t e r s , G . G., C h a n , B . G . a n d K e l l y , S. Η., s u b m i t t e d f o r p u b l i c a t i o n . C a r l s s o n , J., A x e n , R., B r o k l e h u r s t , K. a n d C r o o k , E. Μ., E u r . J. B i o c h e m . (1974) 44, 189-194. O k o s , M. R., Ph.D. D i s s e r t a t i o n , O h i o State U n i v e r s i t y , 1975. O l s o n , A l f r e d C. a n d S t a n l e y , W i l l i a m L., J. Agr. F o o d C h e m . (1973) 21, 4 4 0 - 4 4 5 . B o u n d y , J. A., S m i l e y , K. L., S w a n s o n , C. L. a n d H o f r e i t e r , (1976), s u b m i t t e d f o r p u b l i c a t i o n . G r u l k e , Eric A. a n d O k o s , M a r t i n R., u n p u b l i s h e d results. S a m e j i m a , H. and Kimura, Κ., " E n z y m e E n g i n e e r i n g , " V o l u m e 2, Ε. K. Pye a n d L. B . W i n g a r d , E d i t o r s , p . 131-135, P l e n u m Press, N e w Y o r k , 1974. C h i b a t a , I . , " E n z y m e E n g i n e e r i n g , " V o l u m e 3, P l e n u m Press, to be p u b l i s h e d . S h i m i z u , S . , M o r i o k a , H., T a n i , Y . a n d O g a t a , K., J. F e r m e n t . T e c h n o l . (1975) 53, 7 7 - 8 3 . H a r r i s o n , R . A. P., A n a l . B i o c h e m . (1974) 61, 5 0 0 - 5 0 7 .

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

7.

OLSON

55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71.

72.

73.

74. 75. 76. 77. 78. 79. 80.

AND

KORUS

Immobilized Enzymes

129

O ' D r i s c o l l , K . F., A d v a n c e s i n B i o c h e m . Eng., to be published. O ' D r i s c o l l , K . F., H i n b e r g , I., K o r u s , R . a n d K a p o u l a s , A., J. P o l y m e r Sci. : S y m p o s i u m N o . 46 (1974) 2 2 7 - 2 3 5 . M a e d a , Η., B i o t e c h n o l . B i o e n g . (1975) 17, 1571-1589. T o s a , K . a n d Shoda, Μ., B i o t e c h n o l . B i o e n g . (1975) 17, 481-497. W a t e r l a n d , L. R., M i c h a e l s , A. S., a n d R o b e r t s o n , C. R., A m e r . Inst. C h e m . E n g . J o u r n a l (1974) 20, 5 0 - 5 9 . W a t e r l a n d , L. R., R o b e r t s o n , C . R . a n d M i c h a e l s , A. S., C h e m . E n g . C o m m u n . (1975) 2, 3 7 - 4 7 . K o r u s , R . A. a n d O l s o n , A. C., J. F o o d Sci., a c c e p t e d for p u b l i c a t i o n , 1976. K o r u s , R . A. a n d Olson, A. C., B i o t e c h n o l . B i o e n g . , a c c e p t e d f o r p u b l i c a t i o n , 1976. K o b a y a s h i , H. a n d S u z u k i , H., J. F e r m e n t . T e c h n o l . (1972) 50, 6 2 5 - 6 3 2 K o b a y a s h i , H. an (1976) 18, 37-51. T o s a , Τ., Mori, T . a n d C h i b a t a , I., A g r . Biol. C h e m . (1969) 33, 1053-1059. Tosa, Τ., Mori, Τ., Fuse, N. a n d C h i b a t a , I., B i o t e c h n o l . B i o e n g . (1967) 9, 603-615. A n o n y m o u s , C h e m . E n g . (1970) 77, 4 4 - 4 6 . M e r m e l s t e i n , Ν . Η., F o o d T e c h n o l . (1975) 29 (6), 2 0 - 2 5 . A n o n y m o u s , Food P r o c e s s i n g ( 1975) 36 ( 11), 2 6 - 2 8 . S h i m i z u , J. and K a g a , T. (1972) U.S. P a t e n t 3664927. Scott, D., " E n z y m e s i n F o o d P r o c e s s i n g , " s e c o n d e d i t i o n , G . R e e d , E d i t o r , 4 9 6 - 4 9 8 , A c a d e m i c Press, N e w Y o r k , 1975. R a c k i s , J. J., S e s s a , D . J., S t e g g e r d a , F. F., S h i m i z u , T., A n d e r s o n , J. a n d Pearl, S. L., J. F o o d S c i . (1970) 35, 6 3 4 - 6 3 9 . Slott, S . , M a d s e n , G . a n d N o r m a n , Β. Ε., " E n z y m e E n g i n e e r i n g 2," Ε. K . P y e a n d L. B. W i n g a r d , E d i t o r s , 343-350, P l e n u m Press, N e w Y o r k , 1974. B e r n f e l d , P . a n d W a n , J., S c i e n c e (1973) 142, 6 7 8 - 6 7 9 . B a r k e r , S. A., S o m e r s , P . J., E p t o n , R . a n d McLaren, J. V., C a r b o h y d . R e s . (1970) 14, 287-296. S t r u m e y e r , D. Η., C o n s t a n t i n i d e s , A. a n d F r e u d e n b e r g e r , J., J. F o o d S c i . (1974) 39, 498-502. L e d i n g h a m , W . M. a n d H o r n b y , W. Ε., FEBS L e t t . (1969) 5, 118. B u t t e r w o r t h , T. Α., W a n g , D. I. C. a n d S i n s k e y , A. J., B i o t e c h n o l . B i o e n g . (1970) 12, 615-631. W y k e s , J. R., D u n n i l l , P . a n d Lilly, M. D., Biochim. Biophys. A c t a (1971) 250, 5 2 2 - 5 2 9 . L i n k o , Y., S a a r i n e n , P. a n d Linko, Μ., B i o t e c h n o l . B i o e n g . (1975) 17, 153-165.

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

130 81. 82. 83. 84. 85. 86. 87. 88. 89. 90. 91. 92. 93. 94. 95. 96. 97. 98. 99. 100. 101.

102.

103. 104. 105. 106.

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

M a r s h a l l , J. J. and W h e l a n , W. J., C h e m . Ind. (1971) 1971 (25), 701-702. R e i l l y , P e t e r , J., 1976, p e r s o n a l c o m m u n i c a t i o n . M a r t e n s s o n , K a j , B i o t e c h n o l . B i o e n g . (1974) 16, 567577. M a r t e n s s o n , K a j , B i o t e c h n o l . B i o e n g . (1974) 16, 5 7 9 591. M a r t e n s s o n , K a j , B i o t e c h n o l . B i o e n g . (1974) 16, 15671587. B a c h l e r , M. J., S t r a n d b e r g , G. W. a n d S m i l e y , K. L., B i o t e c h n o l . B i o e n g . (1970) 12, 8 5 - 9 2 . S m i l e y , K. L., B i o t e c h n o l . B i o e n g . (1971) 13, 309-317. W i l s o n , R. J. H. a n d Lilly, M. D., B i o t e c h n o l . B i o e n g . (1969) 11, 349-362. K a y , G. and Lilly, M. D., B i o c h i m . B i o p h y s . A c t a (1970) 198, 276-285. O ' N e i l l , S. P., D u n n i l l B i o t e c h n o l . B i o e n g . (1971 337-352. C h r i s t i s o n , J., C h e m . I n d . (1972) 1972 (5), 215-216. P a r k , Y. K. a n d Lima, D . C., J. F o o d Sci. (1973) 38, 358-359. B a u m , G., B i o t e c h n o l . B i o e n g . (1975) 17, 253-270. Walton, H. M. a n d E a s t m a n , J. Ε., B i o t e c h n o l . B i o e n g . (1973) 15, 951-962. M a e d a , Η., Y a m a u c h i , A. a n d S u z u k i , Η., B i o c h i m . B i o p h y s . A c t a (1973) 315, 18-21. M a e d a , Η., S u z u k i , Η., Y a m a u c h i , A. a n d S a k i m a e , A., B i o t e c h n o l . B i o e n g . (1974) 16, 11517-1528. S t r u m e y e r , D. Η., C o n s t a n t i n i d e s , A. a n d F r e u d e n b e r g e r , J., J. F o o d Sci. (1974) 39, 4 9 8 - 5 0 2 . M a r s h , D. R., Lee, Y. Y. a n d Tsao, G . T., B i o t e c h n o l . B i o e n g . (1973) 15, 4 8 3 - 4 9 2 . M a r s h , D. R., P h . D . D i s s e r t a t i o n , Iowa State U n i v e r s i t y , A m e s , Iowa, 1973. S o l o m o n , B . a n d Levin, Y., B i o t e c h n o l . B i o e n g . (1975) 17, 1323-1333. W e e t a l l , Η. Η., H a v e w a l a , Ν . Β., G a r f i n k e l , Η. Μ., B u e h l , W . M. and B a u m , G., B i o t e c h n o l . B i o e n g . (1974) 16, 169-179. L e e , D. D., Lee, Y. Y., R e i l l y , P . J., C o l l i n s , Ε . V., Jr. and T s a o , G. T., B i o t e c h n o l . B i o e n g . (1976) 18, 253267. L e e , D. D., Lee, Y. Y. a n d Tsao, G. T., D i e S t a r k e (1975) 27, 384-387. M a r s h , D. R. and Tsao, G. T., B i o t e c h n o l . B i o e n g . (1976) 18, 349-362. S a r d i n a s , J. L., Process B i o c h e m . (1976) 11, 10-17. B a r i c o s , W. Η., C h a m b e r s , R . P. a n d C o h e n , W., E n z y m e T e c h n o l o g y D i g e s t (1975) 4, 3 9 - 5 3 .

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

7.

OLSON

107. 108. 109. 110. 111. 112. 113. 114.

115. 116. 117. 118. 119.

AND

KORUS

131

Immobilized Enzymes

B o w e r s , Larry D . a n d Carr, P e t e r W., Anal. C h e m . (1976) 48, 5 4 4 A - 5 5 9 A . W e e t a l l , Η. Η., Anal. C h e m . (1974) 46, 6 0 2 A - 6 1 5 A . M a n d e l s , M. a n d S t e r n b e r g , D., J. F e r m e n t . T e c h n o l . (1976) 54, 267-286. W i l k e , C. R. and Y a n g , R. D., A p p l i e d P o l y m e r S y m p o s i u m N o . 28 (1975) 175-188. K i r k , Τ. Κ., B i o t e c h n o l . B i o e n g . S y m p . (1975) 5, 139150. A n d r e s , C., F o o d P r o c e s s i n g ( 1976) 37 (4), 100. P i t c h e r , W. Η., Jr., C a t a l . Rev.-Sci. E n g . (1975) 12, 37-69. S u n d a r a m , P. V. a n d Pye, Ε. K , " E n z y m e E n g i n e e r i n g ," V o l u m e 2, Ε. K. P y e a n d L. B. W i n g a r d , Jr., E d i t o r s , 449-452, P l e n u m Press, N e w Y o r k , 1974. O ' N e i l l , S. P., B i o t e c h n o l . B i o e n g . (1972) 14, 675-678. L e e , Y. Y., Fratzke, B i o t e c h n o l . B i o e n g . (1976) 18, 3 8 9 - 4 1 3 . K o b a y a s h i , T. and Laidler, K. J., B i o c h i m . B i o p h y s . A c t a (1973) 302, 1-12. K o r u s , R. A. and O'Driscoll, K. F., C a n . J. C h e m . E n g . (1974) 52, 7 7 5 - 7 8 0 . K o r u s , R. A. a n d O'Driscoll, K. F., B i o t e c h n o l . B i o e n g . (1975) 17, 4 4 1 - 4 4 4 . One of u s , RAK, was a n NRC-ARS P o s t d o c t o r a l A s s o c i a t e d u r i n g the p r e p a r a t i o n of this p a p e r .

Research

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

8 Conversion of Aldehydes to Alcohols in Liquid Foods by Alcohol Dehydrogenase C. ERIKSSON, I. QVIST, and K. VALLENTIN SIK, The Swedish Food Institute, Fack, S-40021 Göteborg, Sweden

Enzymic aldehyde-alcohol conversion Aliphatic aldehydes and alcohols are frequently found in the volatile fraction of most kinds of foods, e.g., dairy products, meat, poultry, fish, edible oils, vegetables, potato, fruits, and berries. Aldehydes and alcohols can be formed in a food in the normal or abnormal metabolism of tissues and microorganisms, in Strecker degradation during food processing or in lipid oxidation during storage of fresh, dried or frozen foods or food components. The formation of certain odor-potent aldehydes and alcohols from unsaturated fatty acids under the influence of enzymes and hemoproteins, particularly in vegetables, has been summarized earlier (1) . Aliphatic aldehydes, alcohols and esters can be transformed into each other as scheduled by the reaction route. 12 R-CHO . R-CH OH χ R-COO-R Step 1 is catalyzed by the enzyme alcohol dehydroge nase (ADH) in the presence of reduced or oxidized ni cotinamide-adenine dinucleotides (NADH, NAD ), while in step 2 formation of an ester can be achieved by ester synthetase systems, in the presence of a carboxylic acid (R -COOH), a reaction that is less well known for these types of esters. In step 2 the hydro lysis of aliphatic esters involves carboxylic-ester hydrolases. Green pea skins contain such a hydrolase, which was purified by ammonium sulfate fractionation, gel chromatography and two consecutive steps of ion exchange chromatography. This enzyme was found to hydrolyze ethyl-, η-propyl-, η-butyl-, n-pentyl, n-hexyl-, and η-hex-trans-2-enylacetate in increasing 2

+

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

8.

ERIKSSON

E T AL.

Aldehyde-Alcohol Conversion in Liquid Foods

133

rate order, while etylpropionate, -buturate, -valerate, and - h e x a n o n a t e were n o t h y d r o l y z e d b y t h i s enzyme (Yamashita, I . , and E r i k s s o n , C , unpublished results). alco alip alco from The

E a r l i e r investigations have revealed that plant hol dehydrogenases have a wide s p e c i f i c i t y over hatic straight chain saturated and unsaturated h o l s and aldehydes. This is true for the enzyme p e a (2) , o r a n g e (_3 ) , p o t a t o (k) , a n d t e a (5.) · equilibrium constant of the reaction Alcohol

+ NAD ^==± +

Aldehyde

+ NADH

+ H

+

was f o u n d t o f a v o r a l c o h o l f o r m a t i o n at t h e pH a n d coenzyme r e l a t i o n s that are normally found i n plant material. The e q u i l i b r i u m constant also varies due to the nature of the a l c o h o l - aldehyde p a i r (2). This variation shows t h e c a l c u l a t e d m o l p e r c e n t a g e of n-hexanal and n-hex-trans-2-enal, each i n e q u i l i b r i u m with the cor responding alcohol for two d i f f e r e n t pH a n d NAD/NADH values. Table

I.

Composition of aldehyde-alcohol equilibrium mixtures. Theoretical figures. 100 100 10 10 NAD/NADH PH 6.0 6. 0 7-0 7· 0 1.4 0. l4 0. i4 n-Hexanal (%) 0 .014 n-Hexanol \^>) 99 .986 98.6 9 9 . 86 99- 86 n-Hex-trans-2-enal ($) n - H e x - t r a n s - 2 - e n o l (°/o)

1 .4

98 . 6

12. 5 87. 5

12. 5 87- 5

58 42

Other aldehyde-alcohol pairs w i l l produce s t i l l other figures a c c o r d i n g to chain l e n g t h , presence of d o u b l e bonds (number and p o s i t i o n ) , b r a n c h i n g etc. Odor

properties

of

aldehydes

and

alcohols

The importance of the above knowledge f o r the flavor o f a f o o d was f u r t h e r studied in experiments, where the odor d e t e c t i o n concentration i n water of n-hexanal, η - h e x - t r a n s - 2 - e n a l , n-hex-2,^-dienal, n-hept-trans-2-enal, n-oct-trans-2-enal, and the c o r r e s p o n d i n g a l c o h o l s was d e t e r m i n e d (6). It was found that the odor d e t e c t i o n concentration of both the aldehydes and the a l c o h o l s d e c r e a s e d w i t h the chain length of C^-Cg saturated compounds, while it i n c r e a s e d w i t h the number o f d o u b l e bonds i n C5 a l d e hydes and a l c o h o l s . T h i s c a n be seen i n T a b l e I I , which also shows t h a t t h e o d o r d e t e c t i o n concentration

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

134

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

of alcohols t h a t of the Table

II.

Compound

i s i n a l l cases aldehydes.

significantly

higher

than

Odor d e t e c t i o n c o n c e n t r a t i o n (ODC) o f some aldehydes and a l c o h o l s (Figures from reference 6) ODC ODC ( M ) ODC aid.

n-Hexan-1-ol n-Hexanal η-H ex- trains-2-en - 1- ο 1 η-Hex-trans-2-enal η - H e x - t r a n s - 2 , t r a n s - 4 - d i e n - 1 - oo l n-Hex-trans-2,trans-4·^dienal n-Hept-trans-2-enn-Hept-trans-2-ena n-0ct-trans-2-en-1-ol n-Oct-tran s-2-enal

5 7 5 6 4 6 5

4.8 1.9 6.7 3.2 4 22..4 5.0

X X X X X X

10 10 10 10 10 10

6.6 2.9

X X

10 8 10

259 21 49

231

The r e s u l t s c o n c e r n i n g the e q u i l i b r i u m and odor properties p r o v i d e d the background of f u r t h e r experi ments to r e d u c e the amount o f a l d e h y d e s i n f o o d s by c o n v e r t i n g them to a l c o h o l s by the a d d i t i o n o f A D H . T h i s r e a c t i o n can f o r i n s t a n c e be a p p l i e d to liquid f o o d s , whose p o l y u n s a t u r a t e d f a t t y a c i d s , although present i n small amounts, have o x i d i z e d sufficiently to g i v e r i s e to o f f - f l a v o r due to a l d e h y d e formation. Such a p p l i c a t i o n studies are d e s c r i b e d l a t e r i n this article. Due t o t h e c o n t r i b u t i o n o f s e v e r a l aldehydes to this kind of off-flavor and to d i f f e r e n c e s in their r e a c t i o n r a t e and e q u i l i b r i u m c o n c e n t r a t i o n as well as i n odor d e t e c t i o n c o n c e n t r a t i o n o f b o t h aldehydes and alcohols, such a r e a c t i o n w i l l , or should, not always merely quench the aldehyde odors. The result might t h e r e f o r e be a changed odor s e n s a t i o n from a complex mixture of d i f f e r e n t aldehydes and a l c o h o l s . T h i s s e n s a t i o n m i g h t t h u s n o t o n l y d e p e n d on presence of o d o r b u t a l s o an p o s s i b l e q u a l i t y c h a n g e s , that o c c u r when a l d e h y d e s a r e c o n v e r t e d t o a l c o h o l s o r vice versa. F o r t h i s r e a s o n an a t t e m p t was made t o find out whether c o r r e s p o n d i n g a l d e h y d e s and a l c o h o l s also differ i n odor quality. Water s o l u t i o n s of n - h e x - t r a n s - 2 - e n o l and n - h e x trans-2-enal of varied strength, as w e l l as f o u r dif f e r e n t m i x t u r e s o f t h e s e compounds were s u b j e c t e d to descriptive odor a n a l y s i s . In performing t h i s analysis a s p e c i a l l y t r a i n e d p a n e l w i t h 12 m e m b e r s w a s u s e d .

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

8.

ERIKSSON

E T AL.

135

Aldehyde-Alcohol Conversion in Liquid Foods

T h e p a n e l was t h e same a s t h e o n e u s e d e a r l i e r for d e s c r i p t i v e odor a n a l y s i s of soy and rape seed p r o t e i n (7_) · T h e o d o r n o t e s u s e d i n t h e a n a l y s i s w e r e selected from the l i t e r a t u r e and from other i n v e s t i g a t i o n s in o u r l a b o r a t o r y on o d o r p r o p e r t i e s of pure chemicals. T h e s e o d o r n o t e s w e r e p r e - t e s t e d w i t h some o f the samples to be a n a l y z e d . F i n a l l y the i n t e n s i t y o f thir teen odor notes ( T a b l e I I I ) were e s t i m a t e d , u s i n g a 0 - 9 point scale. T h e maximum s c o r e , 9> represents the strongest i n t e n s i t y one w o u l d e x p e c t i n n o r m a l , everyday l i f e , w h i l e the minimum s c o r e , 0, meant ab sence of that p a r t i c u l a r odor note. The samples, containing varying concentrations of either n-hex-trans-2-enol or η - h e x - t r a n s - 2 - e n a l or mix t u r e s o f t h e m , w e r e p r e s e n t e d a s 20 m l p o r t i o n s a t r o o m temperature i n 50 m l E r l e n m e y e r f l a s k s provided with clear-fit stoppers. Detaile fication of the compounds and sample p r e p a r a t i o n have been p u b l i s h e d elsewhere (6). Table

III.

Selected

odor

notes for

n-hex-trans-2-enol Odor strength Sharp pungent Green, l i k e grass Green, leafy Sour, acid Fruity, citrus Fruity, other In the a n a l y s i s were e v a l u a t e d (Table session three differen each sample had been Table

IV.

1 2 3 4 5 6 7 8 9 10

No.

water

solutions

of

n-hex-trans-2-enal.

Sweet Musty Floral Nut-like Oily, fatty Rubber Candle-like

samples a l t o g e t h e r 10 d i f f e r e n t IV) i n random order. In each u n t i l t samples were a n a l y z e d , evaluated four times.

Concentration trans-2-enol samples used

Sample

and

in and for

mg p e r

l i t r e

(ppm) of

n-hex-

n-hex-trans-2-enal in ten odor d e s c r i p t i o n analysis.

n-Hex-trans-2-enol

n-Hex-tran s-2-enal

10 40 100 -

0.5 5 20 0.5 5 0.5 5

—

10 10 40 4o

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

136

ENZYMES

IN FOOD A N D B E V E R A G E

PROCESSING

A c o m p u t e r p r o g r a m was u s e d t o c a l c u l a t e t h e mean and standard deviation o f the i n t e n s i t y of each odor note f o r each sample and a l s o to t e s t significant dif f e r e n c e s between the samples. I t was f o u n d t h a t t h e most t y p i c a l o d o r n o t e s of n - h e x - t r a n s - 2 - e n ο 1 were "Rubber" and " C a n d l e - l i k e " while those of n-hex-trans-2-enal were " F r u i t y , other" and "Green, l e a f y " and to a l e s s e r extent "Green, like g r a s s " and " F l o r a l " . Odor notes which were perceived e q u a l l y f o r b o t h compounds were "Sour, a c i d " , "Fruity, c i t r u s " and " O i l y , f a t t y " (cf. Table III). The r e s u l t s are e x e m p l i f i e d i n F i g u r e 1 where the changes i n six of the i n i t i a l l y selected thirteen odor n o t e s as i n f l u e n c e d by c o n c e n t r a t i o n are shown. It is o b v i o u s t h a t an i n c r e a s e in concentration of the single compounds e x p e c t e d l y i s f o l l o w e d b y an i n c r e a s e i n per ceived odor i n t e n s i t notes (samples 1-3 an mixtures ( s a m p l e s 7-10) except for the odor note "Rub ber" which decreased i n i n t e n s i t y on i n c r e a s i n g the aldehyde concentration alone (cf. sample 7 and 8, 9 and 10 r e s p e c t i v e l y ) . Most p r o b a b l y , the odor note " R u b b e r " , p r o d u c e d p r i m a r i l y b y t h e a l c o h o l , was mas ked by the increased i n t e n s i t y of other odor notes i n troduced by the i n c r e a s e d aldehyde concentration. In c o n c l u s i o n , a c o n v e r s i o n between this parti c u l a r a l d e h y d e and a l c o h o l w i l l r e s u l t i n an overall change i n odor i n t e n s i t y with l i t t l e change i n odor quality. Application

studies.

B e e r , m i l k and apple j u i c e are well-known examples of l i q u i d foods which have been r e p o r t e d to develop o x i d a t i v e f l a v o r due to l i p i d o x i d a t i o n , a l t h o u g h b o t h beer and a p p l e j u i c e c o n t a i n o n l y m i n o r amounts of l i p i d s . At t h i s stage these products contain alde hydes l i k e n-hexanal, n-hex-trans-2-enal, n-hepttrans-2-enal, etc. I n i t i a l studies revealed that the p r o d u c t s m e n t i o n e d c o n t a i n e d no a c t i v e ADH and no or v e r y s m a l l amounts o f NAD and NADH. I t was also found that n-hexanal added to beer, m i l k , and apple j u i c e c o u l d r a p i d l y and e f f e c t i v e l y be c o n v e r t e d into n - h e x a n o l by the a d d i t i o n o f ADH and NADH. M i l k was c h o s e n as t h e f i n a l o b j e c t of investiga t i o n s i n c e the pH o f a p p l e j u i c e , b e e r and m i l k is a r o u n d 3·5> 4 . 1 , and 6 . 2 , r e s p e c t i v e l y , a n d on t h e fact t h a t p a r t i c u l a r l y A D H , but a l s o NADH, i s l e s s stable a t a p H l o w e r t h a n 6. T h e m i l k u s e d was p r o d u c e d b y a fistulated cow t h a t h a d b e e n f e d s u n f l o w e r o i l in +

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

ERIKSSON E T A L .

Aldehyde-Alcohol Conversion in Liquid Foods

Intensity

Intensity

"FRUITY, CITRUS"

"FRUITY,OTHER"

Intensity

"GREEN, LEAFY

"GREEN,UKE GRASS"

Sample

Sample

Intensity

Intensity

"FLORAL"

"RUBBER"

Sample

0

12 3

M L

4 56

J l l i

7 8 910

.Sample

Figure 1. Mean panel intensities of selected characteristic odor notes used in descriptive analysis of 10 water solutions containing n-hex-trsais-2-enol (samples 1-3), n-hex-trans-2-enal (samples 4-6), and mixtures of the two compounds (7-10). The concentration of the odor substances are listed in Table IV.

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

138

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

order to i n c r e a s e the p r o p o r t i o n of polyunsaturated f a t t y acids i n the milk f a t . Such milk undergoes rapid l i p i d o x i d a t i o n i m m e d i a t e l y a f t e r m i l k i n g , much larger amounts of n - h e x a n a l and s i m i l a r aldehydes b e i n g formed than i n ordinary milk. The sunflower o i l was admini strated t o t h e cow t h r o u g h a t u b i n g d i r e c t l y i n t o the abomasum i n o r d e r t o p r e v e n t the unsaturated fatty acids from b e i n g hydrogenated i n the rumen, which is the case a f t e r o r a l i n t a k e of unsaturated f a t . The fatty acid fraction of the milk produced contained 2k°/o o l e i c , 19°/o l i n o l e i c a n d λ°/ο l i n o l e n i c a c i d . The m i l k was p a s t e u r i z e d a t 6 3 ° C f o r 30 m i n , 2 . 5 mg o f tetracycline ( D u m o c y c l i n , Dumex, C o p e n h a g e n , Denmark) b e i n g added per l i t r e of milk to i n c r e a s e the m i c r o biological stability. T h e s t a b i l i z e d m i l k was then d i v i d e d i n t o two b a t c h e s , one w h i c h was a l l o w e d to c o n t a i n the o r i g i n a and another where th proximately Λ°/ο ( r e d u c e d f a t milk). The f o l l o w i n g two e x p e r i m e n t s were made. In the f i r s t experiment one hundred ml p o r t i o n s of f u l l fat milk and reduced fat m i l k were s t o r e d f o r k days at 4°C i n s t e r i l i z e d stoppered flasks. E v e r y day f o u r samples each of e i t h e r whole milk or reduced fat m i l k were withdrawn f o r gas chromatographic n-hexanal a n a l y s i s . I n two of t h e s e s a m p l e s f r o m e a c h b a t c h , 3 mg o f y e a s t A D H (300 U / m g , B o e h r i n g e r , M a n n h e i m , W . G e r m a n y ) a n d 50 m g o f N A D H (789ε β - N A D H , B o e h r i n g e r , M a n n h e i m , W . G e r m a n y ) per 100 m l w e r e a d d e d 30 m i n p r i o r t o t h e n-hexanal analysis. T h e r e m a i n i n g two s a m p l e s o f e a c h b a t c h were not t r e a t e d and u s e d as c o n t r o l s . The r e a c t i o n t o o k p l a c e at 20°C. The n - h e x a n a l a n a l y s e s were per f o r m e d on d a y s 1 a n d 3 w i t h t h e w h o l e m i l k and on days 2 and k w i t h the r e d u c e d f a t milk. In t h e s e c o n d e x p e r i m e n t 3 m g o f A D H a n d 50 m g o f NADH p e r 100 m l w e r e a d d e d t o b o t h t h e w h o l e m i l k and reduced fat m i l k on t h e s e c o n d d a y a f t e r m i l k i n g a n d pasteurizing. The samples were then s t o r e d f o r 9 days a t k C· C o n t r o l s w i t h no enzyme and coenzyme a d d e d were run p a r a l l e l . T h e n - h e x a n a l a n a l y s e s were made o n d a y s 3> 5> 79 a n d 9 w i t h b o t h t h e enzyme-coenzyme treated and the u n t r e a t e d milk. F o r n - h e x a n a l d e t e r m i n a t i o n t w o 100 m l s a m p l e s of m i l k a n d 200 m l o f d i s t i l l e d w a t e r f o r d i l u t i o n w e r e transferred on e a c h o c c a s i o n t o a p e r v i o u s l y described head space sampling device connected to a Perkin-Elmer 900 g a s c h r o m â t o g r a p h (8). Three hundred ml of heads p a c e g a s was f o r c e d t h r o u g h a p r e c o l u m n c o n c e n t r a t i o n a c c e s s o r y by a i r pressure f o r 30 m i n w h e r e b y t h e volat i l e o r g a n i c compounds i n the head space gas were c o n -

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

8.

ERIKSSON

E T AL.

Aldehyde-Alcohol Conversion in Liquid Foods

139

densed i n a cool trap. The condensed m a t e r i a l was allowed to enter the i n j e c t o r o f the gas chromâtοgraph, kept at 110°C b y s u d d e n h e a t i n g o f t h e t r a p w i t h an oil b a t h at 140°C. T h e s e p a r a t i o n was p e r f o r m e d on an tubular stainless steel c o l u m n , Ο . 7 6 mm I . D . χ 181 mm, c o a t e d w i t h S F 9 6 / l g e p a l CO88O ( 9 5 / 5 ) . The oven tem p e r a t u r e was p r o g r a m m e d 2 0 - l 4 0 ° C a t 2°/min after an i n i t i a l 3 min i s o t h e r m a l p e r i o d , and the n i t r o g e n gas f l o w was 12 m l / m i n . T h e F I D s i g n a l was f e d i n t o an Infratronics CRS-101 electronic integrator, with d i g i tal print-out equipment. The i n t e g r a t o r values of the h e x a n a l peak ( p r e v i o u s l y i d e n t i f i e d by mass spectro metry) were used to r e p r e s e n t the n - h e x a n a l content of the head space. The r e s u l t s g i v e n i n T a b l e s V a n d V I show that boih discontinuous (first experiment) and continuous (second experiment) treatmen either reduced the concentratio formed n-hexanal instantaneously or kept i t reduced throughout the experiment. In the gas chromatograms one c o u l d a l s o see t h a t the n - h e x a n o l peak r o s e along w i t h the d e c l i n e of the n - h e x a n a l peak and t h a t other aldehydes and a l c o h o l s behaved s i m i l a r l y . The i n i t i a l n - h e x a n a l c o n c e n t r a t i o n was i n t h e r a n g e of 0.5 1 ppm, as compared to e a r l i e r studies on m i l k with added n-hexanal. Table

V.

Concentration of over milk stored of added a l c o h o l coenzyme (NADH).

n-hexanal i n the headspace a t k°C. Immediate effect dehydrogenase (ADH) and (integrator values).

Days

Τ Skim no

no

30

min

5

860

910

130

125

milk

addition

Whole

milking

3

milk

ADH+NADH, Whole

after

milk

addition

Skim

2

810

85Ο

100

120

milk

ADH4-NADH,

30

min

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

ENZYMES

140 Table

V I .

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

Concentration o f n-hexanal i n the headspace o v e r m i l k s t o r e d a t k°C f o r 9 d a y s . Long term e f f e c t o f a l c o h o l d e h y d r o g e n a s e (ADH) and c o e n z y m e (NADH) a d d e d on d a y 2. (integrator values). Days

Skim no

no

added

day

2

800

910

1030

125

125

125

76Ο

820

875

100

100

100

1035

125

milk

addition

Whole

milking

milk

ADH+NADH

Whole

after

milk

addition

Skim

PROCESSING

930

milk

ADH+NADH

added

day

2

130

The f u r t h e r a p p l i c a t i o n of enzymic conversion of aldehydes and a l c o h o l s b y t h e A D H - N A D H system must probably await the development of suitable reactors. In m a t e r i a l c o n t a i n i n g enough NADH o r N A D i n correct concentration ratios this coenzyme content can be u t i l i z e d i n the reaction. When, however, the c o enzyme i s i n s u f f i c i e n t o r l a c k i n g , coenzyme from an external source, such as spent brewer's y e a s t , must be u s e d . By a standard procedure (9) we f o u n d that b r e w e r ' s y e a s t c o n t a i n s a b o u t 2 . 5 mg N A D + N A D H / g d r y yeast i n a NAD /NADH ratio f r o m a p p r o x i m a t e l y 2/1 t o 10/1 d e p e n d i n g o n t h e w a y o f p r e p a r a t i o n . A high pro p o r t i o n o f NADH can be obtained by r e d u c t i o n ofN A D either chemically, electrolytically (_K)) o r e n z y m a t i c a l iy. S i n c e t h e number o f m o l e c u l e s o f coenzyme needed w i l l b e t h e same a s t h e n u m b e r o f c o n v e r t i b l e aldehyde or a l c o h o l m o l e c u l e s , i t w i l l i n most c a s e s be b o t h i m p r a c t i c a l and uneconomical to apply the A D H - N A D ( H ) technique without disposal of re-usable enzymecoenzyme systems. I m m o b i l i z a t i o n o f enzymes and c o enzymes on s u i t a b l e carriers seems t o b e a f r u i t f u l evolution to enable future conversions of this type. In t h e c a s e o f A D H a n d N A D H b o t h t h e enzyme a n d a c o enzyme a n a l o g u e have been i m m o b i l i z e d on S e p h a r o s e k B, which allowed the formation of a binary ADH-NADH complex. T h e i m m o b i l i z e d b i n a r y complex was a l s o shown t o b e f u n c t i o n a l i n t h e same w a y a s t h e soluble one (11). T h e o x i d i z e d a n d r e d u c e d coenzymes c a n e a s i l y be t r a n s f e r r e d into each other i n s i t u by a p p l y i n g

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

8.

ERIKSSON E T A L .

Aldehyde-Alcohol Conversion in Liquid Foods

alternative substrates. and r e g e n e r a t e coenzyme and membrane t e c h n i q u e

Other suggestions to are microincapsulation (13)·

141

retain ( 12)

Abstract. Enzymic interconversion of a l i p h a t i c aldehydes, alcohols, and carboxylic esters i s b r i e f l y surveyed. Odor properties are presented, both quantitative ones, as odor detection concentration and q u a l i t a t i v e ones, as odor descriptions of certain aldehydes and a l c o h o l s , which are normally found i n foods after lipid oxidation. Experiments were performed to reduce the amount of preformed aldehydes, p a r t i c u l a r l y n-hexanal i n milk containing polyunsaturated f a t , by addition of alcohol dehydrogenase and NADH. Acknowledgment. The authors are indebted to Mrs. M. Knutsson, M. Sc., Astra-Ewos AB, Södertälje, Sweden, f o r d e l i v e r i n g polyunsaturated cow's milk for t h i s study. Literature cited. (l)

E r i k s s o n , C., i n "Industrial Aspects of B i o chemistry", e d . , Spencer, B., V o l . 30, part I I , p. 865, North Holland/American E l s e v i e r , Amsterdam, 1974.

(2)

E r i k s s o n , C.E., J. Food

(3)

Bruemmer, J.H. and Roe, B., J. Agr. Food Chem. (1971) 19, 266. Davies, D.D., Patil, K.D., Ugochukwu, E . N . and Towers, G.H.N., Phytochemistry (1973) 12, 523. Hatamaka, A. and Oghi, T., Agr. Biol. Chem. (1972) 36, 2033. E r i k s s o n , C.E., Lundgren, Β and V a l l e n t i n , Κ., Chemical Senses and Flavor (1976) 2, 3.

(4) (5) (6)

Sci.

(1968) 33,

525.

(7)

Q v i s t , I . and von Sydow, Ε., Lebensm.-Wiss. u. Technol. (1976) i n press.

(8)

von Sydow, Ε., Andersson, J., Anjou, Κ., Karlsson, G., Land, D. and G r i f f i t h s , Ν., Lebensm.-Wiss. u. Technol. (1970) 3, 11. Klingenberg, Μ., i n "Methoden der enzymatischen Analyse", e d . , Bergmeyer, H . U . , V o l . 2, p. 1975, Verlag Chemie, Weinheim, 1970.

(9)

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

142

(10)

(11) (12) (13)

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

Alizawa, Μ., Coughlin, R.W. and Charles, Μ., Biotechnology and Bioengineering (1976) 18, 209. Gestrelius, S., Månsson, M-O. and Mosbach, Κ., Eur. J. Biochem. (1975) 57, 529. Campbell, J. and Ming Swi Chang, T., Biochim. Biophys. Acta (1975) 397, 101. Chambers, R.P., Ford. J.R., A l l e n d e r , J.H., Baricos, W.H. and Cohen, W., i n "Enzyme E n gineering", ed., Kendall Pye, Ε., and Wingard, J r , L. B., V o l . 2, p. 195, Plenum Press, New York, 1974.

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

9 Physiological Roles of Peroxidase in Postharvest Fruits and Vegetables NORMAN F. HAARD Department of Biochemistry, Memorial University of Newfoundland, St. Johns, Newfoundland, Canada, A1C5S7

Peroxidase (E.C. 1.11.1.7 hydroge was initially reported as a c o n s t i t u e n t of mushroom e x t r a c t s i n 1855 (1) and was l a t e r named by L i n o s s i e r i n 1898 (2). Since t h i s time i t has been the subject of continued i n t e r e s t , experimentat i o n and s p e c u l a t i o n (3). Peroxidase appears to be ubiquitous to the living s t a t e having been i d e n t i f i e d i n animal, p l a n t , microbial and viral systems. Peroxidase is a common c o n s t i t u e n t of higher p l a n t s and may occur a t concentrations up to s e v e r a l percent on a f r e s h weight b a s i s . There is extensive l i t e r a t u r e d e s c r i b i n g changes in peroxidase a c t i v i t y and isoenzyme spectra as a f u n c t i o n of ontogenic change, c o n d i t i o n s of s t r e s s (e.g. water deficit, freeze i n j u r y , chill i n j u r y , h y p e r s e n s i t i v i t y , pathogen i n t e r a c t i o n , hyperoxygenicity, etc.) and as a s p e c i f i c c h a r a c t e r i s t i c of plant v a r i e t i e s and c u l t i v a r s . Although thousands of experimental studies with peroxidase have been publ i s h e d , we have a relatively poor grasp of the f u n c t i o n ( s ) and metabolic c o n t r o l ( s ) of t h i s enzyme in v i v o . Today, there a r e many who b e l i e v e that peroxidase is a v e s t i g e of the past, having no e s s e n t i a l f u n c t i o n in higher p l a n t s . A l t e r n a t i v e l y , other i n d i v i d u a l s f i n d it tempting to link peroxidase with a myriad of important events which include r e s p i r a t o r y c o n t r o l , gene c o n t r o l , hormone metabolism and the b i o s y n t h e s i s and biodegradation of a wide range of secondary plant metabolites. There have been ext e n s i v e s t u d i e s d e s c r i b i n g the a c t i o n of peroxidase on substances which y i e l d b r i g h t c o l o r s on o x i d a t i o n , but which have no apparent p h y s i o l o g i c a l r o l e . Unfortunately, there has been f a r too little done to understand the p h y s i o l o g i c a l l y r e l e v a n t subs t r a t e s f o r t h i s enzyme. I r r e s p e c t i v e of t h e i r p h y s i o l o g i c a l r o l e , it has been w e l l e s t a b l i s h e d that peroxidase can c o n t r i b u t e to d e t e r i o r a t i v e changes i n f l a v o r , texture, c o l o r and n u t r i t i o n i n properly processed f r u i t s and vegetables (4-6). I t has g e n e r a l l y been observed that peroxidase is q u i t e s t a b l e to adverse c o n d i t i o n s encountered during food processing - such as elevated temperature, f r e e z i n g , i o n i z i n g r a d i a t i o n , dehydration and even when inacti143 In Enzymes in Food and Beverage Processing; Ory, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

144

ENZYMES

IN FOOD A N D

BEVERAGE

PROCESSING

vated by such treatments i s capable of r e - n a t u r a t i o n (7). Regene r a t i o n of peroxidase a c t i v i t y can pose a s e r i o u s l i m i t a t i o n to a food p r o c e s s i n g o p e r a t i o n . For example, f r u i t s and vegetables subjected to h i g h temperature - short time treatments (HTST) are p a r t i c u l a r l y prone to peroxidase r e g e n e r a t i o n and a s s o c i a t e d q u a l i t y change during storage. Regeneration of peroxidase occurs w i t h i n hours or days f o l l o w i n g thermal processing and may occur even a f t e r s e v e r a l months i n f r o z e n f r u i t s and vegetables. Peroxidase regeneration appears to i n v o l v e r e f o l d i n g of the polypeptide chain (8) and may r e l a t e to the f a c t that peroxidase contains a conjugated carbohydrate moiety. The r e s i s t a n c e of peroxidase to t h e r mal i n a c t i v a t i o n , together with i t s u b i q u i t y and d i r e c t i n v o l v e ment i n d e t e r i o r a t i o n of food q u a l i t y , has l e d to the wide use of peroxidase a c i t i v i t y as an index of processing e f f i c a c y by blanching and other heat, treatments. Peroxidase i s a l s o of u t i l i t y to the food t e c h n o l o g i s t . For example, i t i s used e x t e n s i v e l dase f o r the d e t e c t i o n a c t i o n the hydrogen peroxide generated by the glucose oxidase c a t a l y z e d conversion of glucose to g l u c o n i c a c i d i s used i n the p e r o x i d a t i c r e a c t i o n to produce a r e a d i l y measurable chromophore from a hydrogen donor such as O - d i a n i s i d i n e . The coupled r e a c t i o n of glucose oxidase and peroxidase may a l s o f i n d use i n food processes designed to r i d a system of r e s i d u a l glucose or oxygen (9). A l s o , the f i n d i n g that isoenzyme p r o f i l e s of peroxidase may be h i g h l y s p e c i f i c f o r d i f f e r e n t p l a n t t i s s u e s a l s o leads to the sugg e s t i o n that peroxidase be used as a means of checking a d u l t e r a t i o n or contamination i n p l a n t e x t r a c t i v e s such as f l o u r s and protein isolates. In t h i s t r e a t i s e , I w i l l examine s e v e r a l l i n e s of experiment a t i o n designed to understated the p h y s i o l o g i c a l r o l e of peroxidase i n postharvest f r u i t s and vegetables. In most case, harvested f r u i t s and vegetables undergo ontogenic change s i m i l a r to that which occurs on the parent p l a n t . A c c o r d i n g l y ; t h e r e a c t i o n s to be discussed are somewhat d i f f e r e n t from those o c c u r r i n g i n processed foods where the s t r u c t u r e and f u n c t i o n a l c a p a b i l i t y of the t i s s u e has been g r o s s l y d i s r u p t e d . I t i s c l e a r that at l e a s t part of the mystery surrounding peroxidases i s due to the presence of unusua l l y l a r g e numbers of isoenzyme species and to the observation that peroxidase can c a t a l y z e a v a r i e t y of r e a c t i o n s , i n some cases with apparently low s u b s t r a t e s p e c i f i c i t y . For t h i s reason i t i s important to review some general information on t h i s enzyme. Classification Peroxidases have been c l a s s i f i e d as i r o n c o n t a i n i n g p e r o x i dases and f l a v o p r o t e i n peroxidases ( 3 ) . The i r o n enzymes are f u r ther subgrouped i n t o ferriprotoporphyrin peroxidases and verdoperoxidases. The f i r s t group a l l c o n t a i n f e r r i p r o t o p o r p h y r i n I I I as a p r o s t h e t i c group and e x h i b i t a red-brown c o l o r when h i g h l y p u r i -

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

9.

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Peroxidase in Postharvest Fruits and Vegetables

145

f i e d . The f e r r i p r o t o p o r p h y r i n type peroxidases are common i n higher p l a n t s and have a l s o been i d e n t i f i e d i n animals (e.g. t r y p tophan p y r r o l a s e , t h y r o i d i o d i n e peroxidase) and microorganisms (e.g. yeast cytochrome C p e r o x i d a s e ) . The verdoperoxidases c o n t a i n an i r o n porphyrin p r o s t h e t i c group which d i f f e r s from f e r r i p r o t o porphyrin I I I and which i s not removed on treatment with a c i d i c acetone as occurs with the former group of peroxidases. Examples of t h i s enzyme are lactopeioxidase found i n m i l k and myeloperoxidase from myelocytes. F l a v o p r o t e i n peroxidases, c o n t a i n FAD as p r o s t h e t i c group, and have been p u r i f i e d from microorganisms (e.g. Streptococcus f a e c a l i s ) a n d animal t i s s u e s . At t h i s time we know r e l a t i v e l y l i t t l e about the l a t t e r two groups of p e r o x i dase i n higher p l a n t s . However, the f e r r i p r o t o p o r p h y r i n I I I peroxidases from h o r s e r a d i s h , Japanese r a d i s h and t u r n i p have been f a i r l y w e l l c h a r a c t e r i z e d . The h o r s e r a d i s h enzyme has a molecul a r weight of approximately 40,000 and contains one f e r r i p r o t o porphyrin I I I group per bonds are taken up i n i n t e r a c t i o One of the remaining c o o r d i n a t i o n bonds appears to be a s s o c i a t e d with a c a r b o x y l group of the p r o t e i n and the other i s coordinated to an amino group or to a water molecule. The h o r s e r a d i s h enzyme and apparently other sources of peroxidase (10) c o n t a i n conjugated carbohydrate which appears to impart unusual s t a b i l i t y to the molecule. The h o r s e r a d i s h enzyme i s s t a b l e i n s o l u t i o n from pH 4 to 12, r e t a i n s 50% of i t s a c t i v i t y a f t e r h e a t i n g at 100 C f o r 12 minutes and i s s t a b l e to low water a c t i v i t y and f r e e z i n g . Because of t h e i r c h a r a c t e r i s t i c a b s o r p t i o n maxima i n the U.V. and i n the Soret r e g i o n the peroxidases are o f t e n c h a r a c t e r ized by the r a t i o of a b s o r p t i o n at 403 and 275 nm or the R.Z. value ( R e i n h e i t s z a h l ) . A h i g h l y p u r i f i e d isoenzyme of h o r s e r a d i s h peroxidase may have an R.Z. value greater than 3.0. Catalytic Properties Four general types of c a t a l y t i c a c t i v i t y have been found i n a s s o c i a t i o n with peroxidases. These are the p e r o x i d a t i c , o x i d a t i c , c a t a l a t i c and h y d r o x y l a t i o n r e a c t i o n s . The p e r o x i d a t i c r e a c t i o n , more g e n e r a l l y thought to be of most p h y s i o l o g i c a l s i g n i f i c a n c e , has been studied more e x t e n s i v e l y than the other three reactions. P e r o x i d a t i c Reaction. In g e n e r a l , p e r o x i d a t i c r e a c t i o n s occur i n the presence of a wide v a r i e t y of hydrogen donors, i n c l u d i n g p - c r e s o l , q u a i c o l , r e s o r c i n o l , benzidine and 0 - d i a n i s i dine. C e r t a i n peroxidases appear to have a greater a f f i n i t y f o r s p e c i f i c hydrogen donors such as NADH, g l u t a t h i o n e or cytochrome C (11). One approach to e l u c i d a t e hydrogen donors of p h y s i o l o g i c a l importance i s the use of a f f i n i t y chromatography (12). The p r e f e r r e d oxidant f o r the p e r o x i d a t i c r e a c t i o n i s hydrogen peroxide although other peroxides are e f f e c t i v e s u b s t r a t e s . In post-

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

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harvest t i s s u e s the p e r o x i d a t i c r e a c t i o n of most obvious importance at t h i s time i s l i g n i f i c a t i o n which can profoundly i n f l u e n c e the toughening of vegetables such as beans and asparagus. I t has a l s o been suggested that the p e r o x i d a t i c r e a c t i o n f u n c t i o n s to protect the c e l l u l a r m i l i e u from peroxides which may cause an imbalance i n redox p o t e n t i a l and damage membranes, enzymes, e t c . (13). O x i d a t i c Reaction. The o x i d a t i c r e a c t i o n r e q u i r e s the presence of molecular oxygen and a s u i t a b l e hydrogen donor. Examples of s o - c a l l e d redogenic hydrogen donors are i n d o l e - 3 - a c e t i c a c i d (IAA), o x a l a c e t i c a c i d , dihydroxyfumaric a c i d , a s c o r b i c a c i d and hydroquinone. The IAA oxidase f u n c t i o n of peroxidase appears to be extremely important i n postharvest f r u i t s and vegetables. There i s a l s o recent evidence that c y t o k i n i n s are o x i d i z e d by peroxidase. I t may a l s o be that the o x i d a t i c f u n c t i o n may cont r i b u t e to d e t e r i o r a t i v t i o n and o x i d a t i o n of e s s e n t i a idases which are e f f i c i e n t i n p e r o x i d a t i c r e a c t i o n with c e r t a i n s u l f h y d r y l reagents w i l l convert them to forms capable of o x i d a t i c c a t a l y z e d o x i d a t i o n of hydrogen donors such as membrane l i pids (14). I t i s tempting to speculate that peroxidase f u n c t i o n s i n such a d e t e r i o r a t i v e way i n senescing or s t r e s s e d f r u i t s and vegetables s i n c e increases i n peroxidase a c t i v i t y i n v a r i a b l y precede or accompany the h y p e r s e n s i t i v e r e a c t i o n . C a t a l a t i c and Hydroxylation Reactions. In the absence of hydrogen donor, peroxidase can convert hydrogen peroxide to water and oxygen although t h i s r e a c t i o n i s some 1000 times slower than the p e r o x i d a t i c and o x i d a t i c r e a c t i o n s . F i n a l l y i n the presence of c e r t a i n hydrogen donors, such as dihydroxyfumaric a c i d , and molecular oxygen, peroxidase can c a t a l y z e h y d r o x y l a t i o n of a v a r i e t y of aromatic compounds notably t y r o s i n e , phenylalanine, p - c r e s o l and benzoic a c i d . The metabolism of phenolic substances i s of p a r t i c u l a r importance to the q u a l i t y of postharvest f r u i t s and vegetables i n that they may act as e f f e c t o r s of hormone metabo l i s m , intermediates i n l i g n i n b i o s y n t h e s i s and may r e s u l t i n d i s c o l o r a t i o n r e s u l t i n g from t h e i r enzymic or non-enzymic oxidation. Isoenzymes The occurrence of m u l t i p l e forms of peroxidase was f i r s t noted by T h e o r e l l (15) working with h o r s e r a d i s h r o o t s . This t i s sue contains seven major isoenzyme forms, which are s i m i l a r i n molecular weight and amino a c i d composition but may be separated by i o n exchange chromatography or e l e c t r o p h o r e s i s (16). The c a t i o n i c species c o n t a i n greater numbers of a r g i n i n e residues while the a n i o n i c species are r i c h i n glutamic a c i d , phenylalanine and t y r o s i n e . Other sources of isoperoxidases are s i m i l a r to 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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horseradish species i n t h e i r constancy of molecular weight and d i f f e r e n c e s i n e l e c t r o p h o r e t i c behavior. Peroxidase from r i p e n ing banana f r u i t i s composed of at l e a s t twelve isoenzymes which have i s o e l e c t r i c p o i n t s ranging from approximately 3.3 up to 9.5 (17). T h i s wide range i n P j makes i t impossible to i s o l a t e and p u r i f y a l l isoenzymes by one technique s i n c e c e r t a i n species are i n v a r i a b l y l o s t by p r e c i p i t a t i o n or adsorption phenomena. There are many s t u d i e s showing changes i n isoperoxidase with r e spect to growth, d i f f e r e n t i a t i o n , age and r e a c t i o n to environment a l s t r e s s e s . We w i l l come back to some s p e c i f i c examples of such change i n postharvest systems. While isoenzyme forms o f t e n d i f f e r i n c a t a l y t i c e f f i c i e n c y (18), f a r too l i t t l e work has been done to e l u c i d a t e the physiol o g i c a l s i g n i f i c a n c e of these m u l t i p l e forms. One must c e r t a i n l y t e s t the p o s s i b i l i t y that isoperoxidases are an a r t i f a c t of the i s o l a t i o n and p u r i f i c a t i o n procedures employed (19). I t has a l s o been shown that c e r t a i species during plant developmen t r a c t i o n (20). There have been, however, numerous experiments employing i n h i b i t o r s of t r a n s c r i p t i o n and t r a n s l a t i o n , deuterium oxide l a b e l and i n c o r p o r a t i o n of labeled-amino a c i d s which demonstrate the de nova synthesis of new isoperoxidases during the p l a n t ' s l i f e c y c l e (21,22). Cellular Localization There are s e v e r a l r e p o r t s shwoing that peroxidase i s l o c a l ized i n v a r i o u s s e c t o r s of the c e l l i n c l u d i n g ribosomes (23), nucleus (24), nucleolus (24), mitochondria (25), c e l l w a l l s (26) and i n the i n t e r c e l l u l a r spaces (27). Peroxidase may be bound to given c e l l p a r t i c u l a t e s by i o n i c i n t e r a c t i o n or by covalent bonding (26). In most cases, only c e r t a i n isoenzyme species are found i n a s s o c i a t i o n with a c e l l u l a r o r g a n e l l e or f r a c t i o n . Unf o r t u n a t e l y i t i s o f t e n d i f f i c u l t to d i s t i n g u i s h l o c a l i z a t i o n r e p r e s e n t a t i v e of the n a t i v e c e l l and that which a r i s e s as a r e s u l t of c e l l u l a r d i s r u p t i o n . I t i s , however, i n t r i g u i n g to spec u l a t e that nature's r a t i o n a l e f o r m u l t i p l e molecule forms of peroxidase l i e s i n t h e i r a f f i n i t y f o r s p e c i f i c microenvironments w i t h i n and between the c e l l s . Such microcompartmentation may provide an added dimension of s p e c i f i c i t y to the peroxidases. In a d d i t i o n , i t i s known that a s s o c i a t i o n of peroxidase with c e l l surfaces can profoundly a f f e c t t h e i r c a t a l y t i c p r o p e r t i e s . This phenomenon, c a l l e d a l l o t o p i c c o n t r o l , w i l l be i l l u s t r a t e d i n the f o l l o w i n g case s t u d i e s . C o n t r o l of L i g n i n B i o s y n t h e s i s L i g n i f i c a t i o n may occur i n postharvest f r u i t s , vegetables, c e r e a l s and pulses and have a profound i n f l u e n c e on the e d i b l e q u a l i t y of the t i s s u e . The presence of l i g n i n and a s s o c i a t e d

American Chemicaf Society Library 1155 16th St. H. W. In Enzymes in Food and Beverage Processing; Ory, R., et al.; Washington, D. C. 20036 ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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TABLE I . Comparison of browning and g r i t c e l l formation i n d i f f e r e n t pear c u l t i v a r s .

Cultivar

TABLE I I .

Browning

Sclereids

40.0

++ +

++ +

52.5

++ +

++ +

97.5

++++++

+

22.5

+

Total polyphenolics

+++++ +

T o t a l recoverabl fruit. Peroxidase a c t i v i t y , assayed with hydrogen peroxide as oxidant and O - d i a n i s i d i n e as H-donor, has the u n i t s A 460 nm/min/mg Ν a t 25 C. 'Yuzuhada f r u i t e x h i b i t excessive l i g n i f i c a t i o n r e l a t i v e to ' B a r t l e t t ' f r u i t . Data from (34). 1

peroxidase days before harvest

'Bartlett'

'Yuzuhada'

0.62

TABLE I I I .

35

1.19

28

1.28

1.18

21

0.84

0.82

14

0.89

0.52

7

1.10

0.63

0

1.18

0.82

Soluble peroxidase from developing pear f r u i t . Peroxidase a c t i v i t y , assayed with hydrogen perox ide as oxidant and O - d i a n i s i d i n e as Η-donor, has the u n i t s A 460 nm/min/mg Ν a t 25 C. 'Yuzuhada' f r u i t have excessive l i g n i f i c a t i o n compared t o ' B a r t l e t t f r u i t . Data from (34). 1

peroxidase 'Bartlett'

'Yuzuhada'

21

0.65

0.39

14

0.81

0.29

7

1.08

0.37

0

1.18

0.57

days before harvest

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f i b r o u s m a t e r i a l s can reduce the d i g e s t a b i l i t y c o e f f i c i e n t of prot e i n s by as much as eighty percent. Toughening of vegetables, such as asparagus and beans, i s due to l i g n i f i c a t i o n of f i b r o v a s c u l a r t i s s u e which occurs s h o r t l y a f t e r harvest (28). Simil a r l y , c e r t a i n f r u i t s c o n t a i n aggregates of h i g h l y l i g n i f i e d c e l l s ( s c l e r e i d s ) which impart a g r i t t y t e x t u r e to the pulp (29). The extent of l i g n i f i c a t i o n i n such cases i s markedly i n f l u e n c e d by v a r i e t y or c u l t i v a r type, c u l t i v a t i o n p r a c t i c e s and postharvest h a n d l i n g . Peroxidase i s involved with c o n j u n c t i o n of phenylpropanoid u n i t s during the d e p o s i t i o n of l i g n i n (30). While other enzymes p a r t i c i p a t e i n l i g n i n b i o s y n t h e s i s , evidence p o i n t s to the peroxidase c a t a l y z e d r e a c t i o n as an important l e v e l of c o n t r o l (31). T i s s u e c u l t u r e s t u d i e s have shown that l i g n i n formation occurs only when peroxidase i s a s s o c i a t e d with c e l l surfaces and that calcium ions e l i c i t s o l u b l i z a t i o n of the w a l l bound enzyme (32, 33). The concept that peroxidas subject to a l l o t o p i c c o n t r o s o l v i n g a problem encountered by f r u i t growers. "Yuzuhada D i s o r d e r " . Pear f r u i t may e x h i b i t a p h y s i o l o g i c a l d i s o r d e r c a l l e d "Yuzuhada" which i s symptomized by excessive s c l e r e i d development (29). "Yuzuhada" i s transmitted as a hered i t a r y c h a r a c t e r i s t i c but may develop i n any c u l t i v a r which i s subjected to adverse growing c o n d i t i o n s . Our survey of some f o r t y s e l e c t i o n s of pear f r u i t i n d i c a t e d an i n v e r s e r e l a t i o n s h i p between f r e e p h e n o l i c substances, noteably c h l o r o g e n i c a c i d , and the degree of s c l e r e i d formation (Table 1) (34). This observation suggested any given pear s e l e c t i o n w i l l i n v a r i a b l y e i t h e r be h i g h l y s u b j e c t to enzymic browning or to l i g n i f i c a t i o n and the most acceptable s e l e c t i o n s were intermediate i n l e v e l s of f r e e phenoli c s and i n l i g n i n content. From these f i n d i n g s we hypothesized that m i n e r a l n u t r i t i o n and l o c a l i z a t i o n i n the developing f r u i t determine the extent of peroxidase a s s o c i a t i o n with the l i g n i n template(s) which, i n t u r n , d e l i m i t s the conjugation of phenylpropanoid u n i t s i n t o l i g n i n . The t h e s i s was supported by our study of peroxidase d i s t r i b u t i o n i n s o l u b l e and p a r t i c u l a t e p o o l s . Comparison of peroxidase i n a pear s e l e c t i o n extremely prone to s c l e r e i d development with a l e s s s u s c e p t i b l e v a r i e t y at v a r i o u s stages of f r u i t development revealed that peroxidase l o c a l i z a t i o n and not i t s net a c t i v i t y were p o s i t i v e l y r e l a t e d to s c l e r e i d f o r mation. Although t o t a l peroxidase was c o n s i s t e n t l y higher i n the pear s e l e c t i o n e x h i b i t i n g minimal l i g n i f i c a t i o n (Table 2), i t was p r i m a r i l y l o c a l i z e d i n the s o l u b l e phase (Table 3 ) . A l t e r n a t i v e l y , a r e l a t i v e l y h i g h f r a c t i o n of peroxidase was a s s o c i a t e d with c e l l p a r t i c u l a t e s i n the pear s e l e c t i o n e x h i b i t i n g "Yuzuhada" d i s o r d e r (Table 4). Histochemical examination of these f r u i t f o r peroxidase confirmed the observation that wall-bound peroxidase was more extensive i n the f r u i t e x h i b i t i n g excessive l i g n i f i c a t i o n . The calcium content of the pulp was a l s o c o n s i s t e n t with

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

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TABLE IV. C e l l w a l l and membrane-bound peroxidase from developing pear f r u i t . Peroxidase a c t i v i t y assayed with hydrogen peroxide as oxidant and O - d i a n i s i d i n e as H-donor has the u n i t s A 460 nm/min/mg Ν at 25°C. "Yuzuhada f r u i t e x h i b i t excessive l i g n i f i c a t i o n r e l a t i v e t o B a r t l e t t f r u i t . Data from (34). 11

f

f

peroxidase days before harvest

'Yuzuhada'

'Bartlett'

21

0.19

0.43

14

0.09

0.23

7 0

TABLE V.

Calcium content of developing pear f r u i t . C a l cium was determined by atomic absorption spec trophotometry on pulp. Note lower l e v e l s of calcium during e a r l y stages of development where l i g n i f i c a t i o n i s i n i t i a t e d . Data from (34). calcium ( p p m ) 'Yuzuhada'

days before harvest

'Bartlett'

41

46

16

34

56

30

27

68

32

21

56

40

14

54

36

7

48

36

0

52

52

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our t h e s i s (Table 5). The lower calcium content of "Yuzuhada" f r u i t throughout e a r l y development may have provided an environment conducive to peroxidase a s s o c i a t i o n with l i g n i n template. The i n f l u e n c e of calcium on peroxidase r e l e a s e from c e l l w a l l s i s shown i n F i g u r e 1. Although a d d i t i o n a l study i s needed t o confirm t h i s model i t provides an a t t r a c t i v e explanation r e l a t i n g mineral n u t r i t i o n of the t r e e to important q u a l i t y parameters of the fruit. F i b e r Formation i n Postharvest Asparagus. Asparagus spears undergo texture changes that occur during maturation, e i t h e r i n the f i e l d or i n storage, and these changes progress from the butt end of the stem t o the t i p . Toughening of asparagus i s due t o l i g n i f i c a t i o n of f i b r o v a s c u l a r t i s s u e and occurs w i t h i n hours a f t e r harvest i n spears stored a t ambient c o n d i t i o n s (35). In view of the apparent involvement of peroxidase-wall a s s o c i a t i o n s i n developing pear f r u i t see whether s i m i l a r c o n t r o l If so, b r i e f treatment of spears with s o l u t i o n s c o n t a i n i n g c a l cium s a l t s would be expected t o prevent l i g n i f i c a t i o n a f t e r harv e s t . Attempts by us to achieve such b e n e f i c i a l e f f e c t s were uns u c c e s s f u l apparently because of the greater t e n a c i t y of asparagus peroxidase f o r the f i b r o v a s c u l a r bundles. Levels of calcium e f f e c t i v e i n a r r e s t i n g l i g n i n d e p o s i t i o n (0.5M C a C l ) i n v a r i a b l y caused extensive t i s s u e damage which was followed by pathogen i n v a s i o n . The s o l u b l i z a t i o n of peroxidase from asparagus t i s s u e r e q u i r e d approximately f i v e - f o l d higher l e v e l s of Ca++ than was observed f o r pear f r u i t (Figure 2)(28). While we observed no d i f ferences i n the degree of w a l l binding of peroxidase and r e l a t i v e l y l i t t l e d i f f e r e n c e i n e x t r a c t a b l e a c t i v i t y during aging of the spears (Figure 3 ) , the i n i t i a t i o n of r a p i d l i g n i f i c a t i o n was c l o s e l y p a r a l l e l e d by the emergence o f new isoperoxidase species (Figure 4). The i n d u c t i o n of new isoperoxidase species and l i g nin d e p o s i t i o n occurred w i t h i n a few hours a f t e r c u t t i n g and were dependent on p r o t e i n s y n t h e s i s . The emergence of new i s o p e r o x i dase species progresses from the butt end of the spear to the t i p . A s i m i l a r p r o g r e s s i o n from the butt to the t i p i s observed i n the toughening process. The emergence of the prédominent new i s o peroxidase species c o r r e l a t e d c l o s e l y with l i g n i n d e p o s i t i o n i n the three segments of the spear (Figure 5). These f i n d i n g s suggest that
Why i s i t that the simple process of c u t t i n g t r i g g e r s the r a p i d b i o s y n t h e s i s of l i g n i n ? I t has a l s o been observed that the height of cut during harvest d r a m a t i c a l l y i n f l u e n c e s the r a t e of toughening of spears (Figure 6 ) . Spears cut at i n c r e a s i n g heights above the ground l e v e l toughen much f a s t e r than those cut a t below ground l e v e l as was the p r a c t i c e p r i o r to the i n t r o d u c t i o n of mechanical h a r v e s t i n g . Our data i n d i c a t e that the c u t t i n g opera t i o n leads to formation of wound ethylene. Spears cut above

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

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

0!05

0.10

C«C1

2

0.15

0.20

0.25

0.30

i n Extraction Madia (M)

Journal of Food Science

Figure 1. Effect of calcium chloride on peroxidase release from pulp tissue of Bartlett ( ) and Yuzuhada ( ) pear fruit (29)

.2

.4

.6

.8

CaCI ( M ) 2

Journal of Food Science

Figure 2. Solubilization of peroxidase from freshly harvested asparagus spear tissue by calcium chloride. Spears were divided into tip (O - O), mid (-\ and butt (Ή sections prior to extraction (29).

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

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1

1

1

20

Figure 3.

1 Time

1

1

40

After

1

60

1

1

1

80

Harvest HRS.

Recovery of total peroxidase from spear tissue stored at 20°C for up to 72 hr (29)

Figure 4. Densitometric scans (460 nm) of polyacrylamide gels of 10 peroxidase extracts from: (A) spears frozen in liquid nitrogen immediately after harvest; (B) spears stored at 20 C for 24 hr after harvest; and (C) spears stored at 20°C for 48 hr after harvest. Spears were divided into tip (- · -), mid ( ), and butt ( ) sections (29) e

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

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

+

12

24

36

48

12

24

36

48

Τ ime.Hr..

Figure 5. Area R, 0.0-0.1 and F, 0.0-0.5 as a function of storage time at 20°C. One inch represents A/ nm of 0.1 and approximately 1-cm distance along the gel. Tip (O - O), mid (-\ \-), and butt f | sections, respectively, showed increased lag in emergence of isoenzyme species with the indicated R/29). t60

J

1

1

1

5

1

10

1

1

1

15

Distance,cm Journal of Food Science

Figure 6. Effect of height of cut toughening of spears stored at 10°C for 2 hr (A-A; Η h> and 72 hr (Ί -1; Ο - Ο ). Spears were cut at 5 cm above ground level (A-A; W~M) ground level (O-O; -\ \-). Each data point represents an average of readings from four spears (29). o r a t

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

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ground l e v e l produce ethylene at a greater r a t e than those cut below ground l e v e l (Table 6). Moreover, treatment of spears with exogenous ethylene a c c e l e r a t e s the emergence of new isoperoxidase species and, as w e l l , the r a t e of spear toughening (28). Our suggested r o l e of ethylene i n l i g n i f i c a t i o n a l s o lends n i c e l y to previous observations that c o n t r o l l e d atmospheres employing e l e vated carbon d i o x i d e depress the onset of f i b e r development (36). Carbon d i o x i d e i s a competitive i n h i b i t o r of a l l known ethylene mediated events i n p l a n t t i s s u e (37). B i o s y n t h e s i s of S t r e s s Metabolites Sweet potato roots accumulate a v a r i e t y of metabolites i n response to s t r e s s (38). These i n c l u d e phenolic substances, coamarin d e r i v a t i v e s and a f a m i l y of f i f t e e n and nine carbon furanoterpenoids (Figure 7 ) . These furanoterpenoid s t r e s s metabo l i t e s are of i n t e r e s t t i n marketable sweet potatoe Ipomeamorone and ipomeamaronal are hepatotoxins and the nine c a r bon compounds 4-ipomeanol, 1-ipomeanol, 1.4-ipomeadiol and i p o meanine are lung and kidney t o x i n s . Evidence has been provided that these terpenes are f u n g i s t a t i c agents which c o n t r i b u t e to disease r e s i s t a n c e of the sweet potato root (38). E a r l i e r s t u d i e s showed that on i n f e c t i o n with the b l a c k r o t organism (Cerat o c y s t i s f i m b r i a t a ) r o o t s accumulate peroxidase i n the healthy l a y e r of c e l l s adjacent to the zone of i n c i p i e n t i n f e c t i o n (41). These f i n d i n g s l e d to the n o t i o n that a s p e c i f i c isoperoxidase species acts as an e f f e c t o r of p r o t e i n synthesis by v i r t u e of i t s a b i l i t y to modify l y s i n e residues i n h i s t o n e p r o t e i n s , and t h e r e by a c t s to e l i c i t the formation of furanoterpenoids (42). I f t h i s concept i s c o r r e c t i t may help us understand why root t i s s u e accumulates low l e v e l s of furanoterpenoids as a r e s u l t of s t r e s s e s imposed during storage and handling. We a l s o had the idea that a unique isoperoxidase may be used as a simple i n d i c a t o r f o r the presence of s t r e s s metabolites i n root t i s s u e . Ethylene can increase the r e s i s t a n c e of sweet potatoes to i n f e c t i o n by £. f i m b r i a t a (43). Such r e s i s t a n c e i s a l s o a s s o c i ated with increased peroxidase and polyphenoloxidase a c t i v i t y (38). E a r l i e r s t u d i e s i n d i c a t e d that ethylene r e l e a s e d from sweet potato t i s s u e by i n v a s i o n of C. f i m b r i a t a took part i n i n c r e a s i n g peroxidase a c t i v i t y (44). Matsuno and U r i a n i (45) reported that sweet potato r o o t s c o n t a i n 4 major and 3 minor isoperoxidase spec i e s . Black r o t i n f e c t e d species e x h i b i t e d no change i n i s o peroxidase numbers while c u t - i n j u r e d and ethylene t r e a t e d root s l i c e s contained a new c a t i o n i c species c a l l e d component H. Ind u c t i o n of component H was accompanied by the formation of a l i g n i n - l i k e substance on the cut s u r f a c e . These workers provided a d d i t i o n a l evidence that component H was e f f i c i e n t i n the conjugation of phenylpropanoid monomers i n t o l i g n i n . Thus, while cut

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 j u r y and ethylene l e d to increases of c o n s t i t u t i v e enzymes and i n d u c t i o n of a new isoperoxidase, i n f e c t i o n only r e s u l t e d i n i n creases i n the constitutive-enzymes. While these data appear to negate any hypothesis suggesting the involvement of a unique i s o peroxidase i n the e l i c i t o r response we continued with t h i s work because previous s t u d i e s d i d not account f o r isoperoxidases ass o c i a t e d with the c e l l u l a r p a r t i c u l a t e f r a c t i o n s . Our study showed that sweet potato roots c o n t a i n i o n i c a l l y bound peroxidase which increased s l i g h t l y as a r e s u l t of cut i n j u r y and q u i t e d r a m a t i c a l l y as a r e s u l t of ethylene treatment and i n f e c t i o n (Figure 8 ) . The r a t e at which peroxidase a c t i v i t y increased a f t e r i n o c u l a t i o n was dependent on the v i r u l e n c e of the mold. That i s , a more v i r u l e n t c u l t u r e of
In Enzymes in Food and Beverage Processing; Ory, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

Peroxidase in Postharvest Fruits and Vegetables

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/

C H

157

3

Ό

" 7 1

X

C H

3

0 OH

Figure 7. Stress metabolites from sweet potato root ipomeamorone, top—a hepatotoxin and 4-ipomeanol, bot tom—a lung toxin

Time

(days)

Physiological Plant Pathology

Figure 8. Ionically bound peroxidase from con trol (A), cut-injured ethylene-treated (-{-), and infected (O) sweet potato root sections (66)

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

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-16 2 mm along

-15 8mm along gel -

Physiological Plant Pathology

Figure 9. Densitometric scans of soluble isoperoxidases isolated from control, cutinjured, ethylene-treated, and infected sweet potato root sections after eight days' trea ment. Data are representative of three additional trials. A 15 /xL sample was applied to each respective gel. M in densitometric scans indicated position of marker dye. Isoenzyme changes were similar for extracts obtained after a 1- and 3-day treatment. No pe oxidase migrated into the gel when the electrodes were reversed (66).

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 t i s p o s s i b l e that an e l i c i t o r isoenzyme i s present. Some s t u d i e s on the o x i d a t i c f u n c t i o n I 1 1 d e s c r i b e l a t e r i n d i c a t e we may have measured the wrong f u n c t i o n i n t h i s system. C o n t r o l roots c o n t a i n two r e a d i l y d e t e c t a b l e isoperoxidases i n the i o n i c a l l y bound f r a c t i o n (Figure 10). Cut i n j u r y r e s u l t e d i n s t r i k i n g increases i n zone 1 and the appearance of 3 new i s o enzymes ( l a , 3, 4). Changes o c c u r r i n g as a r e s u l t of C. f i m b r i a t a i n f e c t i o n and ethylene treatment were e s s e n t i a l l y i d e n t i c a l to those during cut i n j u r y . Again, the r e l a t i v e increases were d i f f e r e n t . Zone l a from ethylene t r e a t e d t i s s u e increased more dram a t i c a l l y than i n the c o n t r o l while zone 1 was s i m i l a r i n a l l samples. These changes are s i m i l a r to those reported by Matsuno and U r i t a n i (45) and they i d e n t i f i e d these c a t i o n i c zones as component H. As p r e v i o u s l y discussed, theyprovided evidence that component H f u n c t i o n s i n l i g n i n d e p o s i t i o n . The covalent bound peroxidase contained nine r e a d i l y i d e n t i f i a b l e isoperoxidase zones i n c o n t r o l t i s s u e and 3 s u l t of treatments (32) c l e a r l y unique to i n f e c t i o n . From these data we conclude that changes i n isoperoxidase, which c l o s e l y p a r a l l e l s t r e s s metabolite formation i n the i n f e c t e d t i s s u e , are not c a u s a l l y r e l a t e d to t e r pene i n d u c t i o n , F i g u r e 11. More recent s t u d i e s i n our l a b o r a t o r y i n d i c a t e that the IAA oxidase f u n c t i o n of peroxidase shows dramatic increases which occur d i f f e r e n t l y i n ethylene treated or cut i n j u r e d t i s s u e (Figure 12). The r i s e i n IAA oxidase again p a r a l l e l s s t r e s s metabolite formation. These data lead us to suggest that i t i s the o x i d a t i c f u n c t i o n of peroxidase and not the p e r o x i d a t i c funct i o n which may r e l a t e to the e l i c i t o r response. I t i s tempting to speculate that a unique IAA oxidase c a t a l y z e s o x i d a t i o n of IAA to s p e c i f i c metabolites which, i n t u r n , de-repress gene exp r e s s i o n or otherwise a f f e c t s b i o s y n t h e s i s of furanoterpenoids. Our p r e l i m i n a r y data has shown that the o x i d a t i o n products of IAA r e s u l t i n g from c a t a l y s i s by the enzyme i s o l a t e d from i n f e c t e d t i s sue d i f f e r from those reported f o r other sources of IAA oxidase. T

Role of Peroxidase i n F r u i t Ripening Peroxidase a c t i v i t y appears to increase during r i p e n i n g and senescence i n f r u i t s , e.g. mango (46), grape (47), apple (48), pear (49) and banana (50). As peroxidase has been implicated i n numerous events of c l e a r importance i n p l a n t senescence i t i s p o s s i b l e that i t plays a r e g u l a t o r y r o l e . These events i n c l u d e l i g n i n synthesis i n c e r t a i n systems, as described p r e v i o u s l y , ethylene biogenesis (51), membrane i n t e g r i t y (52), r e s p i r a t i o n c o n t r o l (53) and o x i d a t i v e metabolism of auxin (54) and c y t o k i n i n . We have taken an i n t e r e s t i n the involvement of auxin metabolism i n the process of f r u i t r i p e n i n g and have c h a r a c t e r i z e d the i n d o l e - 3 - a c e t i c a c i d oxidase f u n c t i o n of peroxidase from normal r i p e n i n g and mutant c u l t i v a r s of tomato f r u i t . Two views have

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

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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

-16 Omm along gel

16 5mm along gel -

Physiological Plant Pathology

Figure 10. Densitometric scans of ionically bound isoperoxidases isolated from control, cut-injured, ethylene-treated, and infected sweet potato root sections after eight days treatment. Data are representative of three additional trials. A 60 pL sample was applied to each respective gel. M in densitometric scans indicates position of marker dye. Isoenzyme changes were simifor for extracts obtained after a 1- and 3-day treatment No peroxidase migrated into the gel when the electrodes were reversed (66).

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

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-*·· · ••— 15 4 mm along gel

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16 Imm along gel -

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Figure 11. Densitometric scans of covalently bound isoperoxidases isolated from con trol, cut-injured, ethylene-treated, and infected sweet potato root sections after eight days' treatment. Data are representative of three additional trials. A 100 μΣ, sample wa applied to each respective gel. M in densitometric scans indicates position of marker dye. Isoenzyme changes were similar for extracts obtained after a 1- and 3-day treat ment. No peroxidase migrated into the gel when the electrodes were reversed (66).

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

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Figure 12. Change in ratio of oxidatic to per oxidatic function of peroxidase from sweet potato root after treatment with exogenous ethylene or infection with C. fimbriata

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

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emerged regarding the involvement of IAA i n f r u i t r i p e n i n g . On the one hand i t has been argued that IAA serves to repress genes which code f o r enzymes involved with f r u i t r i p e n i n g and subse quent senescing r e a c t i o n s . These r e a c t i o n s i n v o l v e both a n a b o l i c (e.g. pigment synthesis) and c a t a b o l i c events (e.g. p r o t e i n hy d r o l y s i s ) . The suggestion that the presence of auxin acts to r e press r i p e n i n g i s supported by various f i n d i n g s that a p p l i c a t i o n of exogenous IAA to f r u i t causes a t r a n s i e n t delay i n the pro cess (55,56). A d i f f i c u l t y w i t h t h i s view i s that l e v e l s of ex ogenous auxin e f f e c t i v e i n d e l a y i n g r i p e n i n g range from .01 to ImM (Figure 13), while concentrations of IAA i n mature f r u i t are s e v e r a l orders of magnitude lower. One can, however, argue that the a p p l i e d hormone must be introduced at a concentration gradient s u f f i c i e n t to reach a p a r t i c u l a r c e l l u l a r compartment at e f f e c t i v e l e v e l s . However, r e c e n t l y i t was shown that fM l e v e l s of IAA delay r i p e n i n g of avocado f r u i t (57) These s t u d i e s are a l s o complicated by the woun t i s s u e s l i c e s or i n f i l t r a t i o been suggested t h a t , r a t h e r than IAA being a negative e f f e c t o r , o x i d a t i o n product(s) of IAA serves as a p o s i t i v e e f f e c t o r of r i pening (58). This view i s supported by f i n d i n g s that IAA oxidase a c t i v i t y increases during r i p e n i n g of f r u i t (59) and by r e p o r t s that methylene o x i d o l e , an o x i d a t i o n product of IAA, as c a t a l y z e d by the h o r s e r a d i s h enzyme, stimulates f r u i t r i p e n i n g (60). Also, i t i s known that an analogue of auxin, l-(2,4 dichlorophenoxy) i s o b u t y r i c a c i d (CPIBA) may serve to modulate the a c t i v i t y of IAA oxidase from f r u i t i n d i f f e r e n t ways. In banana, where CPIBA r e presses f r u i t r i p e n i n g (61) the analogue i s known to i n h i b i t the IAA oxidase from t h i s t i s s u e . A l t e r n a t i v e l y , CPIBA was shown to a c c e l e r a t e r i p e n i n g of pear f r u i t where i t a l s o a c t i v a t e d IAA oxidase (62)(Figure 14). S i m i l a r l y , i t has been reported that the a c t i v i t y of IAA oxidase at low IAA c o n c e n t r a t i o n i s decreased d r a m a t i c a l l y at c h i l l i n g temperatures which c o i n c i d e n t l y r e s u l t i n impaired or repressed r i p e n i n g (63)(Figure 15). Recently, we have attempted to determine f a c t o r s r e s p o n s i b l e f o r c o n t r o l of IAA oxidase i n r i p e n i n g tomato f r u i t . Our examination of IAA oxidase from normal tomato c u l t i v a r s revealed no l a r g e d i f f e r e n c e s i n k i n e t i c p r o p e r t i e s of the p u r i f i e d enzyme as a f u n c t i o n of r i p e n i n g . S i m i l a r l y , the enzyme from the RIN mutant, a c u l t i v a r which f a i l s to r i p e n , had s i m i l a r k i n e t i c p r o p e r t i e s and recoverable a c t i v i t y as was observed f o r the normal r i p e n i n g c u l t i v a r s . However, i t was observed that components i n the phenolic f r a c t i o n from r i p e n i n g tomato f r u i t served to lower the apparent Κ of the IAA oxidase f o r auxin (Table 7). These data i n d i c a t e that IAA oxidase may be modulated by changes i n the phenolic p o o l . The i n f l u e n c e of phenolics on IAA oxidase i s complicated s i n c e dihydroxy phenols such as c h l o r ogenic a c i d may i n h i b i t the enzyme i n a way manifested by a l a g i n the i n i t i a t i o n of IAA o x i d a t i o n . A l t e r n a t i v e l y , monohydroxyl phenols may a c t i v a t e the r e a c t i o n by decreasing the l a g time, s e r -

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

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

•

^=Ξ=== ^ ^ ^ ^

-

^-^^^

1AA

2.4-D

a 1

1

1

1

1 A A

30

1

1

1

1

Ι

1

2.4-D N

η

ι

Ο

10

b

d 1

I

,

,

1

Postharvest Biology of Fruits and Vegetables

Figure 13. The effect of IAA on chlorophyll breakdown is shown in a and c. The effect of IAA and 2,4-D on softening is presented in b and d. The auxin concentrations in the infiltration solutions were: zero (mannitol con trol) (O); O.OlmM (·); O.lmM (\J); and l.OmU (U) (56;.

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

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Peroxidase in Postharvest Fruits and Vegetables

Ί7

0

Plant Physiology

Figure 14. Substrate-velocity plot of IAA oxidase from pear in the presence of CPIBA. The sigmoidal kinetics of the enzyme (O) become less pronounced in the presence of 0.5mM CPIBA (Φ). The normal or expected hyperbolic kinetics are shown for com parison ( )(62).

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

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TABLE V I .

Ethylene e v o l u t i o n from asparagus spears as a f u n c t i o n of height of c u t . Data from (28).

Time after cutting (min)

Height of cut (cm)

45

-5 0 +5 -5 0 +5 -5 0 +5

90

165

TABLE V I I .

Ethylene evolution μΙ/Kg-Hr 2.05 2.13 3.71 2.61 3.13 4.91 2.61 3.12 5.51

μΙ/Hr-spear 0.0486 0.0400 0.0532 0.0620 0.0590 0.0702 0.0020 0.0688 0.0788

Influence of p o l y p h e n o l i c substances from tomato on k i n e t i c parameters of tomato IAA oxidase.

Source p h e n o l i c s

1^ (mM)

Vmax
None

0.75

21.4

Vendor,Mature green

0.71

17.7

Initial climacteric

0.57

17.0

Climacteric

0.31

10.2

Mutant, aged

0.78

13.3

RIN

2

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

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Time

167

(min)

Figure 16. Comparison of DM AC A test and Salkowski test of tomato IAA oxidase activity. Reaction mixtures,finalvolume is 2.0 mL, contained 0.20mM IAA, O.lOmM DCP, O.lOmM Mn , 50mM sodium phosphate (pH 6.1), and O.lOmL enzyme extract from RIN mutant tomatoes. +2

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

ENZYMES IN FOOD AND BEVERAGE PROCESSING

Stage of Ripening lure 17.

Change in IAA recovered in conjugated and free form from ripening tomato fruit. Data for detached mature green fruit (Vendor).

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

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v i n g as a c o f a c t o r f o r the r e a c t i o n or, as shown i n these data, by otherwise a f f e c t i n g the k i n e t i c p r o p e r t i e s of the enzyme. A most i n t e r e s t i n g feature of the tomato enzyme i s that i t y i e l d s d i f f e r e n t products than horse r a d i s h peroxidase and other sources of the enzyme that have been s t u d i e d . Assay of tomato IAA o x i dase by the dimethylaminocinnamaldhyde procedure (64) or by a f l u o r o m e t r i c method (65) r e v e a l no apparent a c t i v i t y although i t i s c l e a r that IAA i s being o x i d i z e d (Figure 16). We have r u l e d out the formation of methyloxindole, methyleneoxindole and i n d o l e 3-aldehyde although the product e x h i b i t s a s i m i l a r U.V. spectra as the aldehyde. A component with i d e n t i c a l chromatographic p r o p e r t i e s as the p r i n c i p l e product of t h i s r e a c t i o n a l s o accu mulates as a r e s u l t of the o x i d a t i o n of r a d i o l a b e l e d IAA i n r i pening tomato f r u i t . Accordingly we are i n t r i q u e d by the p o s s i b i l i t y that a s p e c i f i c o x i d a t i o n product of IAA serves to modulate the " r i p e n i n g genome". We have a l s o r e c e n t l commitently with r i p e n i n by environmental f a c t o r s which c o i n c i d e n t a l l y a l s o a f f e c t f r u i t r i p e n i n g (Figure 17). I t was a l s o of considerable i n t e r e s t to f i n d the v i n e ripened tomato f r u i t accumulate more o x i d a t i o n pro ducts of IAA during r i p e n i n g apparently because IAA i s c o n t i n u a l l y supplied to the f r u i t a f t e r the i n i t i a t i o n of the process. This may p o s s i b l y i n d i c a t e that the messenger RNA's and t h e i r respec t i v e enzymes associated with r i p e n i n g have a r e l a t i v e l y short h a l f l i f e thus r e q u i r i n g continuous gene expression f o r optimal development of r i p e n i n g i n d i c e s . A l t e r n a t i v e l y , the RIN mutant contained no detectable f r e e IAA, and r e l a t i v e l y low l e v e l s of conjugated IAA. This may mean that f a c t o r s which c o n t r o l the formation and storage of IAA and the conversion of IAA from bound to f r e e forms are an abberant c o n t r o l s i t e i n these f r u i t .

Literature Cited 1. Schoenbein, C., Verh. Naturf. Ges. Basel (1885) 1: 339. 2. L i n o s s i e r , M., Soc. Biol., P a r i s (1898) 50: 373. 3. Saunders, B., Holmes-Siedle, Α., Stark, Μ., "Peroxidase". Butterworths, London, 1964. 4. Blundstone, Η., Woodman, J., Adams, J., "Biochemistry of F r u i t s and t h e i r Products". A.C. Hulme, E d i t o r . v o l . 2, p. 561. Academic Press, New York, 1971. 5. Ben A z i z , Α., Grossman, S., Ascarelli, I., Budowski, P., Phytochem. (1971) 10: 1445. 6. Markakis, P., "Postharvest Biology and Handling of F r u i t s and Vegetables". N. Haard and D. Salunkhe E d i t o r s . p. 62. AVI, Westport, Conn., 1975. 7. Schwimmer, S., J . Food S c i . (1972). 37: 350.

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

170

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

8. Wang, S. and DiMarco, G., J. Food S c i . (1972), 37: 574. 9. Reed, G., "Enzymes i n Food Processing". p. 243. Academic Press, New York, 1975. 10. M o r i t a , Y. and Kameda, Κ., Mem. Res. Inst. Food Sci., Kyoto Univ. (1958) 14: 49. 11. A l t s h c h u l , Α., Abrams, R., Hogness, T., J. Biol. Chem. (1940) 136: 777. 12. Huang, Α., Chism, G., Haard, N., J. Food Sci. (1975) 40: 1327. 13. L e v i t t , J., "Responses of Plants to Environmental S t r e s s e s " . p. 147. Academic P r e s s , New York, 1972. 14. Yamauchi, T., Yamamoto, S., H a y a i s h i , O., J. Biol. Chem. (1975) 250: 7127. 15. T h e o r e l l , Η., Ark. Kemi. Min. Geol. (1943)16A: No. 2. 16. Shannon, L., Kay, E., Lew, J., J. Biol. Chem. (1966) 241: 383. 17. Nagle, N. and Haard, N., J. Food S c i . (1975) 40: 576. 18. S h i n s h i , H. and Noguchi Μ., Phytochem (1975) 14: 1255 19. Keilin, D. and Hartree 20. L i u , E. and Lamport 822. 21. Shannon, L., Uritani, I., and Imaseki, H., Plant P h y s i o l . (1971) 47: 493. 22. A n s t i n e , W., Jacobsen, J., Scandalio, J. and Varner, J., Plant P h y s i o l . (1970) 45: 148. 23. Penon, P., C e c c h i n i , J., Miassod, R., R i c a r d , J., T e i s s e r e , M. and Pinna, Μ., Phytochem. (1970) 9: 73. 24. Raa, J., P h y s i o l . Plant (1973) 38: 132. 25. Darimont, E. and Baxter, R., P l a n t a (1973) 110: 205. 26. Ridge, I. and Osborne, D., J. Exper. Bot. (1970) 21: 843. 27. Haard, Ν., Phytochem. (1973) 12: 555. 28. Haard, N., Sharma, S., Wolfe, R. and F r e n k e l , C., J. Food Sci. (1974) 39: 450. 29. Ranadive, A. and Haard, N., J. Food S c i . (1973) 38: 331. 30. Brown, S., B i o . Sci. (1969) 19: 115. 31. Freundenburg, Κ., " L i g n i n S t r u c t u r e and Reactions". M a r t i n , E d i t o r . American Chemical S o c i e t y , Advances in Chemistry S e r i e s 59, Washington, D.C., 1966. 32. Helper, P., Fosket, D. and Newcomb, Ε., Am. J. Botan. (1970) 57: 85. 33. L i p e t z , J. and Garro, Α., J. Cell B i o l . (1965) 25: 109. 34. Ranadive, A. and Haard, Ν., J. Sci. Fd. Agr. (1973) 38: 331. 35. Sharma, S., Wolfe, R. and Haard, Ν., J. Food S c i . (1975) 40: 1021. 36. L i p t o n , W., Amer. Soc. H o r t - S c i . Proc. (1965) 86: 347. 37. Burg, S. and Burg, Ε., Plant P h y s i o l . (1967) 42: 144. 38. U r i t a n i , I., Ann. Rev. Phytopathology (1971) 9: 211. 39. Coxon, D., C u r t i s , R., and Howard, B., Fd. Cosmet. T o x i c o l . (1975) 13: 87. 40. Wilson, Β., Boyd, Μ., H a r r i s , T. and Yang, D., Nature (1970) 231: 52. 41. Kawashima, K. and Uritani, I., Agr. Biol. Chem. (1963) 27:409.

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

9.

HAARD

Peroxidase in Postharvest Fruits and Vegetables

171

42. Stahmann, M. and Demorest, D., "Fungal P a t h o g e n i c i t y and the p l a n t ' s Response". p. 405. Academic Press, New York, 1973. 43. Stahmann, Μ., C l a r e , B., Woodbury, W., Plant P h y s i o l . (1966) 41: 1505. 44. Imaseki, I., Plant P h y s i o l . (1970) 46: 172. 45. Matsuno, H. and U r i t a n i , I., P l a n t and C e l l P h y s i o l . (1972) 13: 1091. 46. Mattoo, A. and Modi, Α., Plant P h y s i o l . (1969) 44: 308. 47. Ivanova, T., and Ivanova, Α., Ann. Tech. A g r i c . ( P a r i s ) (1968) 17: 333. 48. Kuhive, Α., Dokl. Akod. Nank. Azerb. SSR (1969) 25: 109. 49. Ranadive, A. and Haard, N., J. Food S c i . (1972) 37: 381. 50. Haard, Ν., Phytochem. (1973) 12: 555. 51. Yang, S., Arch. Biochem. Biophys. (1967) 122: 481. 52. Dilley, D., "The Biochemistry of F r u i t s and T h e i r Products". p. 195. Academic Press Ne York 1970 53. Aylward, F. and Haisman 54. Gortner, W. and Kent 55. V e n d r e l l , Μ., A u s t r a l i a n J . Biol. S c i . (1969) 22: 601. 56. F r e n k e l , C., Dyck, R. and Haard, Ν., "Postharvest Biology of F r u i t s and Vegetables". p. 19. AVI, Westport, Conn., 1975. 57. Tingwa, P., and Young R., P l a n t P h y s i o l . (1975) 55: 937. 58. Hoyle, Μ., "Mechanisms of Regulation of Plant Growth". p. 659. Royal Society of New Zealand, W e l l i n g t o n , 1974. 59. F r e n k e l , C., Plant P h y s i o l . (1972) 49: 757. 60. F r e n k e l , C., Plant P h y s i o l . (1975) 56: 647. 61. Haard, N., J . Food S c i . (1973) 38: 639. 62. F r e n k e l , C. and Haard, Ν., Plant P h y s i o l . (1973) 52: 380. 63. Haard, N., J . Food S c i . (1973) 38: 907. 64. Meudt, W. and Gaines, T., P l a n t P h y s i o l . (1967) 42: 1395. 65. Haard, N., Chism, G. and Nagle, N., A n a l . Biochem. (1975) 69: 627. 66. Haard, N. and M a r s h a l l , Μ., P h y s i o l o g i c a l Plant Pathology (1976) 8: 195. Acknowledgements Part of the work reported here was supported by a grant from the United States P u b l i c Health S e r v i c e (USPHS 1226001086A1). The data presented here represents the c o l l a b o r a t i v e e f f o r t s of s e v e r a l i n d i v i d u a l s from our l a b o r a t o r y i n c l u d i n g Dr. A.S. Ranadive, Dr. N. Nagle, Dr. G. Chism, Dr. A. Huang, Dr. S. Sharma, Mr. B. Wasserman, Mr. M. M a r s h a l l and Mr. D. Timbie.

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

10 Enzymes Involved in Fruit Softening RUSSELL PRESSEY 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

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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 . Degeneratio disappearance of membraneou 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

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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 succulen o f parenchyma c e l l s (2J 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

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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 s t r u c t u r e of p e c t i n fro 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 .

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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 more c e l l p e c t i n by endopolygalacturonas 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

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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

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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 Watad (47) presen 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 in green peaches, but a c t i v i t 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 gel f i l t r a t i o n and foun 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 esterase appeared. On 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

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

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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 carboxy a c t i o n with C a and t 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 Potentat a s s o c i a t e d with markedl 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

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

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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 on these f a c t o r s . At enzym 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 consists of endo- and exo-cleavin an exopolygalacturonas 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 +

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

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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 o p t i m a l l y a c t i v e at pH 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 hig l e s s than 2 - f o l d durin g 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 . 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 Addoms, R. Μ., N i g h t i n g a l e 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. 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. Bauer, W. D . , Talmadge Keegstra 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. 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 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.

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 92. McCready, R. M. an 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.

11 Uses of Endogenous Enzymes in Fruit and Vegetable Processing ROBERT C. WILEY Department of Horticulture, Food Science Program, University of Maryland, College Park, Md. 20742

The quality of fres 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

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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 phenoli compound hav engendered t amount o f research i n t e r e s idases and c a t a l a s e s a l s 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

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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 r e q u i r i n g s p e c i a l substrate 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 non-galacturonide materia 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

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

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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

R

R

2

-0.16 0.76

Proceedings of the American Society for Horticulture Science

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 firmnes It appears that a 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

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

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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 coul min period at 35°C. Col 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 +

+

4 5

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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 stimulate used to a c t i v a t e s i t e s f o 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

2 +

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.

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

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Journal of Food Science

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 showing optimum a c t i v i t 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. Temperature 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

Food Technology

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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 under simulated pre-processing processing , 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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Effects of pH on PE specific activity

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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 .

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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 blanch ing temperatures. Mos s o f t f r u i t and/or f r u i 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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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 produce too 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.

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

208

12.

13. 14. 15. 16.

17. 18. 19.

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

29. 30.

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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-processe Van Buren, J.P., Moyer (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.

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

12 Oilseed Enzymes as Biological Indicators for Food Uses and Applications JOHN P. CHERRY Southern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, New Orleans, La. 70179

Research u t i l i z i n biochemical changes in l i v i n g organisms is not new (25,38). Enzyme synthesis is genetically controlled, however, their specific task(s) and feedback control mechanisms are also influenced by other constituents in the cellular environment; i.e., cofactors, substrates and products associated with metabolic processes. Scientists have developed the technology to p a r t i a l l y imitate in vivo conditions i n order to study the active enzymes extracted from their natural environment. Nevertheless, enzymes do falter when abused, becoming excellent indicators of several types of cellular change. Gel electrophoresis is a method developed to qualitatively study enzymes. The background and theory of electrophoretic techniques were discussed by Ornstein (46), and applications of these procedures to analyze and compare proteins and enzymes have been presented by a number of investigators (6,8,24,29,48,55,57). Basically, the method applies an electric charge to separate aqueous extracts of proteins i n a gel matrix, such as polyacrylamide or starch. Protein mobility depends upon a combination of factors, including net charge, molecular size, and conformation. Histochemical staining procedures have been developed to detect the location of enzymes i n gels based on their catalytic activity (39). E l e c t r o p h o r e t i c a l l y detected enzyme changes have been used to c h a r a c t e r i z e Aspergillus-peanut i n t e r r e l a t i o n s h i p s ; in the fungus, development and d i f f e r e n t i a t i o n occurs w h i l e the peanuts undergo senescence (14,18). E l e c t r o p h o r e t i c zymograms of enzymes e x t r a c t e d from various plant and animal t i s s u e s were used in chemotaxonomic studies o f genetic s p e c i a t i o n and genera taxonomy (21,38). These s t u d i e s showed that a great amount of g e n e t i c a l l y c o n t r o l l e d molecular d i v e r s i t y , or enzyme multiplicity, e x i s t s in nature as isozymes; i . e . , g e n e t i c a l l y r e l a t e d enzymes with s i m i l a r substrate specificities but d i f f e r e n t e l e c t r o p h o r e t i c m o b i l i t i e s . Zymograms can a l s o vary because of t i s s u e ontogeny. These reports suggest that, in all types of e l e c t r o p h o r e t i c s t u d i e s , care should be taken to i n s u r e that standard known zymograms are developed f o r

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to be examined p r i o r to any e v a l u a t i o n of treatment

Complexity of I d e n t i f y i n g Isozymes as M u l t i p l e Enzyme Forms Hunter and Markert (32) revealed a vast m u l t i p l i c i t y of e l e c t r o p h o r e t i c a l l y d i s t i n c t bands w i t h enzyme a c t i v i t y . The term "isozyme" was proposed by Markert and M o l l e r (42) to r e f e r to enzymatically a c t i v e p r o t e i n s that are separated e l e c t r o p h o r e t i c a l l y and that c a t a l y z e the same biochemical r e a c t i o n . Markert (39) l a t e r proposed modifying the word isozyme with terms, such as a l l e l i c , n o n a l l e l i c , homopolymeric, conformational, h y b r i d , and conjugated (words which deal w i t h genetic and chemical terminology) . The p r i n c i p a l types of molecular m u l t i p l i c i t y that generate isozyme patterns were reviewed by Markert and Whitt (40). I t i s t h e o r i z e d that enzyme m u l t i p l i c i t y i s created at the molecu l a r l e v e l by various combination polypeptides coded by a l l e l i of v a r i o u s s i z e s ; (c) homo- and heteropolymers; (d) polypeptides s e c o n d a r i l y modified i n various ways; and (e) d i f f e r e n t conformations produced by permutations of polymer subunits, or a l t e r n a t e t e r t i a r y and quaternary c o n f i g u r a t i o n s of p r o t e i n s . Organisms may u t i l i z e both genetic and nongenetic mechanisms to form prope r t i e s of enzymes necessary to f i t s p e c i a l metabolic requirements. Shaw (54) c l a s s i f i e d isozymes i n t o primary types, which i n c l u d e d i s t i n c t molecular e n t i t i e s produced from d i f f e r e n t gen e t i c s i t e s , and secondary types, which r e s u l t from a s i g n i f i c a n t a l t e r a t i o n i n the s t r u c t u r e of a s i n g l e polypeptide. Scandalios (52) has employed the term isozyme to define the heterozygous s t a t e of g e n e t i c a l l y v a r i a n t enzymes. He showed a m u l t i p l i c i t y of a l l e l e s (genes) c a r r y i n g information f o r a s p e c i f i c type of enzyme a c t i v i t y . Crosses between s e v e r a l l i n e s of maize with d i f f e r e n t c a t a l a s e phenotypes revealed s i x a l l e l e s , whereas l e u c i n e aminopeptidase isozymes were c o n t r o l l e d by two separate genetic l o c i . I t was f u r t h e r noted that d i f f e r e n t isozymes were present i n d i f f e r e n t t i s s u e s and i n e x t r a c t s of t i s s u e s at d i f f e r ent stages of development. In 1971, the IUPAC-IUB Commission on Biochemical Nomenc l a t u r e (34) recommended that m u l t i p l e forms of an enzyme poss e s s i n g the same a c t i v i t y , separated and d i s t i n g u i s h e d by s u i t a b l e methods ( p r e f e r a b l y by e l e c t r o p h o r e t i c techniques but also by chromatography, k i n e t i c c r i t e r i a , chemical s t r u c t u r e , etc.) and o c c u r r i n g n a t u r a l l y i n a s i n g l e s p e c i e s , should be g e n e r a l l y defined as " m u l t i p l e forms of the enzyme...". M u l t i p l e forms of enzymes that have been s p e c i f i c a l l y c h a r a c t e r i z e d by genetic d i f f e r e n c e s i n primary s t r u c t u r e should be defined as isoenzyme or isozyme. These include g e n e t i c a l l y independent p r o t e i n s , heteropolymers (hybrids) of two or more noncovalently bound polypeptide chains, and genetic v a r i a n t s ( a l l e l i c ) . Enzymes not f a l l i n g i n t o t h i s l a t t e r category i n c l u d e p r o t e i n s conjugated with other groups,

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p r o t e i n s derived from one polypeptide chain, polymers of a s i n g l e subunit, and conformationally d i f f e r e n t forms. Detecting M u l t i p l e Forms of Enzymes Esterases I n v e s t i g a t i o n s were completed to standardize techniques ( p r o t e i n and enzyme e x t r a c t i o n , p u r i f i c a t i o n , and g e l e l e c t r o p h o r e s i s ) used to examine enzymes (2,3,30,31,41). T h i s provided reproducible q u a l i t a t i v e data f o r comparative purposes; i . e . , u n i f o r m i t y i n i n t e n s i t y of g e l s t a i n i n g and repeatable s p a t i a l arrangement of enzyme bands between experiments. This a l s o e s t a b l i s h e d e l e c t r o p h o r e t i c patterns which serve as stand ards to which enzymes from damaged, processed, or mold-infected substances might be compared. A t y p i c a l example of on-gel e l e c t r o p h o r e t i c c h a r a c t e r i z a t i o n of enzyme a c t i v i t y was shown i n r a b b i t e r y t h r o c y t e s ( F i g 1; 13) using polyacrylamide g e complex array of n o n s p e c i f i e x t r a c t s and f r a c t i o n s . Although the r a b b i t s were considered to be i s o g e n i c , some genetic v a r i a b i l i t y i n esterase bands was noted ( c f . , region 1.5-2.5 cm of gels A and Β i n both membrane and cyto plasmic e x t r a c t s ) . N o n s p e c i f i c esterases were detected by i n c u b a t i n g gels i n a mixture of a- and β-naphthyl acetate. By using on-gel techniques, the esterases were c h a r a c t e r i z e d on the b a s i s of t h e i r s u b s t r a t e s p e c i f i c i t i e s and s u s c e p t i b i l i t i e s to i n h i b itors. Group I esterases were i n h i b i t e d by diisopropylphosphorof l u o r i d a t e (DFP), phenylmethanesulfonyl f l u o r i d a t e (PMSF) and e s e r i n e , but not by mercuric c h l o r i d e or p-chloromercuribenzenes u l f o n i c a c i d (CMBSA) ( F i g . 1). These esterases showed s p e c i i c i t y toward a c e t y l t h i o c h o l i n e i o d i d e and thiophenyl acetate and thus appeared to be composed of a c e t y l c h o l i n e s t e r a s e s . Group I I ( a c e t y l e s t e r a s e s ) c o n s i s t e d of enzymes that hydrolyzed e s t e r s of a c e t i c a c i d and β-naphthyl l a u r a t e and thiophenyl acetate, and was not i n h i b i t e d markedly by any of the i n h i b i t o r s t e s t e d . The enzymes i n Groups I I I a,b, and c, IV and VI, c o n s i s t i n g of slow, intermediate, and r a p i d migrating carboxylesterases, r e s p e c t i v e l y , were h i g h l y a c t i v e toward α-naphthyl butyrate, and were i n h i b i t e d p a r t i a l l y or completely by DFP and PMSF but not by mercurials or t r i - o - t o l y l phosphate. Only Groups I and VI were i n h i b i t e d by e s e r i n e . Groups V and VII showed s p e c i f i c i t y t y p i c a l of a r y l e s t e r a s e s , being p a r t i a l l y i n h i b i t e d by DFP, PMSF, and the m e r c u r i a l reagents. These data revealed that the n o n s p e c i f i c esterases i n hypo t o n i c cytoplasmic or membrane f r a c t i o n s from erythrocytes which could be detected using n o n s p e c i f i c substrates were r e a l l y heteromorphic ( s p e c i f i c e s t e r a s e s ) . Some of the d e f i n i t i o n s f o r d e s c r i b i n g the term isozyme may be a p p l i e d to these m u l t i p l e forms of esterases detected i n e r y t h r o c y t e s . However, u n t i l each of the e l e c t r o p h o r e t i c bands of esterase a c t i v i t y i s r e l a t e d to a s p e c i f i c gene(s) by breeding s t u d i e s , a true d e f i n i t i o n of isozyme cannot be used to describe them.

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Figure 1. patterns of nonspecific esterase activity in membrane and cytoplasmic fractions of rabbit erythrocytes. Methods for preparation of erythrocyte fractions are described by Cherry and Prescott (12). A composite shows the classes of esterase activity (I: acetylcholinesterases; II: acetylesterases; III a, b, and c, IV and VI: carboxylesterases and; V and VII: arylesterases. The bands in region 3.0S.5 cm of gels of the cytoplasmic fraction are hemoglobin.

Figure 2. Polyacrylamide gel electrophoretic patterns of peroxidases in peanut cotyledons at various developing and germinating stages. Gel patterns of the cultivar Spancross are presented as an example of typical standard gels.

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

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A p p l i c a t i o n of Enzymes as B i o l o g i c a l I n d i c a t o r s Peroxidases Peroxidase i s probably the most widely used enzyme as a biochemical i n d i c a t o r of d i s e a s e , c e l l u l a r i n j u r y , trauma damage, i n f e c t i o n , e t c . , i n p l a n t s (4,7,16,23,36,37,47, 51,53). In peanuts, most peroxidase a c t i v i t y has been l o c a t e d i n the albumin f r a c t i o n of the p r o t e i n s (17). Peroxidase has been employed with peanuts as an index of b l a n c h i n g temperatures i n food q u a l i t y c o n t r o l and, with c e r t a i n other enzymes, has been i m p l i c a t e d i n the development of o f f - f l a v o r s during storage (1,28,59). Since most peroxidases seem to be l o c a l i z e d i n a s p e c i f i c storage p r o t e i n f r a c t i o n of peanuts, a n a l y s i s of t h i s f r a c t i o n may be a u s e f u l t o o l i n q u a l i t y c o n t r o l during prepara t i o n of p r o t e i n i s o l a t e s or concentrates. A l t e r n a t i v e l y , the removal of the albumin f r a c t i o n during i s o l a t e p r e p a r a t i o n may prevent or r e t a r d th peroxidase a c t i v i t y . Examination of many developing Spancross peanuts from i n d i v i d u a l p l a n t s grown i n Georgia showed a decrease i n peroxidase a c t i v i t y as seeds matured ( F i g . 2). Zymograms c o n t a i n i n g one band at the o r i g i n , one i n r e g i o n 0.7 cm and three i n r e g i o n 3.0-4.0 cm, were t y p i c a l of most p a t t e r n s of mature seeds. A peroxidase band i n r e g i o n 5.0 cm d i s t i n g u i s h e d immature and low intermediate seeds from high intermediate and mature peanuts. The presence of one major band i n r e g i o n 3.0-4.0 cm, the absence of isozymes at the o r i g i n and/or i n r e g i o n 0.7 cm, and the presence (or absence) of a c t i v i t y i n regions 1.0-2.7 and 4.0-5.0 cm d i s t i n g u i s h e d zymograms of mature seeds. Germinating peanuts showed i n c r e a s e d peroxidase a c t i v i t y i n e l e c t r o p h o r e t i c g e l s a f t e r 2 to 4 days ( F i g . 2) that became e s p e c i a l l y p r e v a l e n t i n r e g i o n 0-3.0 cm between 8 and 18 days germination. An i n c r e a s e i n peroxidase a c t i v i t y was noted i n r e g i o n 3.0-4.0 cm at days 6 and 8, decreasing slowly t h e r e a f t e r to 20 days germination. Esterases Esterases are popular i n e l e c t r o p h o r e t i c s t u d i e s of isoenzymes i n higher p l a n t s (8,10,15,19-21,23,43,44,52). Esterases from peanuts at immature and low intermediate stages of development showed much banding v a r i a t i o n that d i f f e r e d f o r Spancross and Florunner c u l t i v a r s ( F i g . 3). A f t e r the low intermediate stage of peanut development, esterase a c t i v i t y on e l e c t r o p h o r e t i c gels increased i n regions 3.0-4.0 and 5.0-7.5 cm. The two c u l t i v a r s could be d i s t i n g u i s h e d by e s t e r a s e bands i n region 4.0-5.0 cm. E x t r a c t s from overmature seeds of Florunner showed decreased esterase a c t i v i t y on e l e c t r o p h o r e t i c g e l s compared to those of mature seeds; no changes were observed i n overmature seeds of Spancross. I n t r a v a r i e t a l a n a l y s i s of these peanut c u l t i v a r s y i e l d e d both q u a l i t a t i v e and q u a n t i t a t i v e e s t e r a s e v a r i a t i o n s that were l i m i t e d p r i m a r i l y to regions 4.0-5.0 cm of the zymograms. V a r i a t i o n s w i t h i n and between various other

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Figure 3. Typical polyacrylamide gel patterns of esterases in peanut cotyledons of Spancross and Florunner cultivars at various developing stages

Figure 4. Polyacryhmide gels of esterase activity in Florunner peanut cotyledons germinating for various times from 0-20 days

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

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peanut c u l t i v a r s were l i m i t e d to q u a n t i t a t i v e d i f f e r e n c e s i n region 4.4 cm. These s t u d i e s suggest that esterase a c t i v i t y i n peanuts may be u s e f u l i n i d e n t i f y i n g p a r t i c u l a r stages of peanut development to maturity, as w e l l as f o r d i s t i n g u i s h i n g Florunner and Spancross c u l t i v a r s . Both q u a l i t a t i v e and q u a n t i t a t i v e band v a r i a t i o n s appeared i n r e g i o n 2.0-7.0 cm s h o r t l y a f t e r Florunner seeds were germinated ( F i g . 4). These changes i n c r e a s e d markedly a f t e r 4 days, c l e a r l y d i s t i n g u i s h i n g e a r l y germinated cotyledons from those developing between 6 and 20 days. I n t e n s i t y of e s t e r a s e a c t i v i t y i n regions 2.0-2.5 and 4.5-5.0 cm of seeds germinated beyond 6 to 8 days increased q u a n t i t a t i v e l y . E s t e r a s e bands of mature seeds present i n other regions of the g e l , decreased between 2 and 8 days germination. Leucine Aminopeptidases Nearly a l l e l e c t r o p h o r e t i c d e t e c t i o n of v a r i a n t peptidases has i n v o l v e d the use of s y n t h e t i c s u b s t r a t e s such as aminoacylnaphthylamides As w i t h the r e a c t i o n medi s t r a t e i s not s p e c i f i c only f o r c e r t a i n peptidases. Despite the vagueness concerning s u b s t r a t e s p e c i f i c i t y , the l e u c i n e aminopeptidases are very u s e f u l enzymes i n e l e c t r o p h o r e t i c s t u d i e s of p r o t e i n polymorphism, p l a n t h y b r i d i z a t i o n , and polypeptide hydrol y s i s (19,21,22,38,58). Zymograms of l e u c i n e aminopeptidase a c t i v i t y d i s t i n g u i s h e d high intermediate, mature, and overmature peanuts of Spancross and Florunner c u l t i v a r s ( F i g . 5 ) . C u l t i v a r s and stages of seed development were s i m i l a r l y d i s t i n g u i s h e d by l e u c i n e aminopeptidases, as was shown with e s t e r a s e s . Changes i n l e u c i n e aminopeptidase a c t i v i t y i n Florunner cotyledonary e x t r a c t s were examined on gels during extended seed germination up to 20 days ( F i g . 6 ) . The peptidase a c t i v i t y i n these p r e p a r a t i o n s remained c o n s i s t e n t l y high d u r i n g e a r l y stages of germination to day 6, a f t e r which i t decreased. By day 12 most of i t could not be detected. T h i s o b s e r v a t i o n suggested that peptidase a c t i v i t y may be p a r t i a l l y r e s p o n s i b l e f o r the reserve p r o t e i n breakdown during e a r l y stages of germination. I n t e r e s t i n g l y , the fast-moving bands (region 5.0-6.0 cm) showed very l i t t l e a c t i v i t y up to 8 days of germination. However, a f t e r t h i s time, most of the remaining a c t i v i t y was a s s o c i a t e d with these bands, suggesting that they may have been a c t i v a t e d or synthesized de novo during t h i s i n t e r v a l . The breakdown of cotyledonary p r o t e i n s was i n v e s t i g a t e d q u a l i t a t i v e l y by g e l e l e c t r o p h o r e s i s ( F i g . 7). P r o t e i n e x t r a c t s from ungerminated seeds (0 day) c o n s i s t e d mainly of the monomeric (1.5 cm) and dimeric (1.0 cm) components of the major storage g l o b u l i n of peanuts, a r a c h i n . Except f o r i n t e n s i f i c a t i o n of two bands i n r e g i o n 0-0.5 cm, no major changes were observed during the f i r s t 2 days of germination. Major changes i n p r o t e i n comp o s i t i o n became evident at and a f t e r day 4. The a r a c h i n components and the bands i n r e g i o n 2.0-3.5 cm decreased q u a n t i t a t i v e l y

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Figure 5. Polyacrylamide gel patterns cine aminopeptidase activity in developing peanut cotyledons of Spancross and Florunner cultivars

Figure 6. Polyacrylamide gels of leucine aminopeptidases in germinating Florunner peanut cotyledons

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i n the g e l p a t t e r n s , and simultaneously there appeared numerous p r o t e i n s i n region 2.5-7.0 cm. The appearance of small p r o t e i n components i n the g e l patterns i n d i c a t e s h y d r o l y s i s of the major storage p r o t e i n s of peanuts by proteases and peptidases to polypeptides of various s i z e s and to f r e e amino a c i d s , p r i o r to t h e i r t r a n s p o r t to the embryo. A f t e r day 14, no f u r t h e r major changes were noted i n the few remaining p r o t e i n s detectable by g e l e l e c t r o phoresis. This c o i n c i d e d w i t h the observation that a c o n s i s t e n t l y low quantity of p r o t e i n and peptidase a c t i v i t y remained i n the cotyledons between days 12 and 20 a f t e r germination began. Fungal-Caused Seed D e t e r i o r a t i o n Studies have shown that standard enzyme e l e c t r o p h o r e t i c patterns of peanuts are d i s t i n c t l y modified by i n f e c t i o n with various A s p e r g i l l u s species (14,18) Biochemical transformations i n c l u d e d e l e t i o n of som and/or production of ne of peanut enzymes i n d i c a t e that the biochemical mechanisms operative i n the saphrophyte-seed i n t e r r e l a t i o n s h i p f u n c t i o n very e f f i c i e n t l y and s y s t e m a t i c a l l y f o r growth of the fungus at the expense of the seed. This i s based on the observation that i n a d d i t i o n to enzyme changes i n the i n t e r r e l a t i o n s h i p , decomposition of storage g l o b u l i n s of l a r g e molecular weight to small p r o t e i n components and f r e e amino acids occurs i n i n f e c t e d peanuts (9,11,14). The type of enzyme systems included i n studies suggested a degradation of storage p r o t e i n peptides by l e u c i n e aminopeptidases, h y d r o l y s i s of many e s t e r linkages by e s t e r a s e s , hormonal i n t e r a c t i o n , and/or o x i d a t i o n of organic substances w i t h peroxides by peroxidases and oxidases, and decomposition of p o t e n t i a l l y t o x i c hydrogen peroxide by c a t a l a s e s . Many of the peanut enzymes remained a c t i v e during A s p e r g i l l u s invasion. In a number of cases, i n t e n s i f i c a t i o n of a c t i v i t i e s of some enzymes and the production of new bands were c o r r e l a t e d w i t h enzyme bands i n m y c e l i a l t i s s u e of the fungus c o l l e c t e d from the seed s u r f a c e . Q u a n t i t a t i v e and q u a l i t a t i v e v a r i a t i o n s i n g e l patterns of c e r t a i n enzymes a l s o d i s t i n g u i s h e d between fungal t i s s u e from the e x t e r i o r surface of peanuts and that grown on a s y n t h e t i c medium, Czapek's s o l u t i o n (14,18). These v a r i a t i o n s i n banding patterns of t i s s u e s c o l l e c t e d from two separate sources may p o s s i b l y be r e l a t e d to d i f f e r e n c e s i n n u t r i t i o n a l needs of the fungus grown under the two c o n d i t i o n s . Phosphogluconate Dehydrogenase, A l c o h o l Dehydrogenase and A l k a l i n e Phosphatase Examples of enzyme changes i n peanuts i n f e c t e d f o r 4 days with various s t r a i n s of A s p e r g i l l u s f l a v u s capable of producing no, intermediate, and high l e v e l s of a f l a t o x i n are shown i n Figure 8. In general, phosphogluconate and a l c o h o l dehydrogenase banding a c t i v i t i e s detected on s t a r c h e l e c t r o p h o r e t i c gels become d i f f i c u l t to d i s c e r n i n e x t r a c t s of

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Figure 7. Polyacrylamide gel electrophoretic patterns of proteins in extracts Florunner peanut cotyledons

Figure 8. Starch gel electrophoretic patterns of phosphogluconate dehydrogenase, alcohol dehydrogenase, and alkaline phosphatase activity of noninfected and A. flavusinfected peanuts after four days. Controls 1 and 2 represent peanuts at day zero and after incubation for four days at 29°C in a high humidity chamber, respectively. A. flavus strains from the Northern Regional Research Center, USDA-ARS, Peoria, III, were labeled as follows: 1. NRRL 5520, 2. NRRL 5518, 3. A-14152, 4. NRRL 8517, 5. A-62462, 6. NRRL 3353, 7. A-4018b, 8. NRRL 3251, and 9. A-13838.

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peanuts i n f e c t e d w i t h f l a v u s . Small amounts of a c t i v i t y were observed only i n c e r t a i n n o n a f l a t o x i n producing s t r a i n s of A. f l a v u s . A phosphogluconate dehydrogenase band i n region 3.5 cm s p e c i f i c a l l y d i s t i n g u i s h e d peanuts i n f e c t e d with n o n a f l a t o x i n producing A. f l a v u s s t r a i n 1 from a l l other contaminants. In c o n t r a s t , a l k a l i n e phosphatase was detected only i n A. f l a v u s - i n f e c t e d peanuts. Two bands of s i m i l a r a c t i v i t y were noted i n a l l contaminated peanuts, regardless of a f l a t o x i n l e v e l s . Since a l k a l i n e phosphatase i s not normally present i n peanuts, t h i s enzyme could be used as an i n d i c a t o r of A^ f l a v u s contami n a t i o n i n peanut p r o t e i n p r e p a r a t i o n s . T h i s would be e s p e c i a l l y suggestive of compositional changes i n p r o t e i n s or other storage c o n s t i t u e n t s and p o s s i b l e a f l a t o x i n contamination of peanut products that could a f f e c t t h e i r f u n c t i o n a l and n u t r i t i o n a l p r o p e r t i e s and t h e i r use i n food products. Preliminary tests for a l k a l i n e phosphatase a c t i v i t y i n peanut products p r i o r to t h e i r u t i l i z a t i o n as food i n g r e d i e n t q u a l i t y c o n t r o l measure Esterases Region 3.0-7.0 cm contained d e t e c t a b l e changes i n esterase patterns a f t e r A. f l a v u s i n f e c t i o n of peanuts, compared with uninoculated or c o n t r o l seeds ( F i g . 9 ) . Four days a f t e r i n f e c t i o n , esterase a c t i v i t y increased both q u a n t i t a t i v e l y and q u a l i t a t i v e l y i n regions 4.0-5.0 and 6.5-7.5 cm. Enzyme bands of most i n f e c t e d seeds i n regions 3.0-4.0 and 5.0-6.0 cm decreased or were not r e a d i l y detected i n the g e l p a t t e r n s . E a r l i e r s t u d i e s with A^ p a r a s i t i c u s showed s i m i l a r i n t e n s i f i c a t i o n of esterase a c t i v i t y (14,18). These changes with A. f l a v u s were s i m i l a r to those of fungal t i s s u e c o l l e c t e d from the e x t e r i o r s u r f a c e of peanuts i n the e a r l i e r work. Some of the esterase p a t t e r n s d i s t i n g u i s h e d A. f l a v u s s t r a i n s producing no, intermediate, or high l e v e l s of a f l a t o x i n s . Leucine aminopeptidase A n a l y s i s f o r l e u c i n e aminopeptidase a c t i v i t y i n polyacrylamide gels c o n t a i n i n g e x t r a c t s from uninoculated peanut t i s s u e s y i e l d e d f i v e bands i n r e g i o n 4.0-7.5 cm ( F i g . 10). Four days a f t e r i n o c u l a t i o n , each of the 9 A. flavus s t r a i n s produced a zymogram that d i f f e r e d from that of the c o n t r o l s and was d i s t i n c t from a l l other f u n g i . The number of bands i n gels of i n f e c t e d peanuts ranged between 6 and 8, compared to 4 and 5 bands i n c o n t r o l g e l s . In a d d i t i o n , band a c t i v i t y was more intense i n many gels of i n f e c t e d seeds than i n those of the c o n t r o l s . As suggested e a r l i e r i n s t u d i e s with developing and germinating peanuts ( F i g s . 5,6,7), changes i n peptidase a c t i v i t y may be i n d i c a t i v e of p r o t e i n h y d r o l y s i s . Gel e l e c t r o p h o r e t i c patterns of p r o t e i n s i n s o l u b l e f r a c t i o n s showed t h a t each A^ f l a v u s s t r a i n caused changes i n these components during the i n f e c t i o n p e r i o d that were d i f f e r e n t from those of the c o n t r o l ( F i g . 11); no p r o t e i n changes were noted i n g e l patterns of noninfected seeds. In general, p r o t e i n patterns of seeds i n f e c t e d by the various f u n g i showed new p r o t e i n components

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Figure 9. Polyacryhmide gel electrophoretic patterns of esterase activity in noninfected and A. fiavus-infected peanuts. Gel descriptions are given in Figure 8.

Figure 10. Polyacrylamide gel electrophoretic patterns of leucine aminopeptidase activity in noninfected and A. Ravus-infected peanuts. Gel descriptions are given in Figure 8.

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 region 0-1,0 cm p l u s increased m o b i l i t y and poor r e s o l u t i o n of the bands i n region 1.0-2.5 cm as the i n f e c t i o n progressed to day 4. At the same time, bands normally l o c a t e d i n region 2.0-3.5 cm disappeared, and a new group of polypeptides appeared i n r e g i o n 5.5-7.0 cm. S p e c i a t i o n and Genetic

Crossability

S u b s t a n t i a l evidence has been accumulated suggesting that the genetic base, or gene p o o l , of c u l t i v a t e d p l a n t s does not have the reserve germ plasm needed to r e s i s t many of the new problems brought on by p o l l u t i o n , dwindling water s u p p l i e s , i n s e c t s , and p l a n t pathogens. F o r t u n a t e l y , there are w i l d species of most c u l t i v a t e d p l a n t s c o n t a i n i n g untapped genetic resources f o r f u t u r e breeding programs. Wild species contain o l d sources of germ plasm which can be used to decrease "genetic e r o s i o n " of commercial crops by broadenin v a r i e t i e s (45). Once th i s understood, s t u d i e s can begin to determine "bridge s p e c i e s " f o r the i n t r o d u c t i o n of new genetic information i n t o c u l t i v a t e d v a r i e t i e s (56). Gel e l e c t r o p h o r e s i s of enzymes o f f e r s a biochemical approach to the e v o l u t i o n a r y aspects of p l a n t s p e c i a t i o n . Amino a c i d changes w i t h i n a p r o t e i n caused by genetic mutational changes can r e s u l t i n a l t e r e d migration r a t e s when the p r o t e i n s are compared i n a matrix system of polyacrylamide or s t a r c h g e l . Since numerous e l e c t r o p h o r e t i c analyses have shown that species d i f f e r from one another i n band frequencies, the i n d i v i d u a l i t y of each p l a n t species can u s u a l l y be expressed according to i t s enzyme banding p a t t e r n s (9,10,19-22,52). The genus Gossypium i s comprised of about 30 d i p l o i d and three n a t u r a l a l l o t e t r a p l o i d species (5,27,33,50). The d i p l o i d (2n) p l a n t s were d i v i d e d i n t o s i x groups or genomes l a b e l e d A to F; t e t r a p l o i d (4n) species were described 2(AD)η s i n c e they were hypothesized to have o r i g i n a t e d from a cross between p l a n t s from the A and D genomes. A n a l y s i s of species r e l a t i o n s h i p s w i t h i n Gossypium by polyacrylamide g e l e l e c t r o p h o r e s i s of p r o t e i n s and enzymes i n e x t r a c t s of dormant seeds was reported by Cherry et a l . (19-22). The chemotaxonomic comparisons of p r o t e i n and enzyme patterns f o r species w i t h i n and between genomes supported the present c l a s s i f i c a t i o n of Gossypium and the o r i g i n of the natural allotetraploids. In a d d i t i o n , the esterase s t u d i e s showed that much more v a r i a t i o n i n banding patterns e x i s t e d w i t h i n the w i l d species than i n new c u l t i v a r s . Figure 12 presents an example of species w i t h i n the D genome which have esterase banding patterns that are more s i m i l a r to each other than to members of other genome groups (A-C, E-F). E n d r i z z i (26) showed c y t o l o g i c a l l y that G. lobaturn and £ . aridum were c l o s e l y r e l a t e d . Gossypium h a r k n e s s i i was shown to cross f r e e l y with G. armourianum(35). The highest chiasma frequency

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Figure 11. Polyacrylamide gel electrophoretic patterns of proteins in noninfected and A. Rawis-infected peanuts. Gel descriptions are given in Figure 8.

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among the D genome species was produced i n crosses between G. klotzschianum and G^ d a v i d s o n i i (49). These comparisons were f u r t h e r supported by the isozyme patterns of the e s t e r a s e s ; i . e . , the more c l o s e l y r e l a t e d the species were g e n e t i c a l l y , the more s i m i l a r were t h e i r banding p a t t e r n s . On the other hand, G. r a i m o n d i i appeared to produce esterase patterns uniquely d i f f e r e n t from other species of the D genome. Much esterase v a r i a b i l i t y i s observed w i t h i n and between species of the D genome. For example, s i x d i f f e r e n t zymograms (A-F), were observed f o r the G^ t h u r b e r i seeds (268 as compared to 24 f o r a l l other s p e c i e s ) . The presence of s i x esterase g e l patterns i n d i c a t e s that notable genetic v a r i a b i l i t y e x i s t s w i t h i n species at the molecular l e v e l , and i n t r a s p e c i f i c v a r i a b i l i t y increases when many seeds are examined. Of major i n t e r e s t i n t h i s study with G. t h u r b e r i are the esterases l o c a t e d i n a r e l a t i v e l y narrow region of the g e l s 6.5-9.2 cm. Within t h i q u a l i t a t i v e and q u a n t i t a t i v e of the r e l a t i v e frequencies of seed expressing a s p e c i f i c p a t t e r n , from each of the four populations analyzed, i s shown i n Table I . In general, zymograms A-D occur much more f r e q u e n t l y i n n a t u r a l populations than do zymograms Ε and F. These data suggest that banding v a r i a t i o n may be expressed both w i t h i n and between popu lations. The o r i g i n of t h i s banding v a r i a t i o n noted f o r G^ t h u r b e r i (and other s p e c i e s ; F i g . 12) may be due to one or more genetic p o s s i b i l i t i e s i n the p l a n t s : (a) isozymic v a r i a n t s of a s p e c i f i c esterase gene i n homozygous or heterozygous p a i r i n g w i t h i t s p a i r e d gene; (b) a number of d i f f e r e n t and s i m i l a r polypeptide subunits i n various combinations derived from c l o s e l y r e l a t e d n o n a l l e l i c genes; (c) enzymes completely unrelated and not from a common source; and (d) a s e l e c t i v e combination i n v o l v i n g a l l of these p o s s i b i l i t i e s . As to which of these a l t e r n a t i v e s i s most l i k e l y , i t can be noted from the data f o r G. t h u r b e r i that the band i n region 8.0 cm i s the only one present i n a l l of the zymograms and i t i s a l s o present i n greater amounts than any of the other esterases of t h i s s p e c i e s . Thus, a l l of the l a t t e r bands may have a r i s e n from t h i s main esterase through mutation w i t h i n a s i n g l e gene ( i n t r a g e n i c v a r i a t i o n ) or through d u p l i c a t i o n of a s i n g l e gene that then underwent mutation ( i n t e r g e n i c v a r i a t i o n ) . H y b r i d i z a t i o n s t u d i e s of pure p l a n t s that produce each of these esterase bands, however, would be needed to t e s t t h i s hypothesis. Much v a r i a b i l i t y of the esterases can a l s o be noted between species of the D genomes. In a number of cases, s p e c i f i c banding patterns occurred with a low frequency i n one species and a h i g h frequency i n another, whereas the reverse s i t u a t i o n could be obtained with other g e l p a t t e r n s . For example, i n G. h a r k n e s s i i , a zymogram with three esterase bands occurs with a h i g h frequency of 67%. Within t h i s s p e c i e s , a zymogram with four esterase bands

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E N Z Y M E S IN FOOD AND

TABLE I.

Frequency of occurrence

Location (Tucson, A r i z o n a area)

B E V E R A G E PROCESSING

of n o n s p e c i f i c e s t e r a s e p a t t e r n s .

Total Seeds Analyzed

A

Β

Santa C a t a l i n a Mountains

78

0.72

0.00

0.18

0.09

0.03

0.00

Rincon Mountains

72

0.13

0.26

0.08

0.49

0.04

0.00

Santa R i t a Mountains

72

0.81

0.08

0.00

0.11

0.00

0.00

268

0.49

0.10

0.13

0.22

0.04

0.01

Zymograms

Baboquivari Mountains TOTALS

*Zymograms A-F of the n a t u r a l populations of G. t h u r b e r i ( F i g . 12). occurs i n low frequency of 21%. In G. armourianum, the frequencies of these two g e l patterns are reversed (25% versus 50%). Similar comparisons can be made among g e l p a t t e r n s of G^ aridurn, G. lobaturn, G. h a r k n e s s i i and G^ gossypioides. T h i s i n d i c a t e s that d i f f e r e n t combinations of genes coding f o r esterases are p o s s i b l y present w i t h i n the species of t h i s group. The v a r y i n g zymograms may be a r e f l e c t i o n of n a t u r a l s e l e c t i o n pressures operating to produce the most s t a b l e genetic composition f o r each s p e c i e s . These isozyme patterns may a l s o be due to d i f f e r e n t i a l genetic c o n t r o l of seed esterase production and composition i n the p l a n t caused by environmental pressures during a p a r t i c u l a r growing season. A l l o p o l y p l o i d s as Bridge

Species

Examination of enzyme patterns of seeds from a l l o t e t r a p l o i d s formed by genetic breeding s t u d i e s showed that these gels compared c l o s e l y to the a d d i t i v e zymograms of the parent species when the l a t t e r were combined i n a s y n t h e t i c mixture (19,21). T h i s technique can be used to determine p a r e n t a l species of n a t u r a l l y o c c u r r i n g h y b r i d s . Where w i l d h y b r i d species cross with c u l t i v a t e d v a r i e t i e s and form v i a b l e seed, the p a r e n t a l s or bridge species could be determined f o r i n t r o d u c t i o n of v a l u a b l e genetic information i n t o new c u l t i v a r s . An example of such a study i s shown i n Figure 13. Esterases from seeds of a man-made v i a b l e a l l o t e t r a p l o i d 2^20.^), species (AZ 239) were examined by g e l e l e c t r o p h o r e s i s . T h i s a l l o t e t r a -

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

CHERRY

Oilseed Enzymes as Biological Indicators

Figure 12. Polyacrylamide gel electrophoretic patterns of seed esterases from nine species wtihin the D genome of the genus Gossypium. Included are the frequencies of zymograms formed from seeds of each species.

Figure IS. Polyacrylamide gel electrophoretic patterns of seed esterases in a comparison between parentals, their synthetic mixture, and the allotetraploid. Comparisons include G. arboreum var G24 (A genome), G. thurberi (Ό genome), their syn thetic mixture, and their synthetic allotetraploid: 2(A D ) , AZ 239. 2

2

2

1

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i

226

E N Z Y M E S I N FOOD AND

B E V E R A G E PROCESSING

p l o i d was formed from a cross between a v a r i e t y o f arboreurn (A^) and t h u r b e r i (D^). The esterases of a s y n t h e t i c mixture of seed e x t r a c t s formed from arboreum and G^_ t h u r b e r i and the i n d i v i d u a l e x t r a c t s o f these p a r e n t a l s were compared with that of the a l l o t e t r a p l o i d AZ 239. I n the s y n t h e t i c mixture, an a d d i t i v e esterase zymogram was formed. The a d d i t i v e zymogram compared c l o s e l y t o the g e l p a t t e r n of AZ 239. This a l l o t e t r a p l o i d has the p o t e n t i a l of s e r v i n g as a bridge species t o cross with other w i l d p l a n t s shown by c l a s s i c a l genetic techniques and g e l e l e c t r o p h o r e s i s to be c l o s e l y r e l a t e d to G^_ arboreum and G. t h u r b e r i . Conclusions Technology has enabled s c i e n t i s t s to take enzymes out of t h e i r i n v i v o environments to study them i n t e s t tubes Enzyme a c t i v i t y during i s o l a t i o n procedures i s a b i o l o g i c a Nevertheless, enzymes do change i n a c t i v i t y and/or are a l t e r e d s t r u c t u r a l l y when abused. Because o f t h i s they can be e x c e l l e n t i n d i c a t o r s of changes r e l a t e d to food p r o c e s s i n g or p h y s i o l o g i c a l mechanisms. S p e c i f i c task(s) performed by enzymes are genet i c a l l y c o n t r o l l e d and can be used to evaluate genetic s p e c i a t i o n and taxonomy to determine bridge species i n breeding s t u d i e s f o r i n t r o d u c i n g genes o f w i l d species i n t o c u l t i v a t e d v a r i e t i e s . Fungi-host i n t e r r e l a t i o n s h i p s can be determined and evaluated, using enzymes to f o l l o w changes i n development and d i f f e r e n t i a t i o n of the former and a r a p i d form of senescence i n the l a t t e r . F u n c t i o n a l molecules such as enzymes should be s t u d i e d with an understanding o f g e n e t i c and ontogenetic v a r i a b i l i t y . A s u f f i c i e n t number of d i f f e r e n t enzymes should be examined to i n s u r e the development of proper standards f o r reaching appropriate conclusions.

LITERATURE CITED 1. 2. 3. 4. 5. 6. 7. 8.

Acker, L., and H. O. Beutler, Fette Seifen Anstrichm., 67:430 (1965). Augustinsson, Κ. B., B. Axenfors, I . Andersson, and H. Ericksson, Biochim. Biophys. Acta 293:424 (1973). Augustinsson, Κ. Β., Ann. N.Y. Acad. S c i . 94:844 (1961). Baptist, J. N., C. R. Shaw, and M. Mandel, J. Bacteriol. 99:180 (1969). Beasley, J. O., Genet. 27:25 (1942). Berry, J. Α . , and R. G. Franke, Amer. J. Bot. 60:976 (1973). Bhatia, C. R . , and J. P. Nilson, Biochem. Genet. 3:207 (1969). Brewbaker, J. L., M. D. Upadhya, Y. Makinen and T. MacDonald. Physiol. Plant. 21:930 (1968).

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

12.

9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.

22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33.

34. 35. 36.

CHERRY

Oilseed Enzymes as Biological Indicators

227

Cherry, J. P., and L. R. Beuchat, Cereal Chem. 53:750 (1976). Cherry, J. P., Peanut S c i . 2:57 (1975). Cherry, J. P., C. T. Young, and L. R. Beuchat, Can. J. Bot. 53:2639 (1975). Cherry, J. P., and J. M. Prescott, Proc. Exp. B i o l . & Med. 147:418 (1974). Cherry, J. P., and J. M. Prescott, (Unpublished data, 1974). Cherry, J. P., R. Y. Mayne, and R. L. Ory, Physiol. P1. Path. 4:425 (1974). Cherry, J. P., and R. L. Ory, Phytochem. 12:283 (1973). Cherry, J. P., and R. L. Ory, Phytochem. 12:1581 (1973). Cherry, J. P., J. M. Dechary, and R. L. Ory, J. Agric. Food Chem. 21:652 (1973). Cherry, J. P., R. L. Ory, and R. Y. Mayne, J. Amer. Peanut Res. Ed Cherry, J. P., F.R.H Theoret. & Appl. Genet. 42:218 (1972). Cherry, J. P., and F. R. H. Katterman, Phytochem. 10:141 (1971). Cherry, J. P., "Comparative Studies of Seed Enzymes of Species of Gossypium by Polyacrylamide and Starch Gel Electrophoresis," Ph.D. Dissertation, University of Arizona, Tucson (1971). Cherry, J. P., F. R. H. Katterman, and J. E . Endrizzi, Evolution 24:431 (1970). Cubadda, R . , A. Bozzini and E . Quattrucci, Theoret. & Appl. Genet. 45:290 (1975). Davis, B. J., Ann. N.Y. Acad. S c i . 121:404 (1964). Dixon, M., and E . C. Webb, "Enzymes," Academic Press Inc., New York (1964). Endrizzi, J. E., J. Hered. 48:221 (1957). F r y x e l l , P. Α . , Adv. Front. Plant S c i . 10:31 (1965). Gardner, H. W., G. E . Inglett, and R. A. Anderson, Cereal Chem. 46:626 (1969). Gottlieb, L. D . , BioSci. 21:939 (1971). Holmes, R. S., and C. J. Masters, Biochim. Biophys. Acta 151:147 (1968). Holmes, R. S., and C. J. Masters, Biochim. Biophys. Acta 132:379 (1967). Hunter, R. L., and C. L. Markert, Science 125:1294 (1957). Hutchinson, J. Β., R. A. Silow, and S. G. Stephens, "The Evolution of Gossypium and the Differentiation of the Cultivated Cottons," Oxford University Press, London (1947). IUPAC-IUB Commission on Biochemical Nomenclature, J. B i o l . Chem. 246:6127 (1971). Kearny, Τ. Η . , Leaflets Western Bot. 8:103 (1957). Kruger, J. E., and D. E . LaBerge, Cereal Chem. 51:578 (1974).

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

228

37. 38.

39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59.

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

LaBerge, D. E., J. E . Kruger, and W.O.S. Meredith, Can. J. Plant S c i . 53:705 (1973). Manwell, C . , and C. M. A. Baker, "Molecular Biology and the Origin of Species: Heterosis, Protein Polymorphism and Animal Breeding, University of Washington Press, Seattle, Washington (1970). Markert, C. L., Ann. N.Y. Acad. S c i . 151:14 (1968). Markert, C. L., and G. S. Whitt, Experientia 24:977 (1968). Markert, C. L., and R. L . Hunter, Histochem. Cytochem. 7:42 (1959). Markert, C. L., and F. Moller, Proc. Natl. Acad. S c i . 45:753 (1959). Marshall, D. R . , and R. W. A l l a r d , J. Hered. 60:17 (1969). Menke, J. F., R. S. Singh, C. O. Qualset, and S. K. Jain, C a l i f . Agric. 27:3 (1973). M i l l e r , J., Science 182:1231 (1973) Ornstein, L., Ann Pawar, V. S., and Payne, W. J., J. W. Fitzgerald, and K. S. Dodgson, Appl. Microbiol. 27:154 (1974). P h i l l i p s , L . L., Amer. J. Bot. 53:328 (1966). P h i l l i p s , L . L., Evolution 17:460 (1963). Reddy, Μ. Μ., and S. F. H. Threlkeld, Can. J. Genet. Cytol. 13:298 (1971). Scandalios, J. G . , Biochem. Genet. 3:37 (1969). Schipper, A. L., Phytopath. 65:440 (1975). Shaw, C. R . , Intern. Rev. Cytol. 25:297 (1969). Shaw, C. R . , Science 149:936 (1965). Simpson, C. E., and R. L . Haney, Texas Agric. Prog. 19:10 (1973). Smithies, O., Adv. Prot. Chem. 14:65 (1959). Sopanen, T . , and J. Mikola, Plant Physiol. 55:809 (1975). Wagenknecht, A. C., Food Res. 24:539 (1959).

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

13 Enzymes and Oxidative Stability of Peanut Products A L L E N J. ST. ANGELO, JAMES C. KUCK, and ROBERT L. ORY Southern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, New Orleans, La. 70179

Investigations of th food l i p i d s are being conducted in all types of foods -- marine, dairy, meat, and vegetable products. This research is a continuous effort to improve nutritional value of foods and to increase s h e l f - l i f e s t a b i l i t y . Also, because of medical implications of peroxidized lipids i n food products, one can readily understand the reason for the great interest in this area of research. Peanuts (groundnuts) are grown primarily i n India, China, Africa, South America, and the United States. In most of these countries, peanuts are extracted and refined, the oil is processed for food uses, and the meal is sold for animal food. The United States is the only country that consumes the whole nut directly in food products. Here, peanuts are used i n many different food products -- peanut butter, candy, confections, bakery products, salted nuts -- and as a cooking oil. In newer food applications, peanuts are being used as defatted peanut flour and as f u l l - f a t flour (1,2). However, defatted meals still contain significant amounts of residual oil that can give rise to lipid peroxides. Peanuts c o n t a i n approximately 50% oil and a r e a good source of unsaturated f a t t y acids (80%), o f which 20%-37% is polyunsaturated. The unsaturated f a t t y acids can be o x i d i z e d , a f f e c t i n g both the q u a l i t y and f l a v o r o f peanut products. Peroxidation of f a t t y acids has been a t t r i b u t e d to a number o f causes: light, air, heat, t r a c e metals, enzymes, microorganisms, and even the presence o f f r e e f a t t y acids that can a c t as f r e e r a d i c a l catalysts. The q u a l i t y o f raw peanuts during storage and processing, t h e r e f o r e , can be s e r i o u s l y a f f e c t e d by a c t i v a t i o n o f endogenous enzymes that o x i d i z e f a t t y acids o r induce other u n d e s i r a b l e reactions. Peanuts a r e a l s o one o f the most handled crops; they r e q u i r e digging during h a r v e s t i n g , then combining, c u r i n g , s h e l l i n g , drying, blanching, and packaging f o r storage. In each o f these steps, they a r e s u s c e p t i b l e to damage that can a c t i v a t e enzymes and u l t i m a t e l y a f f e c t the q u a l i t y o f the final product.

229

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B E V E R A G E PROCESSING

The three causes o f p e r o x i d a t i o n that are of i n t e r e s t to us are a u t o x i d a t i o n i n a i r and c a t a l y s i s by metals and by enzymes. A u t o x i d a t i o n of unsaturated f a t t y a c i d s i n raw peanuts i s a slow process, sometimes taking weeks or months. In t h i s r e a c t i o n , oxygen i s added across a double bond presumably forming a c y c l i c peroxide that cleaves to a f a t t y a c i d hydroperoxide. This hydro peroxide f u r t h e r decomposes i n t o a c i d s , aldehydes, ketones, and other substances that form during storage or p r o c e s s i n g . These byproducts are a major cause of undesirable odors and f l a v o r s . They can a l s o damage and lower the n u t r i t i v e v a l u e of the f i n a l food product by complexing with amino a c i d s and p r o t e i n s . Assay Methods f o r P e r o x i d a t i o n o f Unsaturated

F a t t y Acids

Several methods are used to f o l l o w the progress of r a n c i d i t y , or to determine s h e l f - l i f e s t a b i l i t y These methods i n c l u d e peroxide value (PV) determination i n d i c a t i o n of malonaldehyd bonyl products, and oxygen consumption. In our s t u d i e s , the con jugated diene hydroperoxide (CDHP) method described p r e v i o u s l y ( 3 ^ ) i s employed to study s h e l f - l i f e s t a b i l i t y o f peanut prod u c t s . This method i s r a p i d , simple, r e q u i r e s only a drop of o i l , and the r e s u l t s c o r r e l a t e w e l l with those of the peroxide v a l u e method (5). B r i e f l y , i t i n v o l v e s e x t r a c t i n g a weighed sample of e i t h e r peanut b u t t e r or chopped peanut kernels with hexane, cent r i f u g i n g , then measuring the a b s o r p t i o n of the hexane supernatant at 234 nm. F i g u r e 1 i l l u s t r a t e s the d i r e c t r e l a t i o n s h i p of CDHP

80

r

CDHP [μ MOLES/Ç PN]

Figure 1. Comparison between peroxide value (PVj and conjugated diene hydroperoxide ( C D H P ) values in peanut butter stored 90 days at 25°C

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

13.

ST. A N G E L O E T A L .

Oxidative Stability of Peanut Products

231

to peroxide values i n 34 peanut samples. CDHP values a r e c a l c u l a t e d i n yMoles/g sample, using 28,000 as the e x t i n c t i o n c o e f f i c i e n t i n s t e a d o f the 24,500 used i n the o r i g i n a l method (5). The equation f o r the conversion i s : PV = -11.44 + 4.302(CDHP); the c o r r e l a t i o n c o e f f i c i e n t f o r the two methods i s 0.9812. Peanut Butter The most important use o f peanuts i n the United States i s peanut b u t t e r . M i l l i o n s o f pounds are used i n t h i s product each year. Extending the s h e l f l i f e o f peanut b u t t e r i s a major concern. When peanuts are homogenized f o r processing i n t o peanut b u t t e r , they become s u s c e p t i b l e to o x i d a t i o n because o f t h e i r high polyunsaturated f a t t The f i r s t step to i t s cause. Several minor c o n s t i t u e n t s o f peanut b u t t e r were i n v e s t i g a t e d as p o s s i b l e c a t a l y s t s o f f a t t y a c i d o x i d a t i o n (6). These i n c l u d e d s a l t , enzymes, and m e t a l l o p r o t e i n s t h a t a r e present i n peanuts. I n each s e r i e s o f t e s t s , the c o n s t i t u e n t s were added to peanut b u t t e r and stored a t room temperature (25°C) f o r 28 days. CDHP values were determined before, during, and a f t e r storage. The most s i g n i f i c a n t increases i n o x i d a t i o n occurred i n samples c o n t a i n i n g f e r r i c o r c u p r i c i o n s , lipoxygenase, t y r o s i n a s e , peroxidase, or c h l o r o p h y l l . The degree o f o x i d a t i o n depended upon t h e i r microenvironment, i . e . , whether or not the m a t e r i a l was added i n an aqueous o r nonaqueous s o l v e n t . In a more complete i n v e s t i g a t i o n that included storage o f the peanut b u t t e r samples f o r 3 months a t room temperature, copper and i r o n s a l t s and three indigenous m e t a l l o p r o t e i n s , t y r o s i n a s e , peroxidase, and lipoxygenase, were found t o be major c a t a l y s t s o f peroxide formation (Figure 2)· Tyrosinase contains copper, peroxidase has i r o n , and lipoxygenase contains i r o n (7^.8)· Since p r e l i m i n a r y evidence suggested that c h e l a t i n g agents such as c i t r i c a c i d and EDTA could r e t a r d the c a t a l y t i c oxidat i v e e f f e c t o f the m e t a l l o p r o t e i n s (5,9.) » a d d i t i o n a l study was conducted on the e f f e c t s o f these agents on f a t t y a c i d o x i d a t i o n i n peanut b u t t e r . Peanut b u t t e r was prepared i n the l a b o r a t o r y w i t h and w i t h out c i t r i c a c i d , EDTA, or a s c o r b y l p a l m i t a t e . A f t e r i n i t i a l CDHP values were determined, the peanut b u t t e r s were s t o r e d i n sealed g l a s s j a r s up to 180 days (5 months)· D u p l i c a t e samples were p e r i o d i c a l l y removed and analyzed f o r CDHP contents. As shown i n A o f F i g u r e 3, peanut b u t t e r c o n t a i n i n g c i t r i c a c i d showed an i n c r e a s e o f 5.7 yMoles o f CDHP a f t e r 140 days storage, compared to 11.3 f o r the c o n t r o l . When EDTA was added to peanut b u t t e r (Figure 3B), the i n c r e a s e was only 5.9 yMoles CDHP, compared to 10.3 f o r the c o n t r o l , a f t e r storage f o r 156 days. The a

n

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Figure 2. Effects of various additives suspended in peanut oil on lipid oxidation during storage for three months at 25°C (6)

TIME (DAYS)

Figure 3. Effect of 0.01% citric acid (A), ethylenediaminetetraacetic acid, 0.01% (B), and ascorbyl palmitate, 0.1% (C) on oxidation of peanut butter during storage at 25°C

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

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e f f e c t of a s c o r b y l palmitate (Figure 3C) was even more s t r i k i n g ; i t completely i n h i b i t e d f u r t h e r p e r o x i d a t i o n , whereas there was an increase o f 7 yMoles CDHP f o r the untreated c o n t r o l . These data r e f l e c t a r e d u c t i o n o f 50% p e r o x i d a t i o n f o r the peanut b u t t e r c o n t a i n i n g c i t r i c a c i d , a 43% decrease f o r the sample cont a i n i n g EDTA, and a 100% r e d u c t i o n f o r the sample with added a s c o r b y l palmitate. C i t r i c a c i d and EDTA are known c h e l a t i n g agents and would presumably bind those metals present i n peanut butter. I t i s g e n e r a l l y accepted that l i n o l e i c a c i d i s one o f the primary substrates i n f a t t y a c i d p e r o x i d a t i o n . The l i n o l e i c / o l e i c a c i d r a t i o i s f r e q u e n t l y used as an index f o r p r e d i c t i n g the s u s c e p t i b i l i t y o f peanut o i l towards o x i d a t i o n (10,11,12)· However, Young and H o i l e y (13) have reported a negative c o r r e l a t i o n between o i l - k e e p i n g time and n i t r o g e n content i n peanuts. In other words, the higher the p r o t e i n content, the lower the s t a b i l i t y . While they suggeste was c o i n c i d e n t a l , our dat t e i n s ( m e t a l l o p r o t e i n s , i n p a r t i c u l a r ) appear to a c t as c a t a l y s t s and thus become an i n t r i c a t e part o f the o x i d a t i v e mechanism. Ascorbyl palmitate was shown by Cort (14) to be a b e t t e r a n t i o x i d a n t than b u t y l a t e d hydroxyanisole (BHA) and b u t y l a t e d hydroxytoluene (BHT) f o r i n c r e a s i n g the s h e l f l i f e o f vegetable o i l s and potato c h i p s . As demonstrated i n F i g u r e 3C, a s c o r b y l palmitate s i g n i f i c a n t l y increased the s h e l f l i f e o f peanut b u t t e r , a high p r o t e i n / o i l - c o n t a i n i n g food. To i n v e s t i g a t e the p o s s i b l e e f f e c t s o f d i f f e r e n t handling and processing steps, s e v e r a l d i f f e r e n t samples o f f r e s h l y prepared commercial peanut b u t t e r s were analyzed f o r i n i t i a l CDHP contents. Results shown i n F i g u r e 4 i n d i c a t e d that no two samples had the same i n i t i a l peroxide content. The CDHP contents of nine d i f f e r e n t samples o f peanut b u t t e r v a r i e d from 3.2 to as high as 15.5 (6), suggesting that the q u a l i t y o f the peanuts before r o a s t i n g and processing v a r i e d considerably, and that some were already i n d i f f e r e n t stages o f p e r o x i d a t i o n . These f i n d i n g s s t r e s s the importance o f q u a l i t y checks f o r peroxide content o f peanuts before processing. Lipoxygenase i n Raw Peanuts The r e s u l t s i n F i g u r e 4 i n d i c a t e that r e a c t i o n s taking p l a c e i n the peanuts p r i o r to processing may a f f e c t the q u a l i t y o f processed peanuts. The b i o l o g i c a l c a t a l y s t considered to be the p r i n c i p a l cause o f p e r o x i d a t i o n o f unsaturated f a t t y a c i d s i n many raw o r unprocessed p l a n t products i s the enzyme l i p o x y genase. The occurrence o f t h i s enzyme i s r a t h e r widespread i n legumes and c e r e a l g r a i n s , but i t s existence i n animal t i s s u e i s s t i l l questionable. The enzyme i s h i g h l y s p e c i f i c f o r the peroxi d a t i o n o f unsaturated f a t t y acids that c o n t a i n the c i s - c i s

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

234

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PROCESSING

«1

ft.

ft.

1

2

3

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Figure 4. Initial

1,4-pentadiene systems; i . e . , l i n o l e i c , l i n o l e n i c , and a r a c h i donic a c i d s , e s t e r s , and t r i g l y c e r i d e s . The presence of lipoxygenase i n raw peanuts has been reported by s e v e r a l i n v e s t i g a t o r s over the past two decades (15-18). S t . Angelo and Ory (17) p a r t i a l l y p u r i f i e d the enzyme by ammonium s u l f a t e f r a c t i o n a t i o n and proceeded to separate i t from a c o e x i s t i n g enzyme, presumably l i n o l e i c a c i d hydroperoxide isomerase. This l a t t e r enzyme, which was f i r s t i d e n t i f i e d by Zimmermann i n f l a x s e e d (19), was r e c e n t l y r e p o r t e d to be a l y a s e (20). F i g u r e 5 represents an e l u t i o n p a t t e r n on Sephadex G-100 that we employed to separate lipoxygenase, "hydroperoxide i s o merase," and peroxidase. The 40% ammonium s u l f a t e f r a c t i o n d e s c r i b e d p r e v i o u s l y (17) was chromatographed i n phosphate b u f f e r . Peak 1 contains the lipoxygenase and the "isomerase." Peak 2 contains an a c t i v e peroxidase. No a c t i v i t y was found i n Peak 3. Aqueous lipoxygenase preparations are pH dependent and are not very s t a b l e , even when s t o r e d below f r e e z i n g f o r s e v e r a l days. Approximately 30% o f the a c t i v i t y i s destroyed by i n c u b a t i n g the enzyme at 36°C f o r 30 minutes. At temperatures above 40°, a l l lipoxygenase a c t i v i t y i s l o s t (17). In F i g u r e 6, a c t i v i t y o f the enzyme i n r e l a t i o n to pH i s presented. The enzyme was e x t r a c t e d with phosphate b u f f e r s from pH 5.8 through 8.4; one p r e p a r a t i o n was e x t r a c t e d with d e i o n i z e d water. The enzyme preparations were allowed to stand a t 4°C f o r 8 days. Over the 8-day p e r i o d , a c t i v i t y was l o s t i n each s o l u t i o n . However, these r e s u l t s i n d i c a t e d that the enzyme was the most s t a b l e f o r about 3 days when kept i n phosphate b u f f e r a t pH 7.4.

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

13.

ST.

Oxidative Stability of Peanut Products

ANGELO E T AL.

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Lipoxygenase i s o l a t e d from peanuts with 40% ammonium s u l f a t e was a c t i v e over a pH range from 4 through 7, w i t h the optimum a t 6.1. Cyanide added at the usual 1 mM i n h i b i t o r c o n c e n t r a t i o n d i d not i n h i b i t the enzyme (17). Recently, Sanders e t al· (18) r e -

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

236

ENZYMES

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AND

BEVERAGE

PROCESSING

ported s e p a r a t i n g three lipoxygenase isozymes i n raw peanuts. Two had pH optima o f 6.2 and the other had an optimum a t 8.3. They a l s o r e p o r t e d that higher concentrations o f cyanide i n h i b i t e d the a c i d i c isozymes. Because the cyanide was added to the r e a c t i o n mixture b e f o r e a d d i t i o n o f the enzyme, i t was d i f f i c u l t to e s t a b l i s h the a c t u a l r e a c t i o n c o n d i t i o n s and pH of the cyanides u b s t r a t e s o l u t i o n . Therefore, we r e i n v e s t i g a t e d the e f f e c t of cyanide on peanut lipoxygenase. Since aqueous s o l u t i o n s of sodium and potassium cyanide are u s u a l l y s t r o n g l y a l k a l i n e , i t was necessary to f i r s t i n v e s t i g a t e the e f f e c t of cyanide on the pH o f assay s o l u t i o n s . The r e s u l t s (Table I) show t h a t , as the CN c o n c e n t r a t i o n i n c r e a s e s , even Table I pH of KCN D i s s o l v e d i n Phosphate B u f f e r and Water

Phosphate B u f f e r (pH) 6.1 6.1 6.1 8.3 8.3 5.7 4.7* 5.5,

(M) 0.01 0.10 0.10 0.01 0.10 0.20 0.10 deionized water

(mM)

(pH)

30 30 300 30 30 300 300 30

9.5 6.8 9.8 10.8 9.7 8.9 9.4 10.5

*NaH P0. 2 4 o

though added i n a c i d i c b u f f e r , the pH of the f i n a l s o l u t i o n s becomes very a l k a l i n e . This suggested that the pH o f the cyanide s o l u t i o n s could be c r i t i c a l f o r lipoxygenase s t u d i e s , and l e a d to erroneous c o n c l u s i o n s . In our more recent peanut isozyme-cyanide experiments, we prepared the isozymes by e x t r a c t i n g raw peanuts with 0.5 M NaCl, 0.05 M T r i s b u f f e r , pH 7.2, and c e n t r i f u g i n g twice a t 29,000 g f o r 10 minutes a t 0°C. The supernatant was d i a l y z e d overnight against d e i o n i z e d water a t 4°C, then r e c e n t r i f u g e d . A pH 6 isozyme was found i n the resuspended p r e c i p i t a t e , and the a l k a l i n e isozyme was i n the c l e a r supernatant f r a c t i o n . In each assay, the r e a c t i o n mixture contained 1 ml of the s u b s t r a t e s o l u t i o n , 1.95 ml of b u f f e r , and 0.05 ml of the enzyme p r e p a r a t i o n s . When cyanide was i n v e s t i g a t e d , a microvolume (25-100 μϊ) of cya nide s o l u t i o n was added to b u f f e r c o n t a i n i n g the enzyme, which was allowed to stand f o r 5 minutes. Then s u b s t r a t e was added and the mixture was immediately assayed f o r lipoxygenase 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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237

as p r e v i o u s l y described (17). The pH o f the r e a c t i o n mixture was a l s o determined immediately f o l l o w i n g the assay* When cyanide s o l u t i o n s were incubated with the a c i d i c enzyme-buffer f o r 5 minutes p r i o r to a d d i t i o n o f s u b s t r a t e , a c t i v i t y s t i l l followed the pH curve, i n d i c a t i n g that i n c r e a s i n g amounts o f cyanide b u f f e r e d w i t h i n the pH optimum range o f the lipoxygenase do not i n h i b i t the pH 6.2 isozyme. I f not s t r o n g l y b u f f e r e d , cyanide can cause an i n c r e a s e i n pH that w i l l e f f e c t lipoxygenase a c t i v ity. Cyanide had no e f f e c t on the a l k a l i n e isomerase. L i n o l e i c A c i d Hydroperoxide

Lyase

A s e q u e n t i a l enzyme i n the l i p i d o x i d a t i o n c h a i n i n germin a t i n g watermelon s e e d l i n g s was r e c e n t l y d e s c r i b e d by V i c k and Zimmermann (%0). T h i s enzyme, a l y a s e that cleaves l i n o l e i c a c i d hydroperoxide, was o r i g i n a l l y thought to be an isomerase (19). The l y a s e a l s o appear D i r e c t GC a n a l y s i s o f a ture, as shown i n F i g u r e 7, demonstrated that methyl l i n o l e a t e was cleaved by the peanut enzyme p r e p a r a t i o n (21)· Cleavage occurs a t the C-13 bond r e l e a s i n g two components; hexanal and at l e a s t one other component, presumably a methyl e s t e r o f a C-12 f a t t y a c i d , but not confirmed with a standard compound. E f f e c t o f L i p i d P e r o x i d a t i o n on P r o t e i n S o l u b i l i t y As noted e a r l i e r , o x i d a t i v e degradation o f unsaturated f a t t y acids produces hydroperoxides and t h e i r secondary breakdown produ c t s , a c i d s , a l c o h o l s , aldehydes, and ketones. These compounds are known to damage p r o t e i n s , enzymes, and amino a c i d s (22,23). Recently, S t . Angelo and Ory (24,25) i n v e s t i g a t e d the e f f e c t s o f l i p i d peroxides on raw and processed peanuts during storage. The o i l and p r o t e i n were separated from peanuts according to the d i a gram shown i n F i g u r e 8. CDHP contents were determined on hexane e x t r a c t s o f the o i l s . The d e o i l e d meal, the s a l t - s o l u b l e prot e i n s , and the s a l t - i n s o l u b l e residues were analyzed f o r p r o t e i n contents. The r e s u l t s o f these s t u d i e s (24) showed that hexane e x t r a c t s from roasted whole peanuts, a f t e r 12 months storage a t 30°C, contained an i n c r e a s e d CDHP v a l u e of 32.4 yMoles/g. The development o f peroxides i n s e v e r a l v a r i e t i e s o f raw and roasted peanuts s t o r e d f o r 12 months a t 4°C a l s o was i n v e s t i gated, see F i g u r e 9. Raw peanuts, plus corresponding r o a s t e d samples, were s t o r e d i n s e a l e d g l a s s j a r s . Data obtained with twelve d i f f e r e n t samples f i t i n t o three general curves: (1) those that d i d not develop peroxides i n the f i r s t 28 weeks and whose CDHP values remained a t 3 yMoles/g o f peanuts throughout the storage p e r i o d ; (2) those that were undergoing p e r o x i d a t i o n , but whose i n i t i a l CDHP values were a l s o 3 yMbles/g; and (3) those that showed p e r o x i d a t i o n taking p l a c e , with s l i g h t l y higher CDHP v a l u e s , i n i t i a l l y 8.5 yMoles/g, and i n c r e a s i n g to 13 yMoles/g a f t e r

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

ENZYMES

•

c

PROCESSING

1

•

^ 0 ^ 40

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

^,

λ 0

1 TIME IN MINUTES

Figure 7. Direct GC analysis of reaction products from oxida tion of methyl linoleate by peanut extract. Curve A represents the profile obtained when the extract was deleted from the reac tion mixture; Curve B, minus the substrate; Curve C , total reac tion mixture: 0.1 mL substrate, 0.04 mL of Tween-20 emulsifier, 15 mL of 0.2M.

PEANUTS (RAW OR ROASTEO)

HEXANE (1:5.W/V); CENTRIFUGE MEAL I

|HEXANE/OIL(I ) I

ΓΗΕΧΑΝΕ

(1:5. W/V); CENTRIFUGE

|HEXANE/OIL(2)| COMBINE (|:IO,W/V) H E X A N E OIL FOR CDHP ANALYSIS NoCI-SOLUBLE PROTEINS DIALYSIS. LYOPHILIZE Να CI-SOLUBLE PROTEINS

Figure 8.

IDEOILED M E A L NaCI (i:iO.W/V); CENTRIFUGE

Na CI-INSOLUBLE RESIDUE DIALYSIS LYOPHILIZE NaCI-INSOLUBLE RESIDUE

Scheme for fractionation of oil and pro tein from peanuts

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

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ET AL.

I

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Figure 9. Effect of cold storage on peroxidation of fatty acids in raw peanuts. Peroxidation was measured as the increase in C D H P content for 12 different lots separated into three groups (25).

52 weeks. These data suggested that the peanuts y i e l d i n g curves Β and C were probably o l d e r than those d e p i c t e d i n curve A, and had already undergone some p e r o x i d a t i o n p r i o r to these storage experiments· When the three p e r o x i d a t i o n curves i n F i g u r e 9 were j o i n e d end-to-end. then the composite shown i n F i g u r e 10 was obtained. These data suggested that raw s h e l l e d peanuts could be s a f e l y s t o r e d a t 4°C f o r a t l e a s t 6 months before enzyme-catalyzed o x i d a t i o n begins. Once t h i s o x i d a t i o n s t a r t s , storage a t 4°C i s unable to prevent i t . Again, the most l o g i c a l explanation f o r l i p i d o x i d a t i o n i n raw peanuts i s lipoxygenase. Furthermore, E r i c k s s o n (26) has reported that pea lipoxygenase i s a c t i v e a f t e r storage f o r s e v e r a l months a t -20°C. When the corresponding roasted peanuts were assayed f o r CDHP content a f t e r storage f o r 1 year a t 4°C, the r a t e o f o x i d a t i o n was 8 times f a s t e r than that i n raw peanuts. CDHP u n i t s i n c r e a s e d from an i n i t i a l v a l u e o f 8 yMoles/g to approximately 40 i n 1 year and to 61 a f t e r 2 years storage. Since r o a s t i n g destroys l i p o x y genase, i t i s obvious that t h i s l i p i d p e r o x i d a t i o n i s c a t a l y z e d by nonenzymic f a c t o r s , most l i k e l y by metals o r m e t a l l o p r o t e i n s (e.g. i n a c t i v a t e d lipoxygenase, peroxidase, o r t y r o s i n a s e )

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

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present i n the peanuts, as shown i n the experiments with peanut butter ( l , ^ ) .

St. Angelo and Ory (24) have shown that with i n c r e a s i n g l i p i d p e r o x i d a t i o n i n raw peanuts, recovery o f s a l t - s o l u b l e f r a c tions i n c r e a s e d , and s a l t - i n s o l u b l e residues decreased c o r r e s pondingly. T h i s phenomenon was i n v e s t i g a t e d f u r t h e r with i n t e n t i o n a l l y damaged (by crushing) peanuts s t o r e d under 3 d i f f e r e n t c o n d i t i o n s : sample 1 s t o r e d i n s e a l e d j a r s a t 4°C; sample 2, i n j a r s covered with cheesecloth (access to a i r ) at 30°C; and sample 3, ground and blended with 10% r a n c i d o i l to promote l i p i d p r o t e i n i n t e r a c t i o n , then s t o r e d at 30°C w h i l e covered with cheesecloth. A f t e r 4 1/2 months, the samples were d e o i l e d with hexane and the absorbance measured a t 234 nm. Defatted meals were then e x t r a c t e d with sodium c h l o r i d e , and the y i e l d s o f s o l uble and i n s o l u b l e f r a c t i o n s determined (24). The r e s u l t s showed that sample 1, s t o r e d a t 4°C, had an absorbance of 0.55; 480 mg of s a l t - s o l u b l e m a t e r i a l was recovered from 1.5 g. For samples 2 and 3, p e r o x i d a t i o n and s a l t - s o l u b l e m a t e r i a l i n c r e a s e d ; absorbance readings were 1.23 and 1.90 and weights of s o l u b l e m a t e r i a l recovered were 550 and 588 mg, r e s p e c t i v e l y . Conversely, as pero x i d a t i o n i n c r e a s e d , the s a l t - i n s o l u b l e residues recovered from the three corresponding samples decreased (562, 537, and 515 mg, r e s p e c t i v e l y ) . I t appears, t h e r e f o r e , that enzyme-catalyzed

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

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l i p i d p e r o x i d a t i o n i n raw peanuts a l s o promotes i n t e r a c t i o n s between the peroxides and p r o t e i n s i n the seed. Subsequent changes i n s o l u b i l i t y of the p r o t e i n s could p o s s i b l y a f f e c t p o t e n t i a l use of d e f a t t e d peanut f l o u r as p r o t e i n supplements i n p r o t e i n - f o r t i f i e d food products. E f f e c t s o f Blanching on Q u a l i t y Another f a c t o r that can a f f e c t q u a l i t y of peanut products i s b l a n c h i n g , the process of removing the skins (testae) that c o n t a i n tannins that may c o n t r i b u t e to o f f - f l a v o r s . There are s e v e r a l methods f o r blanching peanuts, but we examined only two, water and s p i n blanching (27)· In s p i n blanching, the peanuts are f i r s t passed through a s e r i e s of f i n e l y adjusted r a z o r blades to s l i t the skins and then through a dryer at 71°C to lower the moisture content to 5% The kernels are then passe remove the s k i n s . The nut In water blanching, the kernels are s l i t as i n s p i n blanching, sprayed with 86°C water f o r 1 1 / 2 minutes, and the skins are then mechanically removed. F i n a l l y , the kernels are d r i e d to 5% moisture. The e f f e c t s of water and s p i n blanching on o x i d a t i v e s t a b i l i t y of raw and roasted peanuts were compared, using unblanched c o n t r o l s , water blanched, and s p i n blanched peanuts that were d i v i d e d i n t o two p o r t i o n s each. One p o r t i o n of the samples were s t o r e d i n a sealed g l a s s j a r a t 25°C. The remaining p o r t i o n s were roasted at 175°C f o r 20 minutes i n a rôtisserie oven, then s t o r e d i n sealed j a r s , as were the unroasted samples. A l l samples were examined f o r developing CDHP contents at monthly i n t e r v a l s f o r 210 days. There were no s i g n i f i c a n t d i f f e r e n c e s i n s h e l f l i f e of unblanched and s p i n blanched raw peanuts, but water blanched raw peanuts had a s h o r t e r s h e l f l i f e and developed l i p i d peroxides f a s t e r than the s p i n blanched and c o n t r o l peanuts. In roasted peanuts, the r e s u l t s were d i f f e r e n t . The unblanched c o n t r o l s had the s h o r t e s t s h e l f l i f e , and the roasted, water blanched peanuts had the longest s h e l f l i f e of a l l three. Results of the r o a s t i n g experiments suggest that elevated temperatures a p p l i e d during blanching may i n a c t i v a t e some enzymes, but the exact systems i n v o l v e d are s t i l l not understood. The foregoing r e s u l t s i n d i c a t e , that s e l e c t i o n of peanut storage c o n d i t i o n s should be based upon t h e i r pretreatment and intended use to i n s u r e the maintenance o f f r e s h q u a l i t y i n peanuts and i n t h e i r roasted products. I f peanuts are to be s t o r e d without vacuum pack c o n d i t i o n s a f t e r r o a s t i n g , they should be water blanched. On the other hand, i f they are to be s t o r e d raw for long periods before r o a s t i n g , they e i t h e r should be unblanched or s p i n blanched.

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Concluding Remarks O x i d a t i o n o f unsaturated f a t t y a c i d s i n peanut products form compounds that can adversely a f f e c t f l a v o r and q u a l i t y . Lipoxy genase has been suspected as the major cause o f l i p i d o x i d a t i o n , but r o a s t i n g c o n d i t i o n s i n commercial processes should i n a c t i vate t h i s enzyme. I n roasted peanut products, m e t a l l o p r o t e i n s are probably r e s p o n s i b l e f o r l i p i d o x i d a t i o n . I n raw peanuts s t o r e d f o r long periods b e f o r e r o a s t i n g , handling p r a c t i c e s that can damage seeds c o u l d a l s o a c t i v a t e enzymes to form peroxides, aldehydes, ketones, and a l c o h o l s , and shorten the s h e l f l i f e o f the f i n a l products. I t i s evident that improvements must be made i n each o f these handling and processing techniques i f q u a l i t y of the f i n a l product i s to be improved.

Literature Cited 1.

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

Harris, H., Davis Spadaro, J. J., Auburn Univ., Agr. Res. Exp. Sta. B u l l . 431, Auburn, A l a . , 1972. Mitchell, J. H., U. S. Patent 3,689,287 (Sept. 5, 1972). St. Angelo, A. J., Ory, R. L., Brown, L . E., J. Amer. Peanut Res. Ed. Assoc. (1972) 4, 186. St. Angelo, A. J., Ory, R. L., Brown, L . Ε., Oleagineux (1973) 28, 351. St. Angelo, A. J., Ory, R. L., Brown, L . E., J. Amer. O i l Chem. Soc. (1975) 52, 34. St. Angelo, A. J., Ory, R. L., J. Amer. Peanut Res. Ed. Assoc. (1973) 5, 128. Chan, H. W. S., Biochim. Biophys. Acta (1973) 327, 32. Roza, M . , Francke, Α . , Biochim. Biophys. Acta (1973) 327, 24. St. Angelo, A. J., Ory, R. L., J. Amer. O i l Chem. Soc. (1975) 52, 38. Holley, K. T., Hammons, R. O., Univ. of Georgia, Agr. Res. Sta. B u l l . 32, Tifton, G a . , 1968. Worthington, R. E., Hammons, R. O., Oleagineux (1971) 26, 695. Young, C. T., Mason, M. E., Matlock, R. S., Waller, G. R . , J. Amer. O i l Chem. Soc. (1972) 49, 314. Young, C. T., Holley, K. T., Univ. of Georgia, Agr. Res. Sta. B u l l . 41, Experiment, G a . , 1965. Cort, W. M . , J. Amer. O i l Chem. Soc. (1974) 51, 321. Siddiqi, A. M . , Tappel, A. L., J. Amer. O i l Chem. Soc. (1957) 34, 529. D i l l a r d , M. G . , Henrick, A. S., Koch, R. B . , Food Res. (1960) 15, 544. St. Angelo, A. J., Ory, R. L., i n "Symposium: Seed Proteins," Inglett, G. E., E d . , Avi Publishing Co. Inc., Westport, Conn., 1972, p 284.

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

13.

ST. ANGELO ET AL.

18.

Sanders, T. H., Pattee, H. E., Singleton, J. Α . , Lipids (1975) 10, 681. Zimmermann, D. C., Biochem. Biophys. Res. Commun. (1966) 23, 398. Vick, Β. Α . , Zimmermann, D. C., Plant Physiol. (1976) 57, 780. St. Angelo, A. J., Dupuy, H. P., Ory, R. L., Lipids (1972) 7, 793. Karel, M . , Schaich, K . , Roy, R. Β., J. Agr. Food Chem. (1975) 23, 159. Matsushita, J., J. Agr. Food Chem. (1975) 23, 150. St. Angelo, A. J., Ory, R. L., J. Agr. Food Chem. (1975) 23, 141. St. Angelo, A. J., Ory, R. L., Peanut S c i . (1975) 2, 41. Ericksson, C. E., J. Agr. Food Chem. (1975) 23, 126. St. Angelo, A. J., Kuck J. C., Amorim H L., Amorim H V . Ory, R. L., J. Amer

19. 20. 21. 22. 23. 24. 25. 26. 27.

Oxidative Stability of Peanut Products

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14 Enzymes in Soybean Processing and Quality Control JOSEPH J. RACKIS Northern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, Peoria, Ill. 61604

Urease, lipoxygenase are used to measure the degree of heat treatment which is a primary determinant of the functionality of soy proteins. Lipoxygenase, i n raw soy flour, can replace chemicals to improve dough and crumb quality and bleach pigments during breadmaking. This is the only commercial application of the enzymes i n soybeans. The functional importance of lipoxygenases are associated with the formation of lipohydroperoxides coupled with secondary oxidation of the hydroperoxides. Enzyme-active soy flours are used at the 0.7 to 1% level (wheat-flour basis). At higher levels, lipoxygenase a c t i v i t y is responsible for the formation of objectionable flavors i n food systems. Enzymes are assuming greater importance i n modifying the functionality of soy proteins. Proteolytic modification shows promise i n development of a new generation of modified soy proteins for food use. Pepsin-hydrolyzed protein isolates are used as whipping agents for egg white replacement. Another development that may have application i n food processing is the "plastein reaction." Proteolytic enzymes are used to p a r t i a l l y hydrolyze soy protein isolates, and then the hydrolyzate i n the presence of supplemental amino acids is incubated with enzymes to reverse the process and reconstitute protein-like substances (plasteins) with different composition and functional properties. The use of alpha-galactosidases to eliminate soybean flatulence by hydrolysis of the oligosaccharides, raffinose and stachyose, has been described. Soybean protein products are ingredients widely used in processed foods and compete with other protein sources. Economic factors and unique functional properties have helped to establish present markets for soybean proteins. Properties of the proteins have been altered by processing such as heat treatment, alcohol extraction, and texturization by extrusion. Enzyme treatment affords the possibility to develop a broader spectrum of functional properties. 244 In Enzymes in Food and Beverage Processing; Ory, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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A functional property of a food ingredient is one that imparts a desirable change or contributes some favorable quality to a fabricated food during processing or in the finished product, A functional ingredient should also inhibit factors that create undesirable changes with a subsequent loss in quality. Functionality includes: nutritional value, flavor and color characteristics, protein solubility, water-fat absorption, texture, improved baking performance, and regulation of enzyme activity. The present paper will be concerned with the use of soybean enzymes in food processing and the application of other sources of enzymes to modify protein and carbohydrate constituents in soybeans. Measurement of enzyme activity as a quality control specification for the manufacture of edible-grade soy protein products will also be discussed. Enzyme-Active Soy Flour A f u l l range of full-fat and defatted soy flours are used in processed foods (1). These products have widely different functional properties dependent upon the temperature, time, and moisture conditions used in their manufacture. Such moist heat treatment is referred to as toasting. Enzyme-active full-fat and defatted soy flours are produced by omitting the toasting operation. Raw soy flour is a good source of enzymes, including amylases, lipases, urease, and lipoxygenases. However, the lipoxygenase enzyme system is the only one of conmercial importance to the food processing industry. Raw soy flour is one of the richest sources of lipoxygenase. Lipoxygenase. The importance of enzyme-active soy flour is its use as a bleaching agent and dough improver for the production of breads, buns, and other fermented bakery goods. The lipoxygenases are the functional components of these flours. Lipoxygenase (E.C. 1.13.1.1.3) catalyzes oxidation of unsaturated fatty acids in lipids containing cis,cis-l,4pentadiene system by molecular oxygen to hydroperoxides. Linoleic and linolenic acids are the most conmon fatty acid moieties in unsaturated lipids that act as substrates. The name lipoxidase was first coined by Andre and Hou (2) for an enzyme in soybeans that oxidized fat, but now the preferred name is lipoxygenase. Soybeans contain multiple forms of lipoxygenase (isoenzymes) as well as a complex system of other enzymes that further decompose lipid hydroperoxides. The decomposition of the lipohydroperoxides can be classified into six characteristic pathways (3). Although Kies et a l . (4) conclude that carotene oxidase and lipoxygenase are two

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distinct enzymes, i t appears that one of the lipoxygenase isoenzymes possesses similar activity. The importance of lipoxygenase is in coupled secondary oxidations which have a desirable effect in breadmaking. Hydroperoxides are also known to damage membranes, affect biological functions, oxidize protein sulfhydryl groups (5), and produce undesirable grassy/beany flavors (6). Therefore, the level and extent of lipoxygenase activity should be carefully regulated when used in a food system. Lipoxygenase in Breadmaking. Cotton (7) has reported that about / l million pounds of full-fat ancT defatted soy flour is used in baked goods in the united States. However, most of the flours are processed with moist heat which readily destroys enzymes and the primary use of these flours is for functional purposes, other than that associated with enzymatic activity. Th produced in the united A series of patents by Haas and Bohn (8) and Haas (9) describe a process for the preparation of a stabilized agent by mixing raw defatted soy flour with 4 parts of corn flour. The addition of 0.75 to 2% of the corn-soy mixture (based on wheat flour weight) is sufficient for bleaching and dough improvement. Higher amounts can lead to flavor problems in bread. Detection levels are summarized in Table I. Flavor problems in bread occur when enzyme-active soy flour is incorporated at higher levels than that required to improve dough characteristics in order to increase the protein content for nutritional purposes (10). Table I. Flavor Detection Level of Soy Flours in Bread Detection level of soy, Workers Haas (1934) Finney et al. (1950) Ûfelt et al. (1952) Ehle ancT Jahsen (1965) Cotton (1974)

0. Ό

>0.4 <4 >5 >4 2

Wolf (10) In Europe, production of enzyme-active flour has become a specialized business (11). There, raw full-fat soy flour is used as a dough improver in the production of bread by two

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processes: (a) traditional fermented bread and (b) mechanically developed bread. Production figures are not available, but i t is estimated that over 90% of the bread produced in the United Kingdom contains soy flour (12) and that i t is rare to find a bread recipe in England wKTch does not contain enzyme-active soy flour (11). Lipoxygenase Function in Bread. In the traditionally fermented oread process, oxiclative reactions of lipohydroperoxides generated by the soy lipoxygenase enzyme system are reported to bleach wheat pigments, condition and/or oxidize gluten for maximum softness of crumb, reduce the rate of staling, decrease binding of added shortening, and reduce production costs (12). Because of its high protein content, enzymeactive soy flour helps to retain water after baking, thereby keeping the loaf soft and fresh longer The recommended level 0.7% i.e., 2 lb per sac 2 times its weight in extra water. There are also commercially available, composite bread improvers based on soy flour incorporating yeast foods and crumb softening agents. Soy lipoxygenase activity also exerts a beneficial effect on the quality of bread made by the mechanically developed bread process. The bulk fermentation time is replaced by intense mechanical action, referred to as the Chorleywood bread process. A high level of oxidizing agent is required (75 ppm of ascorbic acid) and an addition of 0.7% triglyceride shortening. The optimal level of enzymeactive full-fat soy flour is 0.7% based on flour weight. The beneficial effect of 0.7% enzyme-active soy flour added to a Standard Chorleywood recipe is illustrated in Figure 1. Loaves containing soy flour become softer even after 50-hr storage. Many other advantages are obtained with soy additives (12). Soy lipoxygenase reduces the binding of added shortening with protein during dough mixing (13), increases gluten strength (14), and improves baking performance and keeping quality (lZJ. These effects are also attributed to the coupled peroxidation of polyunsaturated lipids mediated through the formation of hydroperoxides (13). In the presence of air, high-energy mixing leads to a release of bound lipid through a lipoxygenase-coupled oxidation of lipid binding sites of the proteins in the dough. Fullfat enzyme-active soy flour is a valuable source of enzymes required for this oxidation. The lipoxygenase present in wheat flour, in common with purified preparations of soybean lipoxygenase (3), is specific for free linoleic and linolenic fatty acids. However, soy flour contains several lipoxygenases which oxidize free and esterified fatty acids. American Chemicaf Society Library 1155 16th St. H. W. Washington, D. C. 20036 In Enzymes in Food and Beverage Processing; Ory, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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Figure 1. Effect of enzyme-active, full-fat soy flour on softness and storage stability of bread: Curve A, standard Chorleywood process; Curve B, standard plus soy flour (12)

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Results of studies (13) which differentiate wheat and soy flour lipoxygenases in their effect on lipid binding are summarized in Table II. In the absence of free linoleic acid, neither the wheat flour lipoxygenase nor the purified soy lipoxygenase were able to induce the coupled oxidation required to release bound lipid in an air-mixed dough. About 801 of the total lipid remained bound. However, with the addition of a raw soy flour, capable of oxidizing free and esterified linoleate, the amount of lipid binding was greatly reduced. In the presence of added free linoleic acid, maximum release of bound lipid occurs with both the crude or purified soy lipoxygenase extracts. Wheat lipoxygenase was much less effective. When soy flour is added to dough, there is maximum oxidation of linoleic and linolenic acids in a l l the lipids of wheat flour. In control doughs wheat lipoxygenase oxidized only free fatt Table II. Effect of Soy Lipoxygenases on Lipid BindingBound lipid, % of total lipid Crude soy lipoxygenase

Purified soy lipoxygenase

Added enzyme

None

Shortening fat alone

81.0

58.5

80.0

Shortening plus 5% linoleic acid

66.5

39.0

36.2

%at-extracted flour supplemented with triglyceride shortening either alone or with 5% linoleic acid. Daniels et al. (13).

Kleinschmidt et al. (17) have developed a process which uses lipoxygenase activity in soy flour to develop flavor in continually mixed bread processes. A dispersion of enzymeactive defatted soy flour and peroxidized cottonseed or soybean o i l is added to the liquid ferment during baking. When used at the 0.51 level (based on flour), an o i l with a peroxide value of 0.03 moles per kilogram of fat is required to produce an acceptable wheaty/nutty flavor. Depending upon conditions used in baking, additional flavors are generated by a Maillard-type reaction.

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Enzymes in Soybean Processing Precise control of heat treatment is critical in that the degree of enzyme inactivation, improvement in nutritive value, and other functional properties of soy protein products depend upon temperature, time, and moisture conditions (1). A number of official and unofficial methods have been used to evaluate the degree of heat treatment which is the primary determinant of functionality. Some of the measurements used for the control of processing are summarized in Table III. Table III. Property Measurement Used to Control Processing of Edible Soy Flour Property affected Control Nutritive measurement Flavor value Lipoxygenase Peroxidase NSIPDI Urease TI Available lysine

+ + -

+ + + + +

— NSI = nitrogen solubility index, PDI = protein dispersity index, and TI = trypsin inhibitor. Rackis et al. (18). Protein Dispersibility Index (PDI) is the percent of total protein (Ν X 6.25) dispersed under control conditions of extraction and measures the effect of heat treatment on protein solubility (AOCS method BA10-65 (19). Nitrogen Solubility Index (NSI) is the percent of total nitrogen which is soluble in water under controlled conditions of extraction, AOCS method BA11-65 (19). Live Steam Process. The PDI of raw unprocessed soybean meal is in the range of 90 to 95% and that of toasted products, 10 to 20%. A continuous spectrum of PDI values and of enzyme activity can be obtained through controlled heat processing (20). The relationship between changes in PDI and enzyme activity is shown in Table IV. Of the enzymes tested, lipoxygenase was the most sensitive and urease was the most resistant to heat processing. Urease has no effect on the functionality of edible-grade soy products. However, in feed manufacturing, measurement of urease activity is required to insure sufficient destruction of urease activity when soybean meal is used in animal rations containing urea.

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Table IV. Enzyme Activity in Soy Flour Treated with Moist Heat

Enzyme Lipoxidase Urease Diastase (beta) Lipase Protease

Negligible PSI80-100 + + + + +

Light cook PSI 50-80

Moderate cook PSI 20-50

Toasted PSI 0-20

_

_

_

+ + + +

+

-

-

—

—

- PSI = Protein Solubilit Lipoxygenase is an important factor in the generation of flavor compounds from the lipids when soybeans are processed under high-water activity such as in the preparation of soy beverages. To what extent lipoxygenase is responsible for the lipid oxidation at low moisture levels (8-14%) that occurs during processing of soybeans into defatted soy flour, concentrates, and isolates is s t i l l not known. A recent review discusses the relationship between lipoxygenase and flavor of soy protein products (10). Several processes have been usecTto inactivate lipoxygenase in soybeans (Table V). The essential condition in a l l of these processes is heat which inactivates enzymes and insolubilizes most of the proteins and generates a cooked or toasted flavor. Table V. Processes for Inactivating Lipoxygenase Process Grinding soybeans with hot water Dry heating-extrusion cooking Blanching Grinding at low pH-cooking Wolf (10)

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Alcohol Processes. Extraction with aqueous alcohol removes many objectionable flavors and is used commercially to prepare soy protein concentrates. Hexane-alcohol azeotrope extraction of defatted soy flakes can also be used to prepare soy flours and concentrates with flavor scores approaching the blandness of wheat flour (21). A process patent issued recently incorporates both hexane-ethanol and aqueous alcohol extraction to prepare soy products with flavor qualities significantly higher than those prepared by present commercial practices (22). Effect of azeotrope extraction on NSI, lipoxygenase, and peroxidase activity is given in Table VI (18). Over 99% of lipoxygenase activity was destroyed during azeotrope extraction at 56°C, whereas no destruction of peroxidase activity occurred. Since peroxidase is a more stable enzyme, measurement of it activity rathe tha f lipoxygenas activity, may have greate for production of blan y products , peroxidas is a very active enzyme capable of utilizing lipohydroperoxides which can be produced even in the absence of lipoxygenase activity (23). As shown in Table VII, peroxidase activity is much higEer at pH 5.0 than 6.5. Table VI. Enzyme Activity and Nitrogen Solubility of Processed Soy Flakes

Processing conditions NSI Hexane-defatted, raw Azeotrope-extracted (3 hr) Azeotrope - extracted (6 hr) Hexane-defatted, toasted Azeotrope-extracted (3 hr) (toasted) Azeotrope-extracted (6 hr) (toasted)

92.9

Lipoxygenase, ymoles 0 /min/g ?

249

Peroxidase— 1875

88.8

1.8

1585

83.5

0.4

2098

41.3

0.5

244

41.0

—

31.2

0.3

— Absorption units/g, at pH 5.0.

—

70 Rackis et al_. (18).

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Table VII. Peroxidase Activity in Maturing Soybeans Days after flowering

Fresh weight, mg

Dry matter, %

35 40 42 44 48 51 55 68 72 Dehulled, defatted flakes

283 376 452 470 516 577 500 300 235

25.8 31.3 35.3 36.8 37.4 39.6 44.6 74.5

ActivitypH 5.0 pH 6.5

46.2 40.5 42.1 45.2 48.5 48.5 44.0

0.67

14.2 16.4 13.5 18.9 17.4 15.6 16.1 12.1

0.22

-3 — Absorption units per g dry matter X 10 Rackis et al. (23). The in situ inactivation of enzymes in soybeans that are wet-milled or steeped in various concentrations of aqueous alcohol is shown in Figure 2. Less than 1% of the original lipoxygenase activity remains after soaking soybeans in 30 to 801 aqueous ethanol at room temperature (24). Over 70% of the urease activity was destroyed by soaking the beans in 40 to 601 aqueous alcohol. Maximum flavor scores are obtained when soybeans are treated with 40 to 60% alcohol. Lipoxygenase and peroxidase activities of soybeans as related to the beany and bitter flavor intensity values (FIV) of maturing soybeans are illustrated in Figure 3 (23). The beany intensity (Curve D) did not change much with increasing maturity as indicated by the small differences in FIV which varied from 2.0 to 2.7. A different pattern was observed for the bitter flavor (Curve C). The average FIV for bitter was 0.4 in the early stages of maturity. As the beans matured, the FIV for bitter increased to 1.6, an increase of about fourfold. There is a correlation (r = 0.73), significant at the 1% level, between lipoxygenase activity (Curve A) at pH 6.8 and the increase in bitter FIV (Curve C) in maturing soybeans. Lipoxygenase activity measured at pH 9.0 remained relatively constant and at a level much below that at pH 6.8 during maturation. Activity at pH 6.8 represents the isoenzyme, lipoxygenase-2, which oxidizes fatty acids in triglycerides as well as other lipids (3).

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

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0

20

40

60

80

100

Ethyl alcohol. % Cereal Chemistry

Figure 2. Effect of soaking soybeans for 24 hr in various aqueous ethanol solutions on urease, lipoxygenase, Nitrogen Solubility Index (NSI), and flavor score. Curve A, lipoxygenase; Curve B, urease; Curve C, NSI; and Curve D ,flavorscore (24).

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The level of peroxidase activity (Curve B) in maturing soybeans is. very high and remains at a relatively constant level throughout most of the maturation period. Based on our studies of maturing soybeans, we conclude that beany and bitter constituents preexist in immature soybeans. Whether the development of bitterness results from a coupled secondary oxidation reaction involving lipoxygenase and peroxidase remains to be determined. Enzyme-Modified Soy Products Proteolytic Modification. Although commercial soy protein products can f u l f i l l a number of functional requirements in present markets for protein additives, enzymatic hydrolysis is also used to create functional properties for specific applications. Pepsin-digeste or in combination with agents in confections, desserts, candies, and cakes, and as foaming ingredients in foam-mat drying of citrus powders (1). Pepsin-hydrolyzed soy isolates are approved as foaming agents in identity standards for soda water. Patents have been issued for enzyme-modified soy products as egg white replacers (25, 26). The use of enzyme treatment to increase solubility 5Γ heat denatured soy isolates (27) and to increase solubility in acid solutions have been described (28). Modification of the functional properties of soy isolates with other proteinases has been investigated (29, 30). Enzymatic modification increased calcium tolerance, water absorption, emulsification, capacity, and foaming properties. However, emulsification and foaming stabilities were reduced. These functional properties were determined in model systems; usefulness of enzyme-modified soy isolates in actual food systems is yet to be evaluated. Carbohydrate Modification. Raffinose, stachyose, and verbascose cause flatulence in man and animals (31). These oligosaccharides are related by having one or more a-ggalactopyranosyl groups, where the α-galactose units are bound to the glucose moiety of sucrose. α-β-Galactooligosaccharides escape digestion because there is no α-galactosidase activity (E.C. 3.2.1.22) in mammalian intestinal mucosa and because they are not absorbed into the blood. Consequently, bacteria in the lower intestinal tract metabolize them to form large amounts of carbon dioxide and hydrogen. Rate of gas production parallels the formation of monosaccharides resulting from enzymatic breakdown of the oligosaccharides and their intermediate products (Figure 4).

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

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50 45

© © Curve Β Θ -

/

e

GO CO

α» OA

*

/ \ 11.4 Z>

_L

25

35

_L

J_

45 55 Days after flowering

65 Cereal Chemistry

Figure 3. Relationship between lipoxygenase and peroxidase activities and Flavor Intensity Values (FIV) for bitterness and beaniness in maturing soybeans. Curve A, lipoxygenase; Curve B, peroxidase; Curve C, FIV, bitterness; and Curve D, FIV, beaniness (23).

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Enzymatic processes that hydrolyze the oligosaccharides in full-fat and defatted soybean meal have been patented (32, 33). Use of immobilized enzyme systems to continuously convert raffinose and stachyose to monosaccharides has been described (34, 35, 36). As shown in Table VIII, about 4 hr was require3""to compTetely hydrolyze the oligosaccharides in soybean whey (36). Soybean oligosaccharides are also metabolized by lactic acidTacteria during the preparation of feraiented soy milk (37). Plastein Products. When soybean protein is partially hydrolyzed by proteinases (peptidyl-peptide hydrolases E.C. 3.4.4) and the hydrolyzate is then incubated with certain proteolytic enzymes under proper conditions, the hydrolysis is reversed and a "protein-like" substance is formed whose propertie original protein. The ne plastein. The plastein reaction requires at least three specific conditions which are different from those for proteolysis (38). These conditions are summarized in Table IX. In addition, Tow molecular weight peptides are effective substrates for the plastein reaction. Of especial importance is the ratio of hydrophobic-hydrophilic groups of the peptide substrate (39). Arai et al_. (38) define plastein formation as a condensationtransesterïFication reaction giving rise to products of higher molecular weight. Other workers (40) have found that only slight changes in molecular weight distribution occur during plastein formation with enzymatic hydrolyzates of milk whey protein concentrates and fish protein concentrates. Apparently, transpeptidation and hydrolysis had a more important role than condensation reactions during plastein formation. Alternate theories for plastein formation have been reviewed (41). M

ft

Food Applications. Although the mechanism of the plastein reaction is s t i l l not well understood, plastein proteins have interesting properties which may be of value in food processing technology. In a series of research reports, Fujimaki and coworkers conclude that the plastein reaction can be used to prepare bland protein-like substances free of a host of other impurities from crude soy protein isolates (38, 42) and single cell protein concentrates (43). A schematic diagram for preparing purified plastein from a crude protein material is illustrated in Fig. 5.

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

E N Z Y M E S IN FOOD AND

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Table VIII. Glucose Production from Soybean Whey— by A. awamori a-galactosidase— Immobilized in an Amicon Hollow-Fiber Dialyzer— Time, hr 0.5 1.0 2.0 3.0 4.0 4.5 4.5 + solubl

Glucose, mg/ml

,

0.31 0.40 0.44 0.49 0.58 0.62

- Soybean whey (18 liters) was equivalent to II soybean slurry. - Crude enzyme (900 ml) at 25 U/ml. - At 45°C, recirculation rate was 185 ml/min. - Consisted of 10 ml of the 4-5 hr sample plus 2.5 U of crude a-galactosidase. Incubated overnight at 25°C. Smiley et al. (36).

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Time, hr. ACS Symposium Series

Figure 4. Relationship between the enzymatic hydrolysis of stachyose and gas production. Anaerobic culture isolated from the dog colon. Curve A , stachyose; Curve B , disaccharides and trisaccharides; Curve C , monomer sugars; and Curve D , gas production (31).

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Crude Protein |-Containing impurities in bound state Hydrolysis with endopeptidase Hydrolyzate

Impurities in free state

Solvent extraction Impurities

Purified Hydrolyzate Synthesis with endopeptidase Plastein Product Extraction with aqueous ethanol

X

Bland, Colorless Plastein Product

1

Peptides, Amino Acids, Other Components Cereal Foods World

Figure 5.

Process for preparing purified, bland plastein products (38)

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

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Table IX. Differences in Reaction Conditions Between Protein Hydrolysis and Plastein Synthesis Condition

Hydrolysis

Synthesis

Substrate Concentration, % pH for acidic enzymes— SH enzymesAlkaline enzymes—

Polypeptide <5 1-4 4-7 7-10

Oligopeptide 30-50 4-5 4-6 5-6

pH range ES-ratio Temperature, °C Period, hr

Range 1-10 1:100-3:100

4-6 1:100-3:100

24-48

24-96

— g pepsin: pH 1.6 in hydrolysis and pH 4.5 in synthesis. — g papain: synthesis.

pH 5.5 both in hydrolysis and in

— g α-chymotrypsin: pH 7.8 in hydrolysis and 5.3 in synthesis.

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

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

1.18 -------

7.98 ------0.38 2.40 0.20

Hydrolyzate

9.71 -----

9.23 ---

2.14

Zein ^_ Amino acid enriched plastein TryThrLys-

Arai et a l . (38).

— Amino acid composition based on a weight percent. Consult source for changes in composition of its other amino acids for both hydrolyzates and plasteins.

Methionine (Met) Tryptophan (Try) Threonine (Thr) Lysine (Lys)

Amino acid

Soy protein isolate Met-enriched Hydrolyzate plastein

Table X. Amino Acid Enrichment Plasteins Prepared from These Pepsin-Hydrolyzed Substrates-

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In the hydrolysis phase, some of the low-molecularweight flavor constituents and other impurities are removed by selective solvent extraction. Two methods are then used to remove the bitterness of the purified hydrolyzate: (a) further hydrolysis with an exopeptidase and (b) synthesis of plastein protein with an endopeptidase. Various enzymes have been used to debitter crude soy protein (42). The economic feasibility of such a process remains to be evaluated. In addition, no organoleptic evaluations have been made in comparing the acceptability of plastein soy protein and of protein isolates prepared.from hexane :ethanol azeotropeextracted soy flakes (21). The use of the plastein reaction to improve the nutritional value of low-quality proteins by enrichment with essential amino acids has also been proposed (38). By incubating soy protein hydrolyzates with L-methionin ethyl ester th methionine content of th nearly sevenfold (Table IX) Similarly, ethy tryptophan, L-threonine, and L-lysine were used to enrich the essential amino acid content of zein plastein protein. Glutamic acid enrichment was used to form a soy plastein that exhibited high water solubility in the pH range of 113. Arai et a l . (38) illustrates the differences in solubility of glutamic acid-enriched soy plastein and soy protein isolate. Such a product may find application in food processing where protein solubility in acid solutions is a requirement. Presently, only enzymatically hydrolyzed soy protein has such a solubility characteristic (28, 29). Peptide gels with interesting~~rheological properties have been prepared (42). The plastein process may prove useful in preparing products having tailored compositional and functional properties, however, nutritional and consumer labeling problems are some of the many special considerations that will need to be evaluated (43). Literature Cited 1.

2. 3. 4.

5.

American Soybean Association. Proc. World Soy Protein Conf., Munich, Germany, November 11-14, 1973, J. Am. O i l Chem. Soc. (1974) 51, 49A. Andre, Ε., Hou, Κ., C. R. Acad. S c i . (1932) 194, 645. Gardner, H. W., J. Agric. Food Chem. (1975) 23, 129. Kies, M. W., Haining, J. L., Pistorius, E., Schroeder, D. H . , Axelrod, B . , Biochem. Biophys. Res. Commun. (1969) 36, 312. American Chemical Society, Symposium on Effects of Oxidized Lipids on Food Proteins and Flavor, J. Agric. Food Chem. (1975) 23, 125.

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6. 7. 8. 9. 10. 11. 12. 13. 14.

15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31.

32.

ENZYMES IN FOOD AND BEVERAGE PROCESSING

Kalbrener, J. E., Warner, K . , Eldridge, A. C., Cereal Chem. (1974) 51, 406. Cotton, R. H., J. Am. Oil Chem. Soc. (1974) 51, 116A. Haas, L . W., Βοhn, R. M. (to J. R. Short Milling Co.) U.S. Patents 1,957,333 and 1,957,336 (May 1, 1934). Haas, L . W. (to J. R. Short M i l l i n g Co.) U.S. Patents 1,957,334 and 1,957,337 (May 1, 1934). Wolf, W. J., J. Agric. Food Chem. (1975) 23, 136. Pringle, W., J. Am. Oil Chem. Soc. (1974) 51, 74A. Wood, J. C., Food Manuf. Ingredient Survey (January 1967) 11-15. Daniels, N. W. R . , Frazier, P. J., Wood, P. S., Baker's Dig. (1971) 45(4), 20. Frazier, P. J., Leigh-Dugmore, F . Α., Daniels, N. W. R., Russell-Eggitt, P. W., Coppock, J. B . , J. S c i . Food Agric. (1973) 24, Mann, D. L., Morrison Morrison, W. R . , Panpaprai, R., Ibid. (1975) 26, 1225. Kleinschmidt, A. W., Higashiuchi, K . , Anderson, R., F e r r a r i , C. G., Baker's Dig. (1963) 37(5), 44. Rackis, J. J., McGhee, J. E., Honig, D. H., J. Am. O i l Chem. Soc. (1975) 52, 249A. American O i l Chemists' Society, "Official and Tentative Methods," 3rd E d . , The Society, Chicago (1973). Hafner, F . Η . , Cereal S c i . Today (1964) 9, 164. Honig, D. Η . , Warner, Κ. Α., Rackis, J. J., J. Food Sci. (1976) 41, 642. Hayes, L . P., Simms, R. P. (to A. E . Staley Mfg. Co.) U.S. Patent 3,734,901 (May 22, 1973). Rackis, J. J., Honig, D. H., Sessa, D. J., Moser, H. Α., Cereal Chem. (1972) 49, 586. Eldridge, A. C., Warner, Κ., Wolf, W. J., Cereal Chem. (In press). Turner, J. R . , U.S. Patent 2,489,208 (1969). Gunther, R. C., Canadian Patent 905,742 (1972). Hoer, R. Α., Frederiksen, C. W., Hawley, R. L., U.S. Patent 3,694,221 (1972). Pour-El, Α., Swenson, T. C., U.S. Patent 3,713,843 (1973). Puski, G., Cereal Chem. (1975) 52, 655. Pour-El Α., Swenson, T. C., Cereal Chem. (1976) 53, 438. Rackis, J. J., American Chemical Society Symposium Series, Number 15 (1975), Edited by A. Jeanes and J. Hodge, Chapter 13, 207. Ciba-Giegy, A. G., French Patent 2,137,548 (1973).

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

14. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43.

RACKIS

Soybean Processing and Quality Control

Sherba, S. Ε., U.S. Patent 3,632,346 (1973). Delente, J., Johnson, J. Η . , O'Connor, M. J., Weeks, L. Ε., Biotechnol. Bioeng. (1974) 16, 1227. Reynolds, J. H., Ibid. (1974) 16, 135. Smiley, K. L., Hensley, D. E., Gasdorf, H. J., Appl. Environ. Microbiol. (1976) 31, 615. M i t a l , Β. Κ., Steinkraus, Κ. H., J. Food S c i . (1975) 40, 114. Arai, S., Yamashita, M . , Fujimaki, M . , Cereal Foods World (1975) 20(2), 107. A r a i , S., Yamashita, M . , Aso, K . , Fujimaki, M . , J. Food Sci. (1975) 40, 342. Hofsten, B . , Lalasidis, G . , J. Agric. Food Chem. (1976) 24, 460. Eriksen, S., Fagerson I S. J. Food S c i (1976) 41 490. Fujimaki, Μ., Yamashita B i o l . Chem. (Japan) (1970) 34, 483. Fujimaki, Μ., Kato, Η . , A r a i , S., Yamashita, M . , J. Appl. Bacteriol. (1971) 34, 119.

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15 Enzyme Methods to Assess Marine Food Quality FREDERICK D. JAHNS and ARTHUR G. RAND, JR. Department of Food Science and Technology, University of Rhode Island, Kingston, R.I. 02881

How fresh is a fres we can i l l u s t r a t e this point! In examining these f i s h , espec i a l l y the one at the bottom, one might conclude "this fish is putrid." However, this evaluation would be incorrect. This fish may not be fresh, but i t has been stored in ice at 0°C for only a few days and certainly is not spoiled. The belly is a l most completely digested in the lower fish but this fish is 6 days old postmortem. It is also possible to have fish only several hours postmortem in the same condition, because they weren't properly iced. While the tissue in the belly region on the lower fish was digested, a cooked fillet of this fish could be quite acceptable to many people. Note that the fish at the top of the picture was caught at the same time and was stored under the exact same conditions as the one at the bottom, yet somehow their appearance is different. It is this type of example that contributes to the elusiveness of evaluating the quality of seafood, particularly in trying to distinguish the fresh and edible stages. The inedible phase is usually quite clear to anyone. There have been many o r g a n o l e p t i c g u i d e l i n e s s e t f o r judgment o f fish q u a l i t y such as t e x t u r e , appearance, c o l o r , eye transparency, and odor. In many cases t h i s approach may give a general i n d i c a t i o n of q u a l i t y , but they are very s u b j e c t i v e (1)

(2). R e a l i z i n g the drawbacks i n using these criteria, and understanding the economic impact o f properly assessing seafood q u a l i t y , many i n v e s t i g a t o r s have set out to f i n d a more r e l i a b l e means. The most l o g i c a l approach was to view the issue through chemical t e s t s . In general most of the chemical t e s t s appear to have v a r i a b l e success, with l i m i t a t i o n s being placed on them by innate b i o l o g i c a l d i f f e r e n c e s from species to s p e c i e s . Some compounds such as trimethylamine, v o l a t i l e nitrogen bases, ammonia, and volatile reducing substances appear mostly i n the l a t t e r stages o f r e f r i g e r a t e d storage, which l i m i t s t h e i r value in that considerable period p r i o r to t h i s time, as i n d i c a t e d by 266 In Enzymes in Food and Beverage Processing; Ory, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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Dugal (3). There are a number o f other techniques, such as an automated determination o f volatile bases (4), the Torry Fish Freshness Meter and the I n t e l e c t r o n Fish Tester (5)(6)(7) which have attempted t o s i m p l i f y q u a l i t y assessment and have met with some success. However, they are not without problems. For example, the Torry meter seems t o be l i m i t e d t o lean f r e s h fish. Frozen or f a t t y f i s h produce somewhat v a r i a b l e r e s u l t s (8). The automated determination o f volatile bases requires a l a b o r a t o r y situation. The c r i t e r i a o u t l i n e d by Shewan & Jones (9) continues t o be a sound b a s i s f o r a marine food q u a l i t y index. The method s e l e c t e d should detect a compound which: 1. W i l l be absent o r present i n constant amount i n f r e s h l y caught food; 2. Should accumulate o r disappear q u i c k l y at a steady rate during post-harvest aging and s p o i l a g e ; 3. Has the c a p a b i l i t estimated. Enzyme methods which have the advantages o f s p e c i f i c i t y and r a p i d i t y meet t h i s basis q u i t e w e l l . The f i r s t enzyme methods to assess marine food q u a l i t y were reported i n 1964 (10)(11); t h i s was an a n a l y s i s f o r the compound hypoxanthine, a metabolic byproduct o f ATP breakdown, employing an enzyme method developed almost two decades before by Kalckar (12). The i n i t i a l work u t i l i z e d ion exchange chromatography to separate the n u c l e o t i d e bases i n a f i s h t i s s u e e x t r a c t . This enzyme technique then employed xanthine oxidase (EC 1.2.3.2) to convert the hypoxanthine f r a c t i o n t o u r i c a c i d , followed by d i f f e r e n t i a l spectrophotome t r y i n the UV f o r a n a l y s i s . There has been ample demonstration of the usefulness o f hypoxanthine as a measure o f f i s h q u a l i t y

in several papers (3)(10)(13)(14)(15).

Burt e t a l . (16) proposed a method o f automating the xant h i n e oxidase r e a c t i o n f o r hypoxanthine a n a l y s i s using the redox dye 2,6 dichlorophenolindophenol (DIP). This placed the method on a c o l o r i m e t r i c b a s i s , s i m p l i f i e d the equipment requirements, and increased sample c a p a c i t y . Figure 2 i l l u s t r a t e s the i n t e r a c t i o n between hypoxanthine, DIP, and xanthine oxidase. The hypoxanthine concentration can be r e l a t e d to the extent o f dec o l o r i z a t i o n o f the dye. The c o r r e l a t i o n between the automated c o l o r i m e t r i c methods, measuring at 600 nm using DIP, and the manual enzyme method measuring u r i c a c i d appearance a t 290 nm was e x c e l l e n t (16). Beuchat Q5) u t i l i z e d DIP i n a manual enzyme assay t o measure hypoxanthine d i r e c t l y i n a deproteinated e x t r a c t , and c o r r e l a t e d t h i s method with an o r g a n o l e p t i c e v a l u a t i o n o f c a t f i s h (Ictalurus punctatus). Jahns e t a]_. (17) a l s o used a s l i g h t l y modified xanthine oxidase-DIP method t o monitor hypoxanthine concentration i n iced Winter Flounder (Pseudopleuronectes americanus). A steady increase i n hypoxanthine concentration occurred with storage time u n t i l the values l e v e l e d o f f a t about 5 yM/g a f t e r 10 days where o r g a n o l e p t i c e v a l u a t i o n i n d i c a t e d 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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Figure 1. The appearance of red hake after six days of storage in ice (0°C)

DIP reduced

DIP oxidized BLUE Figure 2.

The reduction of DIP by xanthine oxidase in the presence of hypoxanthine

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f i s h were no longer f r e s h . Semi-automated hypoxanthine

analysis

The manual xanthine oxidase c o l o r i m e t r i c method has been f u r t h e r modified f o r quick routine a n a l y s i s in the laboratory by employing an automatic p i p e t and a Spectronic 20 c o l o r i m e t e r , o u t f i t t e d with a f l o w - t h r u c u v e t t e . B r i e f l y , the method i s as follows: 1. A 10 g sample of f i s h i s blended f o r one minute with 90 ml of c o l d 0.6N p e r c h l o r i c a c i d . 2. The homogenate i s then f i l t e r e d through Whatman #2 paper to remove p r o t e i n and t i s s u e d e b r i s . 3. A 10 ml a l i q u o t of the f i l t r a t e i s n e u t r a l i z e d to pH 7.6 with 20% Κ0Η and buffered with 0.1M Phosphate b u f f e r , pH 7 . 6 , making the volume up to 100 ml 4. Approximately This i s c h i l l e to form at the top of the tube. 5. The sample i s then c e n t r i f u g e d i n a c l i n i c a l c e n t r i f u g e f o r approximately 3 minutes to remove KC10-. 6. The c e n t r i f u g a t e i s poured o f f and allowed to temper to 25°C. One ml i s used in the a n a l y s i s f o r hypo xanthine, and contains 10 mg of f i s h . 7. A 4 ml p o r t i o n of buffered xanthine oxidase-DIP s o l u t i o n at 25 C i s i n j e c t e d i n t o the 1 ml sample of f i s h e x t r a c t with an automatic p i p e t . The enzyme reagent i s comprised of 60 ml of 0.15M phosphate b u f f e r (pH 7.6) containing DIP @ 23 yg/ml, and 20 ml of xan thine oxidase s o l u t i o n @ 0.02-0.025 units per ml. 8. The absorbance at 600 nm i s read in the f l o w - t h r u cuvette o f the Spectronic 20 at e x a c t l y 2.5 min. The absorbance change i s compared against a standard p l o t obtained by analyzing known hypoxanthine concentra tions. Employing t h i s method we were able to assess the hypoxan thine values found in Whiting (Merluccium b i l i n e a r i s ) during i c e d storage, as shown in Figure 3. Hypoxanthine was monitored over a 32 day p e r i o d , in order t o evaluate concentration well past the f r e s h and acceptable p e r i o d s . Levels of hypoxanthine increased to a maximum of 6 yM/g between 17 and 21 days. It then grad u a l l y declined by 32 days to s l i g h t l y above 2 yM/g. Figure 4 depicts the r e s u l t s obtained from two separate t r i a l s using Tautog (Tautoga o n i t e s ) . Both t r i a l s showed very s i m i l a r r e s u l t s e x h i b i t i n g maximum hypoxanthine values near 3 yM/g at from 12-14 days in i c e d s t o r a g e . T r i a l one was caught in November and T r a i l two was taken during December. The values again diminish a f t e r peak hypoxanthine formation.

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

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Figure 3. Hypoxanthine concentration in whiting during iced (0°C) storage. Each point represents the mean value for two fish.

TIME(DAYS)

Figure 4. Hypoxanthine concentrations in tautog during two iced (0°C) storage trials. Each point in trial one represents onefish,while trial two shows the mean value for two fish.

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Immobilized xanthine oxidase as an a n a l y t i c a l

tool

Another laboratory method f o r hypoxanthine a n a l y s i s , which i s s t i l l i n the p r e l i m i n a r y stages o f development, i s the use of an immobilized xanthine oxidase column. Very few reports are a v a i l a b l e on the immobilization o f xanthine oxidase. Coughlan and Johnson (18) f i r s t reported on methods of preparation d e s c r i b ing attachment to c e l l u l o s e , Sepharose and g l a s s . However, they u t i l i z e d these preparations only to study the f l a v i n chromophore of the enzyme. P e t r i H i (19) and Singer and Warchut (20), working i n our l a b o r a t o r y , studied xanthine oxidase immobilization i n acrylamide gel and on glass and D u o l i t e * beads as a method f o r measuring hypoxanthine c o n c e n t r a t i o n . The acrylamide gel entrapment demonstrated the p o t e n t i a l of immobilized xanthine oxidase f o r hypoxanthine a n a l y s i s but the enzyme was unstable during continuous a n a l y t i c a l use immobilization on Cornin beads, maximum enzyme attachment occurred i n a s h o r t e r time with the glass than the D u o l i t e . However, the D u o l i t e appeared to r e t a i n higher enzyme a c t i v i t y of the two supports, and thus was chosen to prepare an experimental plug flow r e a c t o r f o r hypoxanthine a n a l y s i s . The immobilized enzyme column was connected to a pump and a continuous flow UV analyzer equipped with a recorder as presented i n Figure 5. The buffered s u b s t r a t e , which could be e i t h e r a sample o f f i s h e x t r a c t , o r a known hypoxanthine s o l u t i o n f o r c a l i b r a t i o n , was pumped i n t o the column. The e q u i l i b r i u m absorbance value f o r the product of u r i c a c i d can be d i r e c t l y r e l a t e d to hypoxanthine c o n c e n t r a t i o n , as i l l u s t r a t e d , over a range o f 0-30 yg/ml. This enzyme d e r i v a t i v e e x h i b i t e d remarkable s t a b i l i t y when u t i l i z e d repeatedly over a period of weeks, and r e t a i n e d high a c t i v i t y during months of r e f r i g e r a t e d storage. This a n a l y t i c a l t o o l has great p o t e n t i a l i n r o u t i n e monit o r i n g o f f i s h and marine food q u a l i t y . The equipment and r e agent requirements are minimal i n comparison to s i m i l a r approaches ( 4 ) ( 1 6 ) . The automatic processing and recording of r e s u l t s allows the i n v e s t i g a t o r time t o prepare samples and e l i m i n a t e s constant manual operation which i s g e n e r a l l y involved i n the standard c o l o r i m e t r i c procedure. Diamine-oxidase as an i n d i c a t o r of marine food s p o i l a g e An enzymatic spoilage t e s t that would help to c l a r i f y the hypoxanthine freshness a n a l y s i s f o r a given sample of f i s h was investigated. S p i n e l l i e t a l . (10) compared the pattern of hypoxanthine formation i n f i s h with some other t e s t s , i n c l u d i n g Total V o l a t i l e Bases (TVB). T h e i r r e s u l t s i n d i c a t e d the TVB *Duolite i s a phenolic-formaldehyde r e s i n product of Diamond Shamrock Co.

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HYPOXANTHINE (/ig/ml) Figure 5.

Immobilized xanthine oxidase for the analysis of hypoxanthine

ο HYPOXANTHINE

^

2

4

6

8

10 12 14 16 18 20 22 24 26 28 30 32 TIME (DAYS)

Figure 6. Hypoxanthine concentration and diamine presence in red hake during iced (0°C) storage. Each point represents the mean value for two fish.

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values d i d not begin to measurably increase u n t i l the hypoxan t h i n e development had peaked. The composition of the TVB material undoubtedly includes diamine compounds, which are produced by m i c r o b i a l s p o i l a g e (2J.). A recent report by Mietz and Karmas (22) s u c c e s s f u l l y f r a c t i o n a t e d and quantitated the diamines from tuna by high-pressure l i q u i d chromatography. T h e i r r e s u l t s confirmed that p u t r e s c i n e , cadaverine, and h i s t a mine a l l increased in concentration as the f i s h reached unaccep table levels. Thus, a n o n - s p e c i f i c enzyme t e s t f o r diamines could i n d i c a t e i f these compounds were present and c l a r i f y whether the hypoxanthine a n a l y s i s was i n d i c a t i n g f r e s h or s t a l e fish. A method f o r determining diamines with the enzyme diamine oxidase (EC 1 . 4 . 3 . 6 ) coupled to the o - t o l i d i n e - p e r o x i d a s e c o l o r i m e t r i c system was developed. The procedure e s t a b l i s h e d i s as follows: 1. A 25 g sample 75 ml of c o l d 2. This i s f i l t e r e d through a Whatman #2 paper. 3. A 25 ml p o r t i o n of the f i l t r a t e i s adjusted to pH 8.5 with 20% KOH and made up to 50 ml with 0.1M borate b u f f e r , pH 8 . 5 . 4. About 12 ml i s placed in a c e n t r i f u g e tube and allowed to c h i l l in a f r e e z e r u n t i l a layer of i c e j u s t begins to form on t o p . 5. The c h i l l e d sample i s c e n t r i f u g e d f o r approximately 3 min in a c l i n i c a l c e n t r i f u g e . 6. The c e n t r i f u g a t e was poured o f f and allowed to temper to ambient temperature. Each 1 ml contains 0.125 g of fish. 7. To 1.0 ml of the c e n t r i f u g a t e , 2.0 ml of diamine o x i dase reagent i s added with an automatic p i p e t , and the mixture i s incubated at 37 C f o r 30 min. The diamine oxidase reagent c o n s i s t s o f - - 1 0 ml of diamine oxidase s o l u t i o n @ 4 units/ml of 0.1M borate b u f f e r (pH 8 . 5 ) , 5 ml of o - t o l i d i n e d i h y d r o c h l o r i d e s o l u t i o n @ 500 yg/ml o f water, and 5 ml o f peroxidase s o l u t i o n @ 226 units/ml of pH 8.5 borate b u f f e r (0.1M). 8. The c o l o r development i s determined on a Spectronic 20 Colorimeter equipped with a f l o w - t h r u c u v e t t e . The r e s u l t s are evaluated with a standard curve prepared from the a n a l y s i s of known putrescine c o n c e n t r a t i o n s . Since the purpose of the diamine t e s t was only to support hypoxanthine a n a l y s i s , the r e s u l t s have been presented only in terms of whether diamines were present or not. Figure 6 presents a graph comparing the pattern of hypoxanthine concentration and diamine values in iced Red Hake (Urophycis chuss). In Red Hake the hypoxanthine values increased to a peak of approximately 4 μΜ/g at 17-18 days followed by the usual downward t r e n d . Posi t i v e diamine values were found only in samples more than 18

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days o l d , or a f t e r the hypoxanthine peak. The hypoxanthine and diamine a n a l y s i s procedures were a l s o compared on iced Winter Flounder, as shown in Figure 7. The c h a r a c t e r i s t i c r i s e and drop in hypoxanthine concentration with storage time i s again evident. The peak value of 7.5 yM/g occurred at approximately 11 days postmortem. In a pattern almost i d e n t i c a l to Red Hake, diamines in winter flounder were detected a f t e r the storage time f o r peak hypoxanthine development. Thus, the diamine a n a l y s i s does show value as a s p o i l a g e i n d i c a t o r with the f i s h t e s t e d . Combining the two enzyme procedures, xanthine oxidase f o r hypoxanthine c o n c e n t r a t i o n , and diamine oxidase f o r diamine detection, can c l a r i f y the i n t e r p r e t a t i o n of f i s h q u a l i t y . Figure 8 i l l u s t r a t e s how these two t e s t s g e n e r a l l y i n t e r a c t . m

Rapid v i s u a l enzyme method The methods that hav quick q u a n t i t a t i v e a n a l y s i greatest need e x i s t s f o r a t e s t of marine food q u a l i t y that w i l l be simple, inexpensive, and lend i t s e l f to usage outside of the l a b o r a t o r y , e i t h e r on board s h i p , dockside, or in the food processing plant. Jahns et al_. (17) proposed a method f o r estimat i n g f i s h freshness which makes use of a s t r i p of absorbent paper impregnated with r e s a z u r i n , xanthine oxidase, and b u f f e r combination. When the enzyme t e s t s t r i p was moistened with flounder e x t r a c t s and allowed to react f o r 5 min at room temperature, the c o l o r change from blue to pink c o r r e l a t e d with the actual l a b o r a t o r y c o l o r i m e t r i c hypoxanthine a n a l y s i s of the same e x t r a c t s . This i s i n d i c a t e d in Figure 8 by the b l u e - p i n k - b l u e progression of c o l o r . As already i n d i c a t e d the hypoxanthine method i s b a s i c a l l y a freshness t e s t . I f the t e s t s t r i p was set to turn pink at a hypoxanthine concentration of 5 yM/g or 70 mg%, which would be an average value f o r the f i v e f i s h analyzed in t h i s study, then a simple c o l o r change from blue to a shade of pink could i n d i cate when f i s h can no longer be considered f r e s h . However, t h i s t e s t does not seem to be useful in estimating s p o i l a g e in s e a food. In the r e s u l t s obtained, the hypoxanthine concentration in a l l f i s h studied always begins to diminish a f t e r the f r e s h ness storage p e r i o d . This makes i t d i f f i c u l t , i f not impossible, to equate hypoxanthine concentration with storage time beyond 10-15 days in iced c o n d i t i o n s . In f a c t , the c o l o r changes from pink back toward b l u e , as the f i s h ages, could be m i s i n t e r p r e t e d . A second v i s u a l t e s t to i n d i c a t e spoilage would compliment the hypoxanthine enzyme t e s t s t r i p . Since diamines appear to develop a f t e r the hypoxanthine peak in a s i g n i f i c a n t q u a n t i t y , another v i s u a l t e s t could be employed on the same s t r i p as i n d i c a t e d in Figure 8. Essent i a l l y the hypoxanthine-diamine i n t e r a c t i o n can be d i v i d e d i n t o three c a t e g o r i e s , as i l l u s t r a t e d in Figure 9. Marine foods could

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

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

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

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be simply and q u i c k l y e v a l u a t e d , based on the r e a c t i o n c o l o r s . Class 1 would be the f r e s h food of highest q u a l i t y , where hypoxanthine and diamine l e v e l s are low, i n d i c a t e d as blue i n the t e s t zones. Class 2 would be intermediate q u a l i t y i n which food i s acceptable but not f r e s h . Hypoxanthine l e v e l s are h i g h , i n d i cated by the pink t e s t zone and diamines are s t i l l low, with a blue t e s t zone. Class 3 occurs when hypoxanthine l e v e l s are now lower and a blue t e s t zone, while diamine l e v e l s have i n c r e a s e d , as i n d i c a t e d by the pink t e s t zone. Marine foods i n the 3rd c l a s s would be c l a s s i f i e d as marginal or not acceptable. It may even be p o s s i b l e to s u b - d i v i d e the 3 t e s t categories f u r t h e r by shades o f blue or pink to increase the s e n s i t i v i t y o f grading. Perhaps even a t h i r d i n d i c a t o r to produce a multi-phase s t r i p might be employed to v i s u a l l y i n d i c a t e the presence of t r i methylamine. This might be accomplished by employing the enzyme t r i m e t h y l ami ne dehydrogenase which Large and McDougall (23) suggested could be use When these enzyme t e s t p o s s i b l e to use these d i r e c t l y on the f o o d , employing the t i s s u e f l u i d to wet the t e s t zones. This would e l i m i n a t e the need to sample and prepare a p r o t e i n - f r e e e x t r a c t . The a p p l i c a t i o n of enzyme methods to marine food a n a l y s i s can provide o b j e c t i v e , inexpensive and r a p i d approaches to the assessment of seafood q u a l i t y . Adaptation of these methods to a v i s u a l basis permits extension of enzyme a n a l y s i s t o non-laborat o r y s i t u a t i o n s , such as dockside or on board s h i p s . Thus, enzymatic a n a l y s i s o f marine foods can be u t i l i z e d f o r q u a l i t y assessment at c r i t i c a l p o i n t s , where t h i s type of determination can have maximum impact f o r grading and purchase. Acknowledgements The authors would l i k e to acknowledge the c o n t r i b u t i o n s of James A. Hourigan, Richard J . C o d u r i , J r . and J e f f r e y L. Howe who provided encouragement and t e c h n i c a l a d v i s e . This research was supported i n part by the College of Resource Development, U n i v e r s i t y of Rhode I s l a n d ; by the Cooperative State Research S e r v i c e , U.S. Department of A g r i c u l t u r e ; and by the NOAA O f f i c e of Sea Grant, U.S. Department of Commerce under grants 04-6-158-44002 and 04-6-158-44085. Contribution No. 1709 of the Rhode Island A g r i c u l t u r a l Experiment S t a t i o n .

Literature cited 1.

Farber, L. G. in "Fish as Food" Vol. 4, pg. 65, G. Borgstrom, ed. Academic Press, NY, 1965.

2.

H i l l i g , F . , Shelton, L. R., J.A.O.A.C. (1962) 45, 724.

Jr.,

and Loughrey, J . H . ,

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

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

278 3.

Dugal, L . C., J. Fish. Res. Bd. Can. (1967) 24, 2229.

4.

Ruiter, Α . , and Weiseman, J. M . , J. Fd. Technol. (1976) 11, 59.

5.

Burt, J. R . , Gibson, D. Μ., Jason, A. C., and Sanders, H. R . , J. Fd. Technol. (1976) 11, 73.

6.

Burt, J. R., Gibson, D. Μ., Jason, A. C., and Sanders, H. R., J. Fd. Technol. (1976) 11, 117.

7.

Connell, J. J., Howgate, P. F., Mackie, I. M . , Sanders, H. R., Smith, G. L., J. Fd. Technol. (1976) 11, 297.

8.

Cheyne, Α . , Fishing News International, December (1975).

9.

Shewan, J. M. and Jones, N. R . , J. S c i . Food Agric. (1957) 8, 491.

10.

S p i n e l l i , J., Eklund 29, 710.

11.

Jones, N. R., Murray, J., and (in part) Livingston, Ε. I. and Murray, C. K . , J. S c i . Food Agric. (1964) 15, 763.

12.

Kalckar, H. M . , J. B i o l . Chem. (1947) 167, 429.

13.

Kassamsarn, B . , Sanz Perez, B . , Murray, J. and Jones, N. R., J. Food S c i . (1963) 28, 28.

14.

S p i n e l l i , J., J. Food S c i . (1967) 32, 38.

15.

Beuchat, L . R., J. Agr. Food Chem. (1973) 21, 453.

16.

Burt, J. R., Murray, J. and Stroud, G. D . , J. Food Technol. (1968) 3, 165.

17.

Jahns, F. D . , Howe, J. L., Coduri, R. J., J r . and Rand, A. G. Jr., Food Tech. (1976) 30, (7), 27.

18.

Coughlan, M. P. and Johnson, D. B . , Biochim. Biophys. Acta (1973) 302, 200.

19.

Petrilli, R. "The use of immobilized xanthine oxidase as a method of measuring hypoxanthine concentrations." M.S. Research Project Report, University of Rhode Island, 1975.

20.

Singer, A. and Warchut, A. "Immobilization of xanthine oxidase for applications as an analytical tool." M.S. Research Project Report, University of Rhode Island, 1975.

21.

Eskin, Ν. A. M . , Henderson, Η. M. and Townsend, R. J., "Bio chemistry of Foods," pg. 191-201, Academic Press, NY, 1971.

22.

Mietz, J. J. and Karmas, Ε . , Inst. Food Tech. Abstracts, Anaheim (1976).

23.

Large, P. J. and McDougall, H . , Anal. Biochem. (1975) 64, 304.

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

16 Chemical and Microbiological Changes during Sausage Fermentation and Ripening S. A. PALUMBO and J. L. SMITH Eastern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, Philadelphia, Pa. 19118

Introduction The production of sausage began as one of man's earliest attempts at food preservation, possibly as far back as 1500 B . C . , when people learned that meat would not spoil i f i t were finely chopped, mixed with salt and spices, and allowed to dry in r o l l s . Salami i s thought to be named after the city of Salamis on the east coast of Cyprus (1), an early producer of such products. With the passage of time, each individual region developed i t s characteristic sausage as evidenced in Italy by the well-known Genoa and the lesser known Milano, Sorrento, Lombardi (2), and Siciliano salamis (1). Sausage production involves three of the oldest forms of food preservation: salting, drying, and smoking. The red or pink color typical of cured meat products, was f i r s t noted in Roman times (3). This color was subsequently found to be due to nitrate, occurring as an impurity in the s a l t , which is reduced by bacteria to n i t r i t e ; the n i t r i t e then reacts with the meat pigment myoglobin to give the pink color, nitrosylmyoglobin. The nitrosylmyoglobin is subsequently denatured to give the stable form, nitrosohemochrome. I t was only in the e a r l y p a r t of the 20th century that v a r i o u s b a c t e r i a (with t h e i r enzymes) were discovered to be the agents r e s p o n s i b l e f o r two changes that occur during production of dry fermented sausage: l a c t i c a c i d production and n i t r a t e r e d u c t i o n . T r a d i t i o n a l l y , the a d d i t i o n of the two b a c t e r i a l types, i.e., lactic a c i d b a c t e r i a and m i c r o c o c c i ( n i t r a t e reducing), was left to chance. Much of the n a t u r a l flora was c o n t r i b u t e d by the processor when l e f t o v e r m a t e r i a l from a previous batch was added ("back-slopping") or by reuse of equipment a f t e r l i m i t e d c l e a n i n g . The meat was handled in some

1

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Research S e r v i c e , U.S. Department of A g r i c u l t u r e .

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f a s h i o n to permit the development of i t s own s t a r t e r c u l t u r e . T h i s o f t e n i n v o l v e d low temperature h o l d i n g of meat, plus s a l t and cure i n the form of n i t r a t e , i n shallow pans to permit the growth of n i t r a t e reducing m i c r o c o c c i with subsequent formation of n i t r i t e to e f f e c t c u r i n g . A f t e r the pan cure, sugars and s p i c e s were mixed i n and the meat mixture was ground and s t u f f e d i n t o c a s i n g s . The s t u f f e d sausages were then h e l d i n the "green room" where l a c t i c a c i d b a c t e r i a fermented the sugars and some d r y i n g occurred. The sausages were then moved i n t o the dry room where f u r t h e r dehydration took p l a c e . Much of the chance has been e l i m i n a t e d i n modern sausage manufacture by two p r o c e s s i n g advances: the use of n i t r i t e alone i n cures, and the a d d i t i o n of l a c t i c a c i d s t a r t e r c u l t u r e s c o n t a i n i n g l a c t o b a c i l l i and/or pediococci. The o r i g i n a l development of s t a r t e r c u l t u r e s f o r use i n dry fermented sausages followed separate routes i n the U.S and Europe ( 4 ) . American sausag thus needed to add onl c u l t u r e , Pediococcus c e r e v i s i a e , was l y o p h i l i z e d (5); now i t i s a v a i l a b l e as a f r o z e n concentrate ( 6 ) . European workers favored c u l t u r e s that would reduce n i t r a t e such as a s t r a i n of Micrococcus, M 53, a v a i l a b l e as "Baktofermente" (Rudolf M u l l e r Co.^ Giessen, Germany). The Europeans changed t h e i r views when they observed that the a d d i t i o n of l a c t o b a c i l l i along with the microc o c c i gave b e t t e r c o l o r development and a somewhat more a c i d product (2). T h i s mixed c u l t u r e i s a v a i l a b l e as "Duplofermente," a l s o from R. M u l l e r . The p r o c e s s i n g of dry fermented sausages occurs i n three p r i n c i p a l steps: f o r m u l a t i o n , fermentation, and d r y i n g or r i p e n i n g . Palumbo e t a l . (8) described a process f o r the prepa r a t i o n of pepperoni as a f u l l y dry sausage. During f o r m u l a t i o n of pepperoni, s a l t , glucose, s p i c e s , and cure ( n i t r a t e or n i t r i t e ) are mixed with the meats and s t u f f e d i n t o c a s i n g s . An aging p e r i o d (before a d d i t i o n of other i n g r e d i e n t s ) of s a l t e d meat i s included to encourage the development of m i c r o c o c c i and l a c t o b a c i l l i (£, 9)· The fermentation p e r i o d a t 35°C and 90% RH i s one to three days, depending on the d e s i r e d pH decrease. After fermentation, the sausages are d r i e d at 12°C and 55-60% RH f o r s i x weeks. Major m i c r o b i o l o g i c a l a c t i v i t y occurs during the second and t h i r d steps. In the second step, m i c r o c o c c i reduce n i t r a t e to n i t r i t e w i t h i n the f i r s t 24 h r . of fermentation (9, 10,) with concomitant formation of n i t r o s y l m y o g l o b i n ; l a c t o b a c i l l i convert glucose to l a c t i c a c i d somewhat more slowly than n i t r a t e i s reduced, o f t e n r e q u i r i n g three days to lower the pH to 4.64.7 from a s t a r t i n g value of 5.6 ( 8 ) . 5

Reference to brand or f i r m name does not c o n s t i t u t e endorsement by the U.S. Department of A g r i c u l t u r e over others of a s i m i l a r nature not mentioned.

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

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During the t h i r d step, d r y i n g (or r i p e n i n g as i t i s known i n the European l i t e r a t u r e ) , the l i p i d s and p r o t e i n s of the sausage are attacked by v a r i o u s b a c t e r i a and t h e i r enzymes. The sausages l o s e 20-40% of t h e i r s t a r t i n g (green) weight (11). In the o l d e r p r o d u c t i o n of sausage, fermentation u s u a l l y occurred i n the green room, g e n e r a l l y held at a somewhat higher temperature than the dry room, and some dehydration occurred t h e r e . The sausage was then moved to the dry room where f u r t h e r d r y i n g occurred, along w i t h d e s i r e d f l a v o r and t e x t u r a l changes. In the United S t a t e s , d r y i n g room times and temperatures f o r sausages of a given diameter are s p e c i f i e d by the requirements f o r t r i c h i n a e i n a c t i v a t i o n (12). Although there are almost as many sausage types as there are sausage makers and geographic areas, some c l a s s i f i c a t i o n of t h e i r types and c h a r a c t e r i s t i c s i s p o s s i b l e . Fermented sausages are u s u a l l y described e i t h e r as semi-dry ( l o s s of 8-15% of s t a r t i n g weight) and f u l l 40% of s t a r t i n g weight) t h u r i n g e r , c e r v e l a t , Lebanon bologna, and pork r o l l ; examples of dry sausage i n c l u d e summer sausage, pepperoni, and v a r i o u s salamis (11). Some v a r i e t i e s , however, such as pepperoni, can be prepared as both dry and semi-dry forms. As a group, the I t a l i a n sausages are q u i t e s p i c y , not smoked, and have moisture contents of 35-40%, while the northern European v a r i e t i e s are smoked and have somewhat higher moisture contents (13). Dry sausages can a l s o be described by the maximum m o i s t u r e / p r o t e i n r a t i o (M/P) expected f o r i n d i v i d u a l v a r i e t i e s (14): salami, 1.9/1; pepperoni, 1.6/1; and Genoa, 2.3/1. (A M/P < 1.6/1 g e n e r a l l y i n d i c a t e s a s h e l f s t a b l e product.) Today, the i d e n t i t y of many dry fermented sausage v a r i e t i e s i s b l u r r e d and processors are marketing products w i t h two or three v a r i e t a l names. A sausage l a b e l l e d "thuringer-cervalat-summer sausage" was observed r e c e n t l y i n a l o c a l market. Most of the enzymes that produce the changes during ferment a t i o n and r i p e n i n g of sausages are considered to be a s s o c i a t e d with b a c t e r i a l c e l l s , l o c a t e d e i t h e r w i t h i n the c e l l s , as i n the case of g l y c o l y t i c enzymes, or o u t s i d e the c e l l s , as i n the case of l i p a s e s and proteases. Because of the nature of meat, i t i s not p o s s i b l e to " p a s t e u r i z e " or " s t e r i l i z e " i t before sausage manufacture. Thus, i t i s not p o s s i b l e to do f o r sausage r i p e n i n g what R e i t e r et a l . (15) d i d f o r cheddar cheese r i p e n i n g ; they e s t a b l i s h e d t h a t , because the m i l k was p a s t e u r i z e d , the added b a c t e r i a l s t a r t e r c u l t u r e , with i t s enzymes, was r e s p o n s i b l e f o r the observed changes such as p r o t e o l y s i s , l i p o l y s i s , and f l a v o r development that occur d u r i n g r i p e n i n g of the cheese. A l t e r a t i o n of Sausage Components by B a c t e r i a and T h e i r Enzymes Fermentation. There are two major changes produced i n sausages by fermentation: sugars are converted i n t o l a c t i c a c i d and n i t r a t e i s reduced to n i t r i t e .

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L a c t i c a c i d production. The major a c t i v i t y of the l a c t i c a c i d b a c t e r i a i s the conversion of sugars, u s u a l l y glucose, to l a c t i c a c i d by the homolactic Embdem-Myerhoff pathway. In t h e i r i n v e s t i g a t i o n of the s t o i c h i o m e t r y o f carbohydrate fermentation i n B e l g i a n salami, DeKetelaere et a l . (16) found that l a c t i c a c i d was the major a c i d formed, with minor amounts of a c e t i c , g e n e r a l l y i n the molar r a t i o of 10:1 l a c t i c to a c e t i c . They a l s o detected t r a c e amounts o f b u t y r i c and p r o p i o n i c . Our data f o r pH decrease and a c i d production during fermentation of porkbeef pepperoni are shown i n F i g . 1. (There i s only a very s l i g h t pH change i n pepperoni during the d r y i n g step (8).) S i m i l a r data were obtained f o r Lebanon bologna during fermentation. The extent to which producers of commercial d r y fermented sausage achieve a low pH (acid) product i s i l l u s t r a t e d i n Table I. Some v a r i e t i e s such as Lebanon bologna and summer sausage have low pH v a l u e s , whil have both higher pH value European dry sausages have pH values i n d i c a t i n g v i r t u a l l y no a c i d production. T h e i r formulations u s u a l l y have l i t t l e or no glucose added. For example, N i i n i v a a r a e t a l . (4) added only three grams glucose to 100 kg meat and obtained a pH of 5.3, while M i h a l y i and Kormendy (17) added no glucose to t h e i r formul a t i o n of Hungarian dry sausage and observed a pH of t h e i r f i n i s h e d sausage of 6.28. Table I pH Values of Some Commercial Fermented/Dry Sausages Sausage/Type

pH Range

Processors 10

Reference 16

B e l g i a n salami

4 .48 -• 5.10

Pepperoni

4 .7 - 6.1

Isterband

4 .6 -

5.7

Lebanon bologna

4 .6 -

4.9

Summer sausage

4 .6 -

5.0

Salami

5 .1 -

5.5

8

10

Thuringer

4 .9 -

5.1

4

10

9 10 6 12

8 32 9 33

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N i t r a t e r e d u c t i o n . Many s t u d i e s have been performed with m i c r o c o c c i , the b a c t e r i a that c a r r y out the r e d u c t i o n . M i c r o c o c c i developed during c o l d (5°C) aging of s a l t e d pork and beef for pepperoni (18) ( F i g . 2) and beef f o r Lebanon bologna (18) and d e c l i n e d during pepperoni fermentation ( F i g . 3). A f u r t h e r d e c l i n e of m i c r o c o c c i occurred during the d r y i n g of pepperoni. About midway through the d r y i n g p e r i o d , when the t i t r a t a b l e a c i d i t y reached 1%, v i a b l e m i c r o c o c c i were no longer detected (8). The a c i d content of the pepperoni was concentrated as the sausages dehydrated i n the dry room, thus d e s t r o y i n g the a c i d s e n s i t i v e m i c r o c o c c i . However, m i c r o c o c c i are u s u a l l y i s o l a t e d from l e s s a c i d pepperoni ( 8 ) . DeKetelaere et a l . (16) s t a t e d that m i c r o c o c c i were present through the r i p e n i n g p e r i o d of B e l g i a n salami, even though i t s a c i d content was above 1%. They u t i l i z e d a s a l t - c o n t a i n i n g medium (S 110 agar), as we d i d i n our study of pepperoni during d r y i n g . However, we a l s o employed the gram s t a i the medium and thus observe remained constant, m i c r o c o c c i c o l o n i e s were replaced by b a c i l l i c o l o n i e s . I t i s important f o r i n v e s t i g a t o r s to v e r i f y the colony types on the s e l e c t i v e agars and not r e l y s o l e l y on the supposed s p e c i f i c i t y of a given medium. The r e d u c t i o n of n i t r a t e occurs w i t h i n the f i r s t 24 hr of the fermentation p e r i o d , u s u a l l y during the f i r s t 2 to 16 hr (10). Zaika et a l . (19) observed that formation of most of the cured meat pigment (nitrosylmyoglobin) occurred w i t h i n the f i r s t 24 hr a l s o . They i n d i c a t e d that the m i c r o c o c c i of the n a t u r a l f l o r a are capable of reducing v a r i o u s l e v e l s of n i t r a t e with s a t i s f a c t o r y n i t r o s y l m y o g l o b i n formation ( F i g . 4 ) . With the lowest l e v e l of n i t r a t e t e s t e d , 100 ppm, pigment conversion was somewhat slow; l e v e l s of 200 up to 1600 ppm n i t r a t e gave e q u a l l y s a t i s f a c t o r y pigment conversion. The need f o r n i t r a t e reducing f l o r a i n fermented sausage production has been superseded by the use of n i t r i t e . Cures c o n t a i n i n g only n i t r i t e are adequate f o r the normal processing of most sausages; however, many sausage makers f e e l that dry fermented sausages must be cured with n i t r a t e . The o c c a s i o n a l l a c k of n a t u r a l f l o r a of m i c r o c o c c i that possess d e s i r e d l e v e l s of n i t r a t e reductase was overcome by N i i n i v a a r a and coworkers (4_) who i s o l a t e d s t r a i n s of m i c r o c o c c i having i n t e n s e n i t r a t e reductase a c t i v i t y , along with other t r a i t s d e s i r a b l e f o r the production of dry fermented sausages. B a c t e r i a or, more s p e c i f i c a l l y , the end products of t h e i r metabolism, can produce two c o l o r d e f e c t s i n fermented sausages: " n i t r i t e burn" and "greening." R e l i a n c e on m i c r o b i a l r e d u c t i o n of n i t r a t e to y i e l d the n i t r i t e necessary f o r the c u r i n g r e a c t i o n s can l e a d to instances i n which v a r i o u s m i c r o c o c c i and non-pathogenic s t a p h y l o c o c c i reduce excessive amounts of n i t r a t e (20). The r e s u l t i n g excess n i t r i t e r e a c t s with the meat pigments to give a greenish d i s c o l o r a t i o n . T h i s d e f e c t u s u a l l y occurs on

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

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Journal of Food Science

Figure 1. Changes in pH and % acid (as lactic) during the fermentation of 35°C (8)

I

2

MSA

CD

2

4

6

8

TIME, DAYS

10 12

0

2

4

6

8

10

TIME, DAYS Journal of Food Science

Figure 2. Changes in microflora plated on various media during aging of salted (3% NaCl) beef and pork at 5°C. Media designations: APT (for total count); Rog (Rogosa SL agar for hctic acid bacteria); MSA (Mannitol salt agar for micrococci); PD (Potato Dextrose agar for yeast) (8)

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

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the product surface where the aerobic b a c t e r i a p r o l i f e r a t e . Another surface c o l o r d e f e c t which a l s o y i e l d s a green pigment i s caused by L a c t o b a c i l l u s v i r i d e s c e n s (21) growing on the surface of cured meat products, e s p e c i a l l y emulsion and low a c i d sausages. The organism produces excessive amounts of H^C^ (22) which r e a c t s with nitrosylhemochrome to give a green pigment. Drying (Ripening). D e i b e l (13) s t a t e d that " E s s e n t i a l l y , the microbiology a s s o c i a t e d with the (fermented sausage) product i s r e s t r i c t e d to the green room" (the fermentation). Palumbo et a l . (8) reported that the t o t a l count and the number of l a c t o b a c i l l i remained e s s e n t i a l l y constant during the d r y i n g p e r i o d of pepperoni, while m i c r o c o c c i d e c l i n e d to undetectable l e v e l s . DeKetelaere et a l . (16) reported that the number of l a c t o b a c i l l i and m i c r o c o c c i remained constant during r i p e n i n g of B e l g i a n salami. Palumbo et a l . (8), Smith and Palumbo (18), and D e i b e l et a l . (10) reported that v a r i o u s commercial dry fermented sausages contained l a r g fermented sausage do c o n t a i n v i a b l e b a c t e r i a . Because of the a c i d i t y and low water a c t i v i t y of these products, the b a c t e r i a cannot m u l t i p l y . However, m i c r o b i a l caused changes do occur during the d r y i n g or r i p e n i n g stage. Recent s t u d i e s on dry fermented sausages have i n d i c a t e d that b a c t e r i a and t h e i r enzymes can a t t a c k v a r i o u s sausage components during the r i p e n i n g p e r i o d , so that while the d i f f e r e n t b a c t e r i a l populations appear to be s t a b l e , important changes are o c c u r r i n g i n the l i p i d and p r o t e i n components of the sausage. The end products of m i c r o b i a l a c t i o n on l i p i d s and p r o t e i n s i n c l u d e compounds that give each i n d i v i d u a l sausage type i t s unique f l a v o r and t e x t u r a l c h a r a c t e r i s t i c s . Recent s t u d i e s , e s p e c i a l l y by v a r i o u s European researchers, have y i e l d e d some i n t e r e s t i n g f i n d i n g s on the f a t e of the l i p i d and p r o t e i n components of dry fermented sausages. Lipolysis. Of the two components present i n dry sausages, f a t i s higher i n concentration than p r o t e i n . In products such as pepperoni f a t can comprise more than 50% of the f i n a l product weight (8). The f a t i n many sausage formulations i s pork f a t . As i n d i c a t e d above, m i c r o c o c c i are important i n reducing n i t r a t e i n dry fermented sausages. Work at our l a b o r a t o r y (23) and Cantoni's (24) i n d i c a t e d that m i c r o c o c c i can degrade f r e s h lard. Smith and A l f o r d (23) found t h a t , of the v a r i o u s m i c r o b i a l c u l t u r e s surveyed, Micrococcus f r e u d e n r e i c h i i was the most a c t i v e i n a t t a c k i n g components of f r e s h l a r d . This organism s u b s t a n t i a l l y increased the peroxide v a l u e , and the 2enals and the 2,4-dienals of f r e s h l a r d ; f u r t h e r , though not as a c t i v e as other c u l t u r e s t e s t e d , M. f r e u d e n r i c h i i a l s o increased the a l k a n a l content of f r e s h l a r d . Thus, M. f r e u d e n r i c h i i markedly increased the carbonyl content of the f r e s h l a r d , probably by c l e a v i n g the unsaturated f a t t y a c i d s at the s i t e of the double bonds.

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

286

ENZYMES

I N FOOD

A N DBEVERAGE

PROCESSING

ALL PORK

BEEF 8 PORK

\ \PD

I 2 TIME, DAYS

0

1 2 TIME, DAYS

3

Figure 3. Changes in microflora plated on various media during the fermentation of a pork-beef and an all-pork pepperoni at 35°C (see Figure 2 for media designations) (8)

Figure 4. Formation of cured-meat pigment and changes in pH in Lebanon bolognas prepared with 100, 200, 400, 800, and 1600 ppm NaNO during fermentation with naturalflora(19J s

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

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Cantoni e t a l . (24), studying the a c t i o n of m i c r o c o c c i on l a r d (Table I I ) , i n d i c a t e d that m i c r o c o c c i are very a c t i v e i n degrading l a r d . The organisms produced l i p a s e s which cleave both long and short chain f a t t y a c i d s from the t r i g l y c e r i d e s . M i c r o c o c c i can degrade long chain unsaturated f a t t y a c i d s as i n d i c a t e d by the formation of carbonyls. The presence of m i c r o c o c c i i n f i n i s h e d sausages such as pepperoni (8), as w e l l as others (16, 18), and the work of Smith and A l f o r d (23) and Cantoni e t a l . (24) on the a c t i o n of pure c u l t u r e s of m i c r o c o c c i on l a r d suggest that the l i p i d s of v a r i o u s sausages should be a c t i v e l y attacked during r i p e n i n g . Data on v a r i o u s European sausage type support t h i s contention. The most complete study appears to be the work of Demeyer e t a l . (25) on Belgium salami; other workers have a l s o studied t h i s a t t a c k (4, 17, 26).

Table I I A c t i o n of M i c r o c o c c i on Pork Fat (from Reference 24) Culture

C

A

- 9n> g FA/100 g f a t C

a

D10

C13

23.1

45.2

87

50

660

667

(FA most e a s i l y r e l e a s e d : oleic, myristic, palmitoleic, linoleic)

V o l a t i l e FA, mg/100 g f a t ( P r i n c i p a l VFA:

Carbonyls,

propionic, acetic)

yM/1 of c u l t u r e

( P r i n i c p a l carbonyls: isovaleraldehyde)

0

propionaldehyde,

a

A f t e r 28 days of c u l t u r e ; FA - :f a t t y a c i d .

b

A f t e r 28 days of c u l t u r e ; VFA -• v o l a t i l e f a t t y a c i d .

c

A f t e r 24 days of c u l t u r e .

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Demeyer £t a l . (25) observed i n c r e a s e s i n the f r e e f a t t y a c i d (FFA) and carbonyls of B e l g i a n salami during r i p e n i n g . T r i g l y c e r i d e s were p a r t i a l l y degraded to FFA ( F i g . 5) and the unsaturated F F A s were degraded i n t o carbonyls. The t r i g l y c e r i d e content decreased with a corresponding i n c r e a s e of FFA's and d i g l y c e r i d e s (DG); though based on l i m i t e d h y d r o l y s i s , these data suggest that the enzymes of the m i c r o f l o r a removed o n l y one f a t t y a c i d from the t r i g l y c e r i d e molecule. In a r a t e study of l i m i t e d d u r a t i o n , they q u a n t i t a t e d and i d e n t i f i e d the F F A s r e l e a s e d ( F i g . 6). Though the a c t u a l changes were r e l a t i v e l y s m a l l , the r a t e of l i p o l y s i s appeared to decease i n the order: l i n o l e i c (18:2) > o l e i c (18:1) > s t e a r i c (18:0) > p a l m i t i c (16:0). The FFA d i s t r i b u t i o n ( F i g . 6) suggests that the microc o c c a l l i p a s e has s p e c i f i c i t y f o r t r i g l y c e r i d e s c o n t a i n i n g l i n o l e i c acid. Demeyer et a l . (25) then went on to q u a n t i t a t e the t o t a l carbonyl content of f a two methods (benzidine observed a s e v e r a l - f o l d i n c r e a s e i n t o t a l carbonyls during r i p e n i n g . They d i d not r e c o r d changes i n i n d i v i d u a l carbonyls, as d i d Halvorson (26) i n h i s study of Isterband, a Swedish fermented sausage. Halvorson observed s i g n i f i c a n t changes i n n-hexanals, n - o c t a n a l s , and some of the 2-alkenals during r i p e n i n g ; these compounds can c o n t r i b u t e to the aroma of the f i n i s h e d sausages. He a l s o detected i n c r e a s e s i n the l e v e l of s e v e r a l short chain branched carbonyls which could a r i s e from amino a c i d s by the Strecker degradation. Though most l i p a s e a c t i v i t y i s a s s o c i a t e d with m i c r o c o c c i , i t has a l s o been reported i n l a c t o b a c i l l i (Fryer et a l . (27); Oterholm et a l . (28)). These i n v e s t i g a t o r s used s t r a i n s of l a c t o b a c i l l i of d a i r y o r i g i n and observed very a c t i v e degradation of t r i b u t y r i n , with r e l a t i v e l y l i t t l e a c t i v i t y against other t r i g l y c e r i d e s . However, the a c t i v i t y of meat l a c t o b a c i l l i against pork f a t ( l a r d ) and other f a t s present i n dry sausages needs f u r t h e r study. P r o t e o l y s i s . A f t e r the l i p i d s , the most p l e n t i f u l component of dry fermented sausages i s p r o t e i n . Less work has been done on the changes i n the nitrogenous compounds r e l e a s e d during sausage r i p e n i n g , although progress has been made. M i h a l y i and Kormendy (17) reported a 7% decrease i n t o t a l p r o t e i n and a 36% i n c r e a s e i n non-protein n i t r o g e n (NPN) o c c u r r i n g from the 10th to the 100th day of r i p e n i n g of Hungarian dry sausage. I t was not p o s s i b l e to determine whether the p r o t e o l y t i c enzymes were of meat or m i c r o b i a l o r i g i n . They a l s o reported changes i n the sacroplasmic and m y o f i b r i l l a r f r a c t i o n s , but s i n c e the methods u t i l i z e d were developed f o r f r e s h (not cured or d r i e d ) meat, the s i g n i f i c a n c e of these changes cannot be p r o p e r l y accessed. 1

1

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

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Sausage Fermentation and Ripening

distribution

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Figure 5. Changes in fatty acid distribution over lipid classes (%) during ripening of Belgian salami: M . G . (monoglycerides), D . G . (diglycerides), T . G . (triglycerides), F . F . A . (free fatty acids), and P.L. (polar lipids) (25)

% of t o t a l in F.F.A.

DAYS Journal of Food Science

Figure 6. Percent of total palmitic (16:0), stearic (18:0), oleic (18:1), and linoleic (18:2) acid present in F . F . A . (25)

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D i e r i c k e t a l . (29) studied the changes i n the NPN f r a c t i o n s during the r i p e n i n g of B e l g i a n salami (Table I I I ) and found an i n c r e a s e of f r e e ammonia from deamination of amino a c i d s and an increase o f f r e e α-amino n i t r o g e n (amino a c i d s ) from the break down of p r o t e i n s . Peptide-bound n i t r o g e n decreased s l i g h t l y , with a decrease i n n u c l e o t i d e n i t r o g e n and an i n c r e a s e i n nucleo side nitrogen. R e l a t i v e changes i n the c o n c e n t r a t i o n of f r e e amino a c i d s (Table IV), f o r the sake of convenience, a r e presented i n three groups: those amino a c i d s showing a l a r g e increase during r i p e n i n g , those a c i d s showing a small i n c r e a s e , and those showing a decrease. Of unusual i n t e r e s t i s the decrease of glutamic a c i d that was added to the sausage mix i n the form of MSG (monosodium glutamate)* Even though i t was added f o r f l a v o r , a considerable p a r t of the glutamate was degraded by the time the sausage was f u l l y ripened and ready f o r consumption

Concentration of Nonprotein Nitrogen Compounds during Ripening of B e l g i a n Salami (from Reference 29) Days of Ripening

NH

3

0

15

36

24*

58

76

Free a-NH^-N

141

234

255

Peptide bound

161

152

145

Nucleot.-N

34

13

12

Nucleos,-N

33

78

83

a-NH -N 2

*mg N/100 g of dry matter.

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

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Table IV Changes i n Free Amino A c i d s During 36 Days of Ripening of B e l g i a n Salami (from Reference Large Increase, >12X

Small Increase, <12X

29)

Decrease

Thre

Asp, A l a , l i e u

Glu

Pro

Ser, V a l , Phen

His

Leu γ-ΝΗ -ΒΑ 2

Orn

D i e r i c k e t a l . (29) observed a decrease i n h i s t i d i n e ( h i s ) , t y r o s i n e ( t y r ) , and o r n i t h i n e (orn) concentrations and corresponding i n c r e a s e s i n the c o n c e n t r a t i o n s of histamine, tyramine, and p u t r e s c i n e , the d e c a r b o x y l a t i o n products of h i s , t y r , and orn. Rice and Koehler (30) attempted to i d e n t i f y the organism(s) that possess t y r and h i s decarboxylase a c t i v i t i e s . They i n v e s t i g a t e d the l a c t i c a c i d s t a r t e r c u l t u r e organisms, i n c l u d i n g s t r a i n s of l a c t o b a c i l l i , s t r e p t o c o c c i , and Pediococcus c e r e v i s i a e . Only the s t r e p t o c o c c i showed t y r decarboxylase a c t i v i t y ; however, s t r e p t o c o c c i are not u s u a l l y s t a r t e r c u l t u r e s f o r fermented sausages and the s i g n i f i c a n c e of t h i s f i n d i n g cannot be adequately evaluated. They d i d not t e s t other organisms such as m i c r o c o c c i that are present i n many fermented sausages i n l a r g e numbers and could c o n c e i v a b l y c a r r y out t h i s metabolic activity. Despite t h e i r i n a b i l i t y to l o c a t e the source of decarboxy l a s e a c t i v i t y , R i c e et a l . (31) surveyed v a r i o u s dry fermented sausages f o r t h e i r histamine and tyramine content (Table V ) . The histamine content of most sausages was low. Emulsion-type products such as bologna weiners were low, w h i l e braunschweiger, because of i t s l i v e r content, was s l i g h t l y higher than ground beef. In c o n t r a s t , tyramine was found i n l a r g e r q u a n t i t i e s i n a v a r i e t y of dry fermented sausages. Those sausages c o n t a i n i n g the higher c o n c e n t r a t i o n s could p r o v i d e s u f f i c i e n t tyramine i n moderate s e r v i n g s to produce p r e s s o r responses i n tyramine susceptible individuals.

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

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Table V Amine Content o f Various Sausages (from Reference 31) Histamine

Avg.

Tyramine

Avg.

Dry sausage

2.87*

Hard salami

Semidry

3.59

Pepperoni

1.89

Summer sausage

184

Braunschweiger

3.60

Genoa salami

534

Ground beef

2.70

Smoked land-Jaeger

396

Lebanon bologna

224

sausage

Bologna

210* 39

Weiners

*yg/g of sausage.

V i r t u a l l y a l l the changes during sausage fermentation and r i p e n i n g are enzyme-catalyzed and most of the enzymes are of b a c t e r i a l o r i g i n . The v a r i o u s types of changes reported here have not a l l been studied i n a s i n g l e sausage type; however, where overlapping s t u d i e s e x i s t , t h e i r r e s u l t s are s i m i l a r . In summary, b a c t e r i a and t h e i r enzymes: ferment sugars to l a c t i c a c i d ; reduce n i t r a t e to n i t r i t e ; degrade t r i g l y c e r i d e s to f a t t y a c i d s and cleave unsaturated f a t t y a c i d s and amino a c i d s to form v a r i o u s carbonyls which can c o n t r i b u t e to f l a v o r . P r o t e i n s are degraded to v a r i o u s simpler n i t r o g e n - c o n t a i n i n g compounds such as amino a c i d s , ammonia, and amines by both meat and m i c r o b i a l enzymes. Literature Cited 1. Pederson, C. S., "Microbiology of Food Fermentations," 153-172, The AVI Publishing Company, Inc., Westport, Connect i c u t , 1971. 2. "Foreign Style Sausage," Sausage and Meat S p e c i a t l i t i e s , The Packer's Encyclopedia, part 3, 168-172, The National Provisioner: Chicago, I l l i n o i s , 1938. 3. Federal Register (1975) 40(218), 52614-52616.

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

16.

4. 5. 6. 7. 8. 9. 10. 11.

12.

13.

14.

15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.

PALUMBO

AND

SMITH

Sausage

Fermentation

and

Ripening

293

Niinivaara, F . P . , Pohja, M . S . , and Komulainen, Saima E., Food Tech. (1964) 18(2), 25-31. Deibel R. H., Wilson, G. D . , and Niven C. F., J r . Appl. Microbiol. (1961) 9, 239-243. Everson, C. W., Danner, W. E., and Hammes, P. A., Food Technol. (1970) 24, 42, 44. Nurmi, E., Acta Agralia Fennica, (1966) 108, 1-77. Palumbo, S. Α . , Zaika, L . L., Kissinger, J. C., and Smith J. L., J. Food S c i . (1976) 41, 12-17. Palumbo, S. Α . , Smith, J. L., and Ackerman, S. Α . , J. Milk & Food Tech. (1973) 36(10), 497-503. Deibel, R. H., Niven, C. F., Jr., and Wilson, G. D . , Appl. Microbiol. (1961) 9, 156-161. MacKenzie, D. S., "Prepared Meat Product Manufacturing," 62-64, American Meat Institute Center for Continuing Education, 1966. Anonymous, Meat an and Poultry Inspectio Program Inspection Service, U.S. Department of Agriculture, Washington, D . C . , 1973. Deibel, R. H., "Technology of Fermented, Semi-Dried and Dried Sausages," Proceedings of the Meat Industry Research Conference, Chicago, I l l i n o i s , March 21-22, 1974; 57-60, American Meat Institute Foundation, Arlington, Virginia, 1974. Anonymous, Chemistry Laboratory Guidebook, Consumer and Marketing Service, Laboratory Services Division of U.S. Department of Agriculture, Washington, D . C . , 1971. Reiter, B . , Fryer, T. F., and Sharpe, M. E., J. Appl. Bact. (1966) 29(2), 231-243. DeKetelaere, Α . , Demeyer, D . , Vandekerckhove, P . , and Vervaeke, I., J. Food S c i . (1974) 39, 297-300. Mihalyi, V. and Kormendy, L., Food Tech. (1967) 21, 13981402. Smith, J. L . and Palumbo, S. Α . , Appl. Microbiol. (1973) 26(4), 489-496. Zaika, L . L., Z e l l , T. E., Smith, J. L., Palumbo, S. Η . , and Kissinger, J. C., J. Food S c i . (1976) 41, 1457-1460. Bacus, J. N. and Deibel, R. H., Appl. Microbiol. (1972) 24(3), 405-408. Niven, C. F., J r . and Evans, J. B . , J. Bact. (1957) 73, 758-759. Niven, C. F., Jr., Castellani, A. G., and Allanson, V., J. Bact. (1949) 58(5), 633-641. Smith, J. L . and Alford, J. Α . , J. Food S c i . (1969) 34(1), 75-78. Cantoni, C., Molnar, M. R . , Renon, P . , and Giolitti, G., J. Appl. Bact. (1967) 30(1), 190-196. Demeyer, D . , Hoozee, J., and Mesdom, J., J. Food S c i . (1974) 39, 293-296.

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

294 26. 27. 28. 29. 30. 31. 32. 33.

ENZYMES IN FOOD AND BEVERAGE PROCESSING

Halvarson, H . , J. Food S c i . (1973) 38, 310-312. Fryer, T. F., Reiter, Β . , and Lawrence, R. C., J. Dairy S c i . (1967) 50, 388-389. Oterholm, Α . , Ordal, Z. J., and Witter, L . D . , Appl. Microbiol. (1968) 16, 524-527. Dierick, Ν . , Vandekerckhove, P . , and Demeyer, D . , J. Food S c i . (1974) 39, 301-304. Rice, S. L . and Koehler, P. E., J. Milk Food Tech. (1976) 39(3), 166-169. Rice, S., Eitenmiller, R. R . , and Koehler, P. E., J. Milk Food Tech. (1975) 38(4), 256-258. Ostlund, K. and Regner, B . , Nord. Vet.-Med. (1968) 20(10), 527-542. Goodfellow, S. J. and Brown, W. L., Food Product Develop. (1975) 9(6), 80 and 82.

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

17 Enzymic Hydrolysis of Ox-Blood Hemoglobin K. J. STACHOWICZ Institute of Fermentation Industry, Rakowiecka 36, Warsaw, Poland C. E . ERIKSSON and S. T J E L L E SIK, The Swedish Food Institute, Fack, S-40021 Göteborg, Sweden

In the s e a r c h f o r n o v e l , cheap p r o t e i n s to be used i n food e i t h e r as s u b s t i t u t e s f o r meat, f o r fortification purposes or as f u n c t i o n a l additives, the b l o o d of s l a u g h t e r e d a n i m a l s has been surprisingly little considered. Even though some whole b l o o d i s used as an i n g r e d i e n t i n a few t y p e s o f foods i n a few a r e a s i n the w o r l d , w i d e r use o f b l o o d is hampered primarily for religious and ethical r e a s o n s and by its characteristic f l a v o r and c o l o r . In a d d i t i o n to this, attempts to use b l o o d in c e r t a i n foods as a combined l p r o t e i n and i r o n fortifier and coloring agent have failed due to the enhancing o f lipid o x i d i a t i o n and off-flavor development c a t a l y z e d by the i r o n - p o r p h y r i n group p r e s e n t i n the hemoglobin (1). T h i s k i n d of catalytic activity may a l s o be i n c r e a s e d by denaturation of hemoproteins e . g . by heat (2). The total amount o f b l o o d r e p r e s e n t s a g r e a t d e a l o f p r o t e i n , which to day is used to o n l y a limited e x t e n t as an animal f e e d . O f t e n , however, it g i v e s rise to s e r i o u s pollution of water recipients and s h o u l d t h e r e f o r e be s u b j e c t to i n t e n s e s t u d i e s c o n c e r n i n g its p r o c e s s i n g into v a r i o u s p r o t e i n p r o d u c t s f o r f o o d u s e . In Sweden most blood is collected under strict h y g i e n i c c o n d i t i o n s i n immediate c o n n e c t i o n w i t h the s l a u g h t e r o f cattle and p i g s . T h i s b l o o d is then stabilized by sodium citrate, t r a n s p o r t e d c o l d to c e n t r a l plants where the plasma is s e p a r a t e d from the red cells. Plasma, e i t h e r liquid, concentrated, f r o z e n , or d r i e d is used i n the manufacture of meat p r o d u c t s , e . g . sausage m a i n l y due to its good e m u l s i fication and b i n d i n g p r o p e r t i e s . The r e d cell fraction is d r i e d and m o s t l y used i n a n i m a l f e e d i n g , a

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

296

ENZYMES IN FOOD AND BEVERAGE PROCESSING

s m a l l p a r t in f o o d . In Sweden about 2000 tons of b l o o d p r o t e i n mainly hemoglobin, is available annually i n the r e d cell fraction. T h i s q u a n t i t y r e p r e s e n t s , howe v e r , l e s s than 1% of the Swedish p r o t e i n consumption to d a y . S i n c e p r o t e i n is overconsumed in our c o u n t r y , p r o d u c t i o n of b l o o d p r o t e i n f o r human nutrition is not r e l e v a n t . Hence, utilization of b l o o d p r o t e i n s s h o u l d be p r i m a r i l y based more on their p o s s i b l e technical f u n c t i o n s i n f o o d like their b i n d i n g p r o p e r t i e s , their use as f l a v o r p r e c u r s o r s , c o n s i s t e n c y r e g u l a t o r s , meat e x t e n d e r s , e t c . I t i s thus b e l i e v e d t h a t a c c e s s to a wide range of b l o o d p r o t e i n p r o d u c t s w i t h differ e n t p r o p e r t i e s s h o u l d be advantageous. The o l d e s t and still most w i d e l y employed approach is the p r e p a ration of a d e c o l o r e d p r o t e i n p r o d u c t from the r e d cell fraction by removal o f the heme group from hemog l o b i n through the a c i d - a c e t o n fications of it. I precipitated w h i l e the hematin remains in the mixed acetone-acid-water phase. Acid

acetone

Hemoglobin

> Apohemoglobin(s)+ hematin

A g r e a t d e a l o f work has been c a r r i e d out to o p t i m i z e t h i s p r o c e s s a c c o r d i n g t o p r o t e i n y i e l d , product c o l o r , f u n c t i o n a l p r o p e r t i e s , solvent r e c y c l i n g , explosion safety, etc. In A u s t r a l i a the c o s t of a f u l l s c a l e p l a n t f o r production of a decolored p r o t e i n product from whole b l o o d has been e s t i m a t e d , on the b a s i s o f extensive p i l o t plant investigations . In our l a b o r a t o r y a n o t h e r method has been a p p l i e d f o r o b t a i n i n g p r o t e i n p r o d u c t s from the r e d c e l l f r a c t i o n , i n c l u d i n g a decolored product. P r e l i m i n a r y work r e v e a l e d t h a t enzymic h y d r o l y s i s of hemoglobin f o l lowed by g e l f i l t r a t i o n or u l t r a f i l t r a t i o n y i e l d e d h y d r o l y s a t e f r a c t i o n s which c o n t a i n e d o n l y s m a l l amounts of heme. These i n v e s t i g a t i o n s w i l l be publised later. However, some l a r g e - s c a l e work was a l s o c a r r i e d out i n o r d e r to produce s i n g l e , o n e - k i l o g r a m b a t c h e s of ox b l o o d p r o t e i n h y d r o l y s a t e f o r e x p e r i mental f o o d u s e . T h i s work w i l l be p r e s e n t e d i n what follows. Pretreatment

of red

cells.

Most p r o t e o l y t i c enzymes t e i n s b e t t e r than n a t i v e ones s t e r i c u n a v a i l i b i l i t y of b o t h tive substrate. The r e d c e l l

a t t a c k denatured p r o due to the i n h e r e n t the enzyme and the n a f r a c t i o n obtained a f t e r

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

17.

plasma

separation

35-kO°/o

of

dry

by

The

hemolyzing

Denaturation by

a l k a l i

formed

treatment

and

ration

effect

to

a

to

fram

ox

of

drawn

up

addition natant

was

content

was

reported ethyl

released

with

by

water.

accomplished

by

per

a l k a l i

denaturation

alcohol

and

( j 5 , 7^)·

protein

was

protein

heat

The

measurement

denatu

of

in

the

the

presence

was

by

red

adding

c e l l

reached.

5

Ν NaOH

fraction

This

pH

was

2 were

1 Ν HC1 and

f i r s t then

diluted

k°/o.

about

performed

hemolyzed

p H 11

1,

further

of

them

pre-investigation

treated

of

samples

of

protein

inorganic i s

h

2k

0.5,

The

cells

enzyme.

u n t i l

to

after

ments.

the

sample

blood

retained

soy

treatment

stirred

a

the

some

denaturation

estimated

proteolytic A l k a l i

after compare

containing

and

d i l u t i n g

dissolved

combined

was

rate

by

previously

applied

hydrolysis a

by

involving

treatment

of

to

l i q u i d

protein within

simply

the

order

viscous

protein

them, of

a

mainly

treatment,

in

methods

forms

matter

constituents.

297

Hydrolysis of Ox-Blood Hemoglobin

STACHOWICZ ET AL.

The

adjusted

to

pH 9

centrifuged.

with

water

d i l u t i o n

to

The a

factor

by

the

super

hemoglobin was

always

9. The in

a

enzyme

pH-stat

Switzerland). k°/o o f

about in

the

of

alcalase

a c t i v i t y

(Mettler Twenty

water

™

(NOVO,

consumed

being

10

min.

The

at

50

and

continuously

p H 9·

By

between

a l k a l i

and

combined

(Table in

I ) .

the

subsequent

Large-scale A l k a l i 1,

(k5-k6.5 was

grade,

owing

out

at

in

a

in

Figuve

both and

at

i t s

1 ml

for

superior

treatment

of

f i r s t

The 0.01

Ν NaOH

difference

p H 11

9

mg

/ u m o l 0H~~

with

was

one

alcohol-heat

scale

denatured, in

pH 9

to

treatment simplicity

was

preferred

operations.

1.

during feed

during

f i l t r a t e d water) and

enzyme

membrane

ture-controlled tration

to

a l k a l i

large

3.3-3-9%

proteolytic

shown

r i e d

ethyl

no

5

in

the

during

out

pH

operations.

hydro l y z e d

Denmark) as

Hence,

r e p r o d u c i b i l i t y ,

added,

method

to

Then

Denmark)

recorded

11,

containing

adjusted

was

treatment

performed

11 + D V

pH-stat.

carried

this

two and

f i r s t the

was

were

10 + D K

substrate

p H 9)

t i t r a t i o n

the

of

DK

Copenhagen, to

found h

of was

vessel

(pre-adjusted

C

ml

hemoglobin,

t i t r a t i o n

measurements

system:

by

means

time

before

volume

protein

of

(NOVO,

p r i n c i p a l l y

enzymic

certain

tank

the

C

blood

hemolyzed

a l c a l a s e ®

reactor The

a

50

ox

from

reaction i n

the

starting

reduction

red the

cells food

Copenhagen, designed was

car

tempera u l t r a f i l

period

that

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

298

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

Γ

Temperature controlled feed tank (1)

Recycling of rejec

Φ

(2)

(3)

<8 (M

U l t r a f i l t r a t i o n module

Ψ Ultrafiltrate tank

(5)

Figure 1. Principal procedure for the enzymic hydrolysis and ultrafiltration of protein from oxblood red cells: (1) enzyme, substrate (2) circu lating pump; (3) inlet pressure regulator and gauge; (4) outlet pressure regulator and gauge; and (5) protein hydrolysate

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

17.

299

Hydrolysis of Ox-Blood Hemoglobin

STACHOWICZ E T A L .

o c c u r r e d when t h e u l t r a f i l t r a t i o n was g o i n g o n . The pH d r o p i n t h e r e a c t i o n m e d i u m was c o m p e n s a t e d for by f r e q u e n t a d d i t i o n o f measured v o l u m e s of 5 Ν NaOH. Table

I.

Effect of pre-treatment on t h e r a t e of enzymic h y d r o l y s i s of b l o o d p r o t e i n . Hydrolysis rate / u m o l OH""/10 m i n

Pre-treatment

22

Control, untreated p H 11 f o r 0.5 h 1 h 2 h 2k h Ethyl alcohol 1 h Freeze-dried Air-dried pH

11,

kk

h,

k3

kS k8

26 a k3

ko

freeze-dried

Enzyme : A l c a l a s e 5 Temperature: 50°C pH: 9.0

mg p e r

20

ml

of

k°/o p r o t e i n

in

water

The u l t r a f i l t r a t i o n d e v i c e was o f t h e hollow fiber type (Romicon I n c . , U S A ) . Three different ultra f i l t r a t i o n m e m b r a n e s , XM 50, PM 10, a n d PM 5 w i t h nominal cut-off values of 50.000, 10.000, and 7.000, respectively, were u s e d . I t was e x p e c t e d t h a t the different characteristics of the membranes and the time of r e a c t i o n would affect the p r o p e r t i e s of the protein hydrolysates. The c i r c u l a t i o n flow and u l t r a f i l t r a t e f l o w were m a i n t a i n e d b y a pump. The i n l e t and outlet pressure of the u l t r a f i l t r a t i o n u n i t were kept con s t a n t and the c i r c u l a t i o n f l o w r a t e a d j u s t e d by means o f two p r e s s u r e r e g u l a t o r s . (See F i g u r e 1.) T h e u l t r a f i l t r a t e was c o l l e c t e d i n 5 1 batches from which samples were taken f o r f u r t h e r analysis. T h e r e j e c t e d m a t e r i a l was c o n t i n u o u s l y r e c y c l e d from the u l t r a f i l t r a t i o n u n i t back to the feed tank. The t o t a l h y d r o l y s i s t i m e r a n g e d f r o m 52 m i n t o 2 h , de p e n d i n g on t h e f l o w c a p a c i t y o f t h e membrane u n i t used and the p r e - h y d r o l y s i s time. The number of split p e p t i d e b o n d s , as measured by the t o t a l consumption o f N a O H , was a b o u t t h e same i n a l l t h r e e experiments. The s t a r t i n g m a t e r i a l and the d i f f e r e n t fractions of the u l t r a f i l t r a t e , c o l l e c t e d at r e g u l a r intervals d u r i n g the h y d r o l y s i s , were s u b j e c t e d to d r y weight and hemin d e t e r m i n a t i o n . Hemin d e t e r m i n a t i o n was b a s e d on t h e f o r m a t i o n o f a p y r i d i n e h e m o c h r o m o g e n

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

300

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

complex i n An average

strong a l k a l i u l t r a f i l t r a t e

volumes

the

of

PROCESSING

under reducing conditions (8). was p r o d u c e d b y m i x i n g equal

u l t r a f i l t r a t e

fractions.

Both

the

start

ing m a t e r i a l and the average u l t r a f i l t r a t e were also a n a l y z e d on t h e i r a s h c o n t e n t . The p r i n c i p a l u l t r a f i l t r a t e contents Tables II - IV. In a l l hydrolysate

and

running

three runs dry matter

conditions

II.

Hydrolysis

and

with

reactor.

alcalase

Compositio

u l t r a f i l t r a t e . Volume

k

start

i n

an

Running

Total dry matter

of

XM

ox

5

conditions.

Total hemin

Dry matter hemin (¥>)

48

1661

blood

membrane

(s)

(i) Protein

i n

respectively. Looking can see that both the tration increased with a small increase while the c o n c e n t r a t i o n i n -

u l t r a f i l t r a t i o n

proteins and

shown

a f a i r l y large, f i n a l y i e l d of was o b t a i n e d ; 83°/o, 73, a n d 72 in

t h e X M 5 0 , PM 1 0 , a n d PM 5 r u n s at the intermediate yields, one dry matter and the hemin c o n c e n t i m e . T h e X M 50 r u n s h o w e d o n l y in b o t h t h e PM 10 a n d PM 5 r u n s Table

are

2.9

U l t r a f i l t r a t e

0.6 0.7 0.5 0.7 0.9 0.9 0.9 1.5

1 .1 1 .2 0.9 1.4 1-5 1.5 1-5 2.9

5 5 5 5 5 5 5 5

170 174 175 184 176 177 180 188

ko ko

1424

12.0

0.8

1416

12.2

0.9

R e c o v e r y (°/o)

89

85

Membrane:

XM 5 0 .

fraction

No.

1

2 3 4 5 6 7 8 Sum,

fractions

Average

f i l t r a t e

Romicon

Cut

25 off:

50.000.

2.46

Area:

Pressure, inlet: 103 k P a ( 1 . 1 k p / c m , 15 l b / i n Pressure, outlet: 69 k P z ( 0 . 7 k p / c m , 10 l b / i n Flux: 89 l / m χ h . Enzyme: A l c a l a s e , 10.1 g.

m

). ).

Temperature: 4 8 . 5 - 51 C . T i m e : Pre-hydrolysis 107 m i n . u l t r a f i l t r a t i o n 13 m i n . T o t a l N a O H c o n s u m p t i o n : 3 · 0 m o l .

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

17.

Table

III.

H y d r o l y s i s and u l t r a f i l t r a t i o n o f ox b l o o d p r o t e i n s w i t h a l c a l a s e i n a P M 10 m e m b r a n e reactor. Composition of s t a r t i n g material and u l t r a f i l t r a t e . Running conditions. Volume

d) Protein

46,5

start

Ultrafiltrate fraction No. 1

10.0 10.0 10.0 1 1 .0

2 3 4 Sum,

301

Hydrolysis of Ox-Blood Hemoglobin

STACHOWICZ ET AL.

Total dry matter

Total hemin

ω

ω

1548 109 259 325 428

Dry matter hemin

38.8

Μ

2.5

0.007 0.007 0.014 0.033

0.006 0.003 0.004 0.007

0.117

0.010

fractions

Average

4i.0

f i l t r a t e

88

R e c o v e r y (°/o)

1121 72

0.3

Membrane: R o m i c o n PM 10. Cut o f f : 10.000. Area: 2.46 m2 ( 2 6 . 5 f t ) . Pressure, inlet: 138 k P z (l.1kp/ cm , 20 l b / i n | ) . Pressure, outlet: 103 k P a ( 1 . 1 kp/ cm , 15 l b / i n ). Flux: 12,7 l / m x h. Enzyme: Alcalase, 22.5 g . Temperature: 49.5 50.5°C T i m e : P r e - h y d r o l y s i s 21 m i n , u l t r a f i l t r a t i o n 64 m i n . T o t a l NaOH c o n s u m p t i o n : 3·0 m o l . p

2

2

c r e m e n t was m u c h l a r g e r f r o m t h e f i r s t to the last f r a c t i o n . The hemin content of the u l t r a f i l t r a t e dry m a t t e r was a l w a y s s i g n i f i c a n t l y l o w e r t h a n t h a t o f the s t a r t i n g m a t e r i a l . The average u l t r a f i l t r a t e dry matter contained 0.9, 0.01, a n d 0.02% o f h e m i n , t h u s a 3 - , 2 5 O - , a n d 1 5 0 - f o l d heme r e d u c t i o n i n t h e XM 5 0 , PM 1 0 , a n d PM 5 r u n s r e s p e k t i v e l y . It s h o u l d be n o t e d t h a t the f i g u r e s o f heme c o n t e n t , particularly in the PM 10 a n d PM 5 e x p e r i m e n t s , a r e somewhat uncertain s i n c e i t i s j u s t on the v e r g e o f d e t e c t a b i l i t y with the method used (8). The three average ultrafiltrates were freezed r i e d a f t e r h a v i n g been n e u t r a l i z e d by the a d d i t i o n of HC1, c i t r i c o r l a c t i c a c i d . The moisture and ash con t e n t were d e t e r m i n e d i n the f r e e z e - d r i e d p r o d u c t s . The results i n T a b l e V show t h a t the d r y m a t t e r contained 13-16% o f a s h . T h e a d d e d N a O H f o r p H - a d j u s t m e n t d u r i n g the r e a c t i o n and the a c i d used f o r n e u t r a l i z a t i o n be f o r e f r e e z e - d r y i n g a c c o u n t f o r a b o u t two t h i r d s o f the

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

302

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

Table

IV.

H y d r o l y s i s and u l t r a f i l t r a t i o proteins with alcalase in a reactor. Composition of sta and u l t r a f i l t r a t e . Running Volume

Total dry matter

U)

(1) Protein

Ultrafiltrate fraction No.

1

2 3 4 5 6 7 8 Sum,

fractions

Average

f i l t r a t e

R e c o v e r y (°/o)

n o f ox b l o o d PM 5 m e m b r a n e rting material conditions.

Total hemin

Dry matter hemin

(e)

W 3.0

52.5

1767

start

PROCESSING

5 5 5 5

73 97 120 143

0.016 0.011 0.012 0.018

0.02 0.01 0.01 0.01

5 5

226 263

0.052 0.081

0.02 0.03

ko

1285

0.252

0.02

ko

1307

0.292

0.02

89

73

0.6

Membrane: R o m i c o n PM 5 . Cut o f f : 7.000. A r e a : 2.18 m (23.5 p ) Pressure, inlet: 138 k P a ( 1 . 4 kp/cm? 20 l b / i n ) . Pressure, outlet: 103 k P a ( 1.1 kp/cm , 15 l b / i n ). Flux: 28 l / m χ h. Enzyme: Alcalase 22.5 g . T e m p e r a t u r e : 50 - 5 1 ° C . Time: Pre-hydrolysis 7.5 min, ultrafiltration 44.5 m i n . T o t a l NaOH c o n s u m p t i o n : 3-2 m o l . f

t

2

2

Table

V.

Some p r o p e r t i e s n e u t r a l i z e d and a t e s f r o m ox b l o was made e i t h e r c i t r i c acid.

Average

Hemin

u l t r a f i l t r a t e

of hydrolyzed, ultrafiltrated, freeze-dried protein hydrolysod red c e l l s . Neutralization with hydrochloric acid or

Moisture (°/o)

Ash

W)

50

0.9

5.8

12.9

PM

10

0.01

4.9

PM

5

0.02

6.1

XM

Color Chloride

Citrate light brown

16.3

dark brown beige

14.0

beige

cream

cream

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

17. STACHOWICZ ET AL. Hydrolysis of Ox-Blood Hemoglobin

303

ash content, while the r e s t o r i g i n a t e s from the mineral content o f the r e d c e l l f r a c t i o n . The c o l o r of the dry p r o d u c t d e p e n d e d on the a c i d u s e d f o r neutralization. H y d r o c h l o r i c a c i d (and l a c t i c a c i d ) gave brown products, whilst c i t r i c a c i d gave c r e a m - c o l o r e d ones. Particularly the product treated with lactic acid was v e r y h y g r o s c o p i c . P r o d u c t s f r o m t h e PM 10 a n d PM 5 runs were somewhat b i t t e r , no s i g n i f i c a n t difference in b i t t e r n e s s b e i n g f o u n d when e a r l y a n d l a t e f i l t r a t e fractions were compared. F u r t h e r work on the use of blood protein hydrolysates w i l l include investigations on the p o s s i b l e c o n t r o l o f the amount o f i n o r g a n i c cons t i t u e n t s and b i t t e r n e s s as w e l l as i n v e s t i g a t i o n s on the f u n c t i o n a l p r o p e r t i e s of these products.

Abstract T h i s paper p r e s e n t lysis under slightly alcaline c o n d i t i o n s can be used to p r e p a r e p r o t e i n h y d r o l y s a t e s w i t h a v e r y low heme c o n t e n t from the p r o t e i n m a i n l y h e m o g l o b i n , i n the r e d cells of c a t t l e b l o o d . The r e a c t i o n s were c a r r i e d out in a h o l l o w f i b e r membrane r e a c t o r , b o t h the r e a c t i o n and the p r o p e r t i e s of the product b e i n g a f f e c t e d by the c h o i c e o f membrane. The appearance o f the p r o d u c t s c o u l d be f u r t h e r a f f e c t e d by the c h o i c e of a c i d f o r neutralization o f the h y d r o l y s a t e . The p r o d u c t s o b t a i n ed had a fairly h i g h content of ash and were somewhat bitter. Literature cited (1) Tappel, A.L., in "Autioxidation and antioxidants" L u n d b e r g , W . O . , ed., Vol. I , p . 3 2 5 , I n t e r s c i e n c e Publ., New York 1 9 6 1 . (2) Eriksson, J.

Am.

(3) Lewis, (4) (5)

C.E., Oil

U.J.,

Olsson,

Chem. S o c . J.

Biol.

P.,

and Svensson,

(1971)

Chem.

48,

(1954)

S.,

442. 206, 109.

A n s o n , M.L., and M i r s k y , A.E., J. Gen. Physiol. ( 1 9 3 0 ) 13, 469. W i l s o n , B.W. in P r o c e s s I n d u s t r i e s o f Australia Impact and growth, N a t i o n a l C h e m i c a l E n g i n e e r i n g C o n f e r e n c e , p. 431, S u r f e r s P a r a d i s e , Q u e e n s l a n d , 1974.

( 6 ) Fukushima,

D.,

C e r e a l Chem.

(1969)

46,

156.

(7)

Fukushima,

D.,

C e r e a l Chem.

(1969)

46,

405.

(8)

P a u l , K.G., Theorell, H., and Åkesson, A c t a Chem. S c a n d . ( 1 9 5 3 ) 7 , 1 2 8 4 .

Å.,

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

18 Utility of Enzymes in Solubilization of Seed and Leaf Proteins E . A. CHILDS, J. L. FORTE, and Y. KU a

b

Department of Food Technology and Science, University of Tennessee, Knoxville, Tenn. 37901

The use o f enzyme leaf p r o t e i n s has been s t u d i e d as a means o f overcoming difficulties p r e s e n t e d by the v a r y i n g c o n d i t i o n o f seed and l e a f m a t e r i a l a v a i l a b l e f o r p r o c e s s i n g . The variations a r e the result o f d i f f e r e n c e s i n the original c o n d i t i o n o f the m a t e r i a l and its p o s t - h a r v e s t treatment. E x t r a c t i o n o f P r o t e i n from O i l s e e d s O i l s e e d aleurin p r o t e i n s a r e found in c y t o p l a s m i c vacuoles. Examples i n c l u d e the 7S and 11S g l o b u l i n s of soybeans ( 1 ) , a r a c h i n from peanuts (2,3), edistin from hemp seed ( 4 ) , vicilin and legumin from pea seeds ( 5 ) , and vicilin and legumin from Vicia f a v a ( 6 ) . Based on the similarity o f amino a c i d sequences as meas u r e d by the D i f f e r e n c e Index s u g g e s t e d by Metzger ( 7 ) , D i e c k e r t and D i e c k e r t (8) have s u g g e s t e d the r e s e r v e p r o t e i n s o f leguminous seeds a r e q u i t e homologous and a r e u s u a l l y hexamers o r t e t r a m e r s o f a disulfide bridged s u b u n i t w i t h an A - S - S - B s t r u c t u r e . These similarities i n (a) c y t o p l a s m i c l o c a t i o n and c o m p a r t m e n t a l i z a t i o n and (b) amino a c i d sequence o f a variety o f o i l s e e d p r o t e i n s o f f e r s a u n i f y i n g view o f the c o n d i t i o n s f a c e d in e x t r a c t i n g p r o t e i n s from o i l s e e d s f o r p r o d u c t i o n o f p r o t e i n concentrates or isolates. Several techniques a r e a v a i l a b l e f o r production o f seed p r o t e i n c o n c e n t r a t e s and i s o l a t e s . Protein i s o l a t es c a n be p r e p a r e d from d e f a t t e d seeds by e x t r a c t i o n i n d i l u t e a l k a l i , f o l l o w e d by i s o e l e c t r i c p r e c i p i t a t i o n o f Current Address: D e d e r i c h C o r p o r a t i o n , P.O. Box 7, Hubertus, WI 53033 Current Address: Biology Division, Oak Ridge N a t i o n a l L a b o r a t o r i e s , Oak Ridge, TN 37830 a

b

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

18.

CHILDS

ET

AL.

Solubilization of Seed and Leaf Proteins

305

the p r o t e i n a t an a c i d i c pH, and d r y i n g t h e p r e c i p i t a t ed m a t e r i a l a f t e r washing. P r o t e i n c o n c e n t r a t e s may be produced from seeds by e x t r a c t i o n o f low m o l e c u l a r w e i ght m o l e c u l e s a t pH 4.0 w i t h the p r o t e i n r e m a i n i n g i n the i n s o l u b l e m a t e r i a l f o r d r y i n g . Sugarman (9) d e v e l oped methodology f o r p r o d u c i n g p r o t e i n c o n c e n t r a t e s from f u l l f a t seeds. An aqueous e x t r a c t i o n i s p e r f o r m ed and the r e s u l t i n g e m u l s i o n broken by s h e a r f o r c e s a t an a p p r o p r i a t e temperature and pH. These methods a r e e f f i c i e n t i f the seed has n o t been heated t o an e x t e n t t h a t w i l l cause d e n a t u r a t i o n of the p r o t e i n . I f d e n a t u r a t i o n has o c c u r r e d , e x t r a c t a b l e p r o t e i n y i e l d s w i l l drop t o l e s s than 20% i n soybeans o r c o t t o n s e e d (10,11). Because most o i l s e e d s a r e a l s o an o i l s o u r c e , they may undergo a hexan t o h e a t and m o i s t u r In s p e c i f i c commodities such as soybeans, h e a t may a l s o be u t i l i z e d t o maximize the n u t r i t i o n a l q u a l i t y o f t h e p r o t e i n (15). Thus the p r o b a b i l i t y o f u t i l i z i n g seeds c o n t a i n i n g denatured p r o t e i n s f o r p r o d u c t i o n of p r o t e i n i s o l a t e s and c o n c e n t r a t e s seems l i k e l y . In c o t t o n s e e d , M a r t i n e z e t a l (12) have suggested the lowered s o l u b i l i t y o f p r o t e i n from h e a t e d seeds i s the r e s u l t o f c y t o p l a s m i c p r o t e i n s " g l u i n g " the c y t o p l asmic v a c u o l e s c o n t a i n i n g the s t o r a g e p r o t e i n s t o g e t h e r so t h e y cannot d i s p e r s e when the c e l l i s r u p t u r e d d u r i ng g r i n d i n g . In o r d e r t o i n c r e a s e the e x t r a c t i o n o f p r o t e i n from h e a t d e n a t u r e d s u b s t r a t e s , t e c h n i q u e s which c h e m i c a l l y o r m e c h a n i c a l l y break the c y t o p l a s m i c p r o t e i n s h o l d i n g the a l e u r i n v a c u o l e s t o g e t h e r must be developed. Should t h e i n s o l u b i l i z a t i o n be the r e s u l t o f the b i n d i n g o f i n d i v i d u a l p r o t e i n s o r s t o r a g e vacuol e s t o o t h e r c e l l u l a r c o n s t i t u e n t s (e.g. f i b e r p o l y m e r s ) , t h e s e bonds would a l s o have t o be b r o k e n . E x t r a c t i o n o f P r o t e i n from

Leaves

L e a f p r o t e i n s o c c u r t h r o u g h o u t the c y t o p l a s m o f i n d i v i d u a l c e l l s w i t h 30-40% o f the p r o t e i n l o c a l i z e d i n the c h l o r o p l a s t (16). E x t r a c t i o n o f t h e s e p r o t e i n s has been a c c o m p l i s h e d by p u l p i n g t h e l e a v e s f o l l o w e d by p r e s s i n g t o s e p a r a t e the p r o t e i n a c e o u s j u i c e from the l e a f f i b e r . The j u i c e i s then h e a t e d t o c o a g u l a t e the p r o t e i n and the c o a g u l a t e d p r o t e i n and j u i c e a r e s e p a r a t e d by a p p r o p r i a t e means (17). When t h e s e c h l o r o p l a s t p r o t e i n s a r e e x t r a c t e d , l a r g e amounts o f pigments a r e a l s o f r e e d from the c e l l u l a r m a t r i x t o y i e l d a green p r o d u c t . The p r e s e n c e o f t h e pigments has lowered l e a f p r o -

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

306

E N Z Y M E S IN

FOOD AND

B E V E R A G E PROCESSING

t e i n c o n c e n t r a t e a c c e p t a b i l i t y as a human f o o d . This has l e d t o development o f p r o c e s s e s w h i c h produce a f i b e r f r e e p r o t e i n - c a r o t e n o i d complex f o r use as an ani m a l f e e d (18,19,20,21,22). R e c e n t l y , a number o f s o l v e n t e x t r a c t i o n and d i f f e r e n t i a l h e a t i n g t e c h n i q u e s have been d e v e l o p e d t o produce c o l o r l e s s o r l o w - c o l o r e d p r o t e i n c o n c e n t r a t e s (23,24,25,26,27,28,29,30,31,32,33). P r o d u c t i o n o f any o f the above p r o d u c t s r e q u i r e s the a v a i l a b i l i t y o f non-heated l e a f p r o d u c t s . Approxi m a t e l y 45% o f the l e a f p r o t e i n can be e x t r a c t e d from f r e s h a l f a l f a l e a v e s (33) w h i l e o n l y 5-15% o f a v a i l a b l e p r o t e i n can be e x t r a c t e d from a l f a l f a d r i e d a t 75-150°C (34) . Approaches t o E x t r a c t i o n o f Heat Denatured P r o t e i n From Seeds and L e a v e s Two e x p e r i m e n t a i n experiments t o i n c r e a s e the y i e l d o f e x t r a c t e d p r o t e i n from h e a t - d e n a t u r e d seeds and l e a f s : enzymatic d i g e s t i o n and u l t r a s o n i c d i s p e r s i o n . C e l l u l a s e s and p r o t e a s e s have been used t o h y d r o l y z e p o r t i o n s o f the p l a n t m a t r i x t o a l l o w more e f f i c i ent d i s p e r s i o n and e x t r a c t i o n o f p r o t e i n s . Hang e t a l (35) showed pea bean p r o t e i n e x t r a c t a b i l i t y was i n c r e a sed by t r e a t m e n t w i t h A s p e r g i l l u s n i g e r c e l l u l a s e and Abdo and K i n g (36) and S r e e k a n t i a h e t a l , (37) reported i n c r e a s e d n i t r o g e n e x t r a c t i o n from soybeans, c h i c k p e a s and sesame f o l l o w i n g c e l l u l a s e t r e a t m e n t . Conversely Lu and K i n s e l l a (32) found a n o n - s i g n i f i c a n t i n c r e a s e i n a l f a l f a meal e x t r a c t a b i l i t y f o l l o w i n g c e l l u l a s e treatment. In our l a b o r a t o r i e s , c e l l u l a s e has n o t i n c r e a s e d p r o t e i n e x t r a c t i o n y i e l d s from a l f a l f a meal, screw e x p r e s s e d c o t t o n s e e d meal, o r hexane d e f a t t e d soybean meal. H y d r o l y s i s o f seed p r o t e i n s has been i n t e n s i v e l y investigated. A r z u e t a l (38) d e s c r i b e d the h y d r o l y s i s o f c o t t o n s e e d cake p r o t e i n s by 10 d i f f e r e n t p r o t e o l y t i c enzyme p r e p a r a t i o n s o f b a c t e r i a l , f u n g a l , p l a n t , and a n i m a l o r i g i n . A l l enzymes h y d r o l y z e d c o t t o n s e e d p r o tein. P r o t e o l y t i c enzymes have a l s o been shown t o hydr o l y z e soybean (39) and sesame meal (37) p r o t e i n s . In none o f t h e s e s t u d i e s was the e f f i c i e n c y o f p r o t e i n e x t r a c t i o n from a food s u b s t r a t e measured. The use o f u l t r a s o n i c energy t o i n c r e a s e s o l u b i l i z a t i o n o f p r o t e i n from h e a t d e n a t u r e d s o u r c e s i s a new development. Wang (10) i n c r e a s e d the e f f i c i e n c y o f p r o t e i n e x t r a c t i o n from a u t o c l a v e d soybean f l a k e s from 16% t o 58% by a p p l i c a t i o n o f u l t r a s o n i c waves. In a d d i t i o n , the u l t r a c e n t r i f u g e p a t t e r n s o f the e x t r a c t e d p r o t e i n s

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

18.

CHiLDS E T

AL.

307

Solubilization of Seed and Leaf Proteins

were t h e same o f t h o s e o f p r o t e i n s e x t r a c t e d from nona u t o c l a v e d samples by c o n v e n t i o n a l t e c h n i q u e s . M o l i n a and La Chance (40) i n c r e a s e d the e x t r a c t a b i l i t y o f p r o t e i n from h e a t t r e a t e d c o c o n u t meal by t r e a t m e n t w i t h a v a r i e t y o f p r o t e o l y t i c enzymes. In those e x p e r i m e n t s , the meal was i n c u b a t e d w i t h the p r o t e a s e and the r e s i d u e e x t r a c t e d w i t h d i l u t e a l k a l i . Y i e l d s o f up t o 80% were o b t a i n e d . In experiments w i t h c o t t o n s e e d meal and a l f a l f a meal (11,41), M o l i n a and L a Chanes's t e c h n i q u e s were modified (Figure 1). I n s t e a d o f s h a k i n g , the samples were s t i r r e d and s e p a r a t i o n o f the s o l i d s u b s t r a t e from the l i q u i d was a c c o m p l i s h e d by g r a v i t y f i l t r a t i o n through c o t t o n organdy r a t h e r than by c e n t r i f u g a t i o n .

25g Meal + Enzyme + 100 ml

liquid

I Stir

30 min.

a t 50°C

I Filter Filtrate (Enzyme F r a c t i o n )

Residue Suspend i n 100 0.75% NaOH Stir

30 min.

ml

of

a t 60oc

Filtrate (NaOH F r a c t i o n ) Figure 1. Flow sheet for proteolytic enzyme-chemical extraction of protein from heat-denatured substrates

P r o t e o l y t i c enzyme p r e t r e a t m e n t i n c r e a s e d e x t r a c t a b i l i t y o f p r o t e i n from h e a t d e n a t u r e d c o t t o n s e e d meal (Table I ) . W i t h o u t enzyme t r e a t m e n t , o n l y 15% o f the c o t t o n s e e d p r o t e i n was e x t r a c t e d by a two-step c h e m i c a l e x t r a c t i o n (11) . F i c i n t r e a t m e n t i n c r e a s e d the amount o f p r o t e i n e x t r a c t i o n a p p r o x i m a t e l y 2.5 t i m e s . Trypsin t r e a t m e n t was most e f f e c t i v e , c a u s i n g an approximate f i v e f o l d i n c r e a s e i n e x t r a c t i o n w i t h g r e a t e r than 65% of p r o t e i n b e i n g e x t r a c t e d .

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

308

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

T a b l e I . E f f i c i e n c y o f a two-step p r o t e o l y t i c enzyme-chemical t e c h n i q u e f o r e x t r a c t i o n o f p r o t e i n from c o t t o n s e e d and a l f a l f a meal p r o t e i n s . A l l d a t a a r e t h e mean o f t h r e e r e p l i c a t e s + s t a n d a r d d e v i a t i o n (From 1 1 ) .

Enzyme None Papain Ficin Trypsin

Total % Kjeldahl Protein Extracted C o t t o n s e e d Meal A l f a l f a Meal 15.1 16.0 42.7 69.4

+ + + +

2.3 2.4 1.8 5.7

21.9 23.6 34.1 60.2

+ + + +

4.6 2.8 11.3 5.7

Without enzyme f a p r o t e i n was e x t r a c t e meal, t r e a t m e n t w i t h p a p a i n d i d n o t markedly improve the e f f i c i e n c y o f e x t r a c t i o n (23.6%). F i c i n treatment i n c r e a s e d t h e amount o f p r o t e i n e x t r a c t e d a l m o s t twof o l d (34.1). T r y p s i n t r e a t m e n t was t h e most e f f e c t i v e , c a u s i n g an approximate t h r e e f o l d i n c r e a s e i n e x t r a c t i o n w i t h g r e a t e r than 60% o f a v a i l a b l e p r o t e i n b e i n g e x t r a cted. Parameters A f f e c t i n g t h e E n z y m a t i c - C h e m i c a l E x t r a c t i o n Process pH. E x p e r i m e n t s have been performed i n which t h e amount o f p r o t e i n e x t r a c t e d d u r i n g t h e enzymatic h y d r o l y s i s s t e p and the d i l u t e a l k a l i e x t r a c t i o n s t e p s were q u a n t i f i e d s e p a r a t e l y as a f u n c t i o n o f t h e pH o f t h e enzyme i n c u b a t i o n . T r y p s i n t r e a t m e n t was more e f f e c t i v e than f i c i n o r b r o m e l a i n a t pH's from 4.0 - 9.5 (Table I I ) . There was f i n i t e b u t l i t t l e v a r i a t i o n amongst the enzyme f r a c t i o n s . T r y p s i n t r e a t m e n t , howe v e r , r e s u l t e d i n a two t o t h r e e f o l d i n c r e a s e i n t h e p r o t e i n c o n t e n t o f t h e NaOH f r a c t i o n r e l a t i v e t o f i c i n or bromelain treatment. T h i s would s u g g e s t t h a t t r y p s i n a c t s on t h e s u b s t r a t e i n a manner which a l l o w s the p r o t e i n t o become more d i s p e r s a b l e i n d i l u t e a l k a l i . A t l e s s than pH 6.5, b r o m e l a i n was t h e most e f f i c i e n t i n t h e enzyme e x t r a c t i o n s t e p i n a l f a l f a meal (Table I I I ) . A t pH's g r e a t e r than 6.5, t r y p s i n and b r o m e l a i n were more e f f e c t i v e than f i c i n . As w i t h c o t t o n s e e d , t h e m a j o r i t y o f t h e p r o t e i n was e x t r a c t e d i n t h e d i l u t e a l k a l i s t e p s u g g e s t i n g the enzymes a r e a c t i n g i n a manner which a l l o w s t h e p r o t e i n t o become more d i s p e r s a b l e i n t h e a l k a l i .

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

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

2. 10+0.7

0.70+0.2

1.40+0.6

3.40+0.2

2.51+0.8

9.0

1. 80+0.9

0.60+0.2

1.30+0.7

3.40+0.4

2.48+0.9

8.0

1. 80+0.9

0.60+0.1

1.30+0.6

2.55+0.4

2.50+0.6

7.0

1.30+0.2

1. 60+1.2

0.40+0.1

1.15+0.7

2.60+0.6

2.40+0.4

6.0

1.10+0.3

1. 30+0.2

0.80+0.2

1.10+0.6

2.30+0.4

2.45+0.5

5.0

1.30+0.3

1.10+0.1

1.20+0.2

0.70+0.2

1. 30+0.6

0.70+0.2

1.20+0.8

2.05+0.1

1.80+0.4

4.0

1.40+0.3

0. 80+0.2

Enz .

0.50+0.2

NaOH F r a c .

Bromelain Frac. NaOH F r a c .

0.75+0.3

Enz.

Ficin Frac.

0.50+0.2

1.20+0.2

Enz.

3.0

pH

Trypsin Frac. NaOH F r a c .

Grams o f p r o t e i n e x t r a c t e d from 25 g o f c o t t o n s e e d meal i n a two s t e p p r o t e o l y t i c enzyme-chemical e x t r a c t i o n t e c h n i q u e u t i l i z i n g t r y p s i n , b r o m e l a i n , o r ficin. E x t r a c t a b i l i t y i s p r e s e n t e d as a f u n c t i o n o f t h e pH o f the enzyme r e a c t i o n media.

Table II

I—»

1

3

•Α

•ή.

CO

δ*

«Η.

r

ο

CO

η

S

3

00

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

0.53+0.1

0.62+0.1

0.95+0.1

0.97+0.1

1.10+0.1

0.72+0.1

0.78+0.1

0.92+0.2

1.00+0.2

1.05+0.2

1.05+0.2

4.0

5.0

6.0

7.0

8.0

9.0

1.22+0.2

0.52+0.2

0.75+0.1

Trypsin Frac. NaOH F r a c .

3.0

Enz.

0.98+0.1

0.49+0.2 0.80+0.1

0.48+0.1

0.98+0.1

0.55+0.2 0.83+0.1

0.92+0.1

0.90+0.2

0.55+0.2

0.53+0.1

0.52+0.1

1.10+0.1

0.82+0.1

0.81+0.1

0.41+0.0

0.68+0.1

1.00+0.1

0.43+0.2

0.80+0.0

0.68+0.1

0.74+0.1

0.39+0.1

0.38+0.0

1.20+0.3

Bromelain NaOH F r a c . Frac.

0.72+0.2

Enz.

0.72+0.1

Ficin Frac. NaOH F r a c .

0.34+0.1

Enz.

Grams o f p r o t e i n e x t r a c t e d from 25 g o f a l f a l f a meal i n a two s t e p p r o t e o l y t i c enzyme-chemical e x t r a c t i o n t e c h n i q u e u t i l i z i n g t r y p s i n , b r o m e l a i n o r f i c i n . E x t r a c t a b i l i t y i s p r e s e n t e d as a f u n c t i o n o f t h e pH o f t h e enzyme r e a c t i o n media.

Table I I I

18.

CHiLDS E T

AL.

311

Solubilization of Seed and Leaf Proteins

One p o s s i b l e h y p o t h e s i s t o e x p l a i n the above d a t a i s t h a t the p r o t e i n s a r e bound t o a polymer l i g a n d a f t e r h e a t treatment and the p r o t e o l y t i c enzyme c l e a v e s the two m o l e c u l e s . A c a n d i d a t e polymer might be d i e t a r y fiber. To t e s t t h i s h y p o t h e s i s , a N e u t r a l D e t e r g e n t F i b e r (NDF) e x t r a c t o f c o t t o n s e e d meal was prepared (Van S o e s t , 1973). C o t t o n s e e d meal samples were a l s o produced from the e x t r a c t i o n r e s i d u e s . I t was n o t e d t h a t the Ν c o n t e n t o f the NDF samples from t r y p s i n t r e a t e d c o t t o n s e e d meal was much lower than t h a t from o t h e r samples (41) . T h e r e f o r e , t r y p s i n was a b l e t o c l e a v e p r o t e i n - f i b e r bonds more e f f i c i e n t l y than o t h e r enzymes. T h i s s t r o n g l y s u g g e s t s , b u t n o t c l e a r l y prove, the s u p e r i o r performance o f t r y p s i n may be based on i t s a b i l i t y to c l e a v e f i b e r - p r o t e i n bonds. T h i s a c t i o n could r e l e a s e non-hydrolyzed p r o t e i n r a t h e tha s o l u b l e fragments; an e x h i b i t i t s normal non-denatured s o l u b i l i t y c h a r a c t e r istics in dilute alkali. T h i s h y p o t h e s i s i s g i v e n add i t i o n a l c r e d e n c e by r e c e n t o b s e r v a t i o n s t h a t 60% o f N i t r o g e n s o l u b i l i z e d from c o t t o n s e e d by t r y p s i n t r e a t ment has a m o l e c u l a r w e i g h t g r e a t e r than 1000 d a l t o n s (42) . R e l a t i o n s h i p o f enzyme c o n c e n t r a t i o n t o p r o t e i n extraction. The r e l a t i o n s h i p o f enzyme c o n c e n t r a t i o n to p r o t e i n e x t r a c t i o n i n c o t t o n s e e d was c u r v i l i n e a r (Table I V ) . Treatment w i t h a t r y p s i n c o n c e n t r a t i o n o f 0.03g/25g meal caused g r e a t e r than 75% o f the p r o t e i n to be e x t r a c t e d . A t g r e a t e r than 0.03g enzyme/25g meal, t h e r e was a lowered r a t e o f i n c r e a s e i n p r o t e i n e x t r a c t ability.

Table

IV

E f f e c t o f T r y p s i n C o n c e n t r a t i o n on the E f f i c i e n c y o f a Two-Step T r y p s i n - C h e m i c a l E x t r a c t i o n o r P r o t e i n From C o t t o n s e e d and A l f a l f a Meal Amount o f Trypsin/25g Substrate 0 0.01 0.03 0.06 0.12 0.24

T o t a l % Kjedahl P r o t e i n Extracted Cottonseed 16.4 18.2 78.4 82.6 86.5

+ + + + +

2.3 1.7 3.1 4.3 6.1

Meal

Alfalfa 21.8 27.3 41.7 57.5 64.3 73.4

+ + + + + +

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

Meal 2.1 1.4 4.8 7.3 2.8 4.8

312

ENZYMES

IN FOOD A N D B E V E R A G E

PROCESSING

The r e l a t i o n s h i p o f t r y p s i n c o n c e n t r a t i o n t o e x t r a c t a b l e p r o t e i n was q u i t e d i f f e r e n t f o r a l f a l f a meal ( T a b l e I V ) . The e f f i c i e n c y o f the p r o c e s s i n c r e a s e d w i t h i n c r e a s i n g amounts o f t r y p s i n up t o 0.24g t r y p s i n / 25g a l f a l f a meal. T h i s i s 8 times the c o n c e n t r a t i o n used i n c o t t o n s e e d meal. E f f e c t o f U l t r a s o n i c Energy on the Enzyme-Chemical Technique Because o f the p o t e n t i a l advantages a s s o c i a t e d w i t h use o f the u l t r a s o n i c t e c h n i q u e (e.g., c a v i t a t i o n o f sample t o c r e a t e new s i t e s f o r enzyme a c t i v i t y , i n creased mixing e f f i c i e n c y , decreased r e a c t i o n times, and r e d u c e d energy e x p e n d i t u r e s ) , e x p e r i m e n t a l s t u d i e s have been performed t o determine the e f f e c t s o f u l t r a s o n i c energy on the nique i n cottonseed soybea e n z y m a t i c - c h e m i c a l - u l t r a s o n i c t e c h n i q u e i n c r e a s e d the e f f i c i e n c y o f t o t a l p r o t e i n e x t r a c t i o n from c o t t o n s e e d meal o v e r enzyme use a l o n e ( T a b l e V ) . In soybean meal, the i n c r e a s e i n e x t r a c t i o n e f f i c i e n c y was a p p r o x i m a t e l y 10%.

Table V S o l u b i l i z a t i o n o f c o t t o n s e e d and soybean meal p r o t e i n by e n z y m a t i c - c h e m i c a l t e c h n i q u e s w i t h and w i t h o u t u l t r a s o n i c energy

Technique T r y p s i n , no ultrasonic energy T r y p s i n , 200 accoustical watts

Total % Kjeldahl Protein Extracted C o t t o n s e e d Meal Soybean Meal

65.66+4.98

80.72+3.85

72.99 + 3.18

91.32

+

4.70

The p r e s e n c e o f u l t r a s o n i c energy a l s o i n c r e a s e s the r a t e o f t h e e n z y m a t i c - c h e m i c a l p r o c e d u r e w i t h o u t u l t r a s o n i c energy, a p p r o x i m a t e l y 120 minutes was r e q u i r e d f o r the e x t r a c t i o n (11,42,4 3 ) . With the a d d i t i o n o f u l t r a s o n i c energy, t h e r e were no s i g n i f i c a n t d i f f e r ences i n the i n the p e r c e n t a g e p r o t e i n e x t r a c t e d f o r s o n i c a t i o n times v a r y i n g from 1 - 1 6 min o f t r y p s i n c o t t o n s e e d meal m i x t u r e s ( T a b l e V I ) . These d a t a s u g g e s t

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

18.

CHILDS ET AL.

313

Solubilization of Seed and Leaf Proteins

t h a t s o n i c a t i o n times o f 1 min a r e adequate. The same was t r u e f o r soybean meal where 90% o f a v a i l a b l e p r o t e i n was e x t r a c t e d i n one minute. Therefore, u l t r a s o n i c energy i n c r e a s e s t h e e f f i c i e n c y o f enzyme s o l u b i l i c a t i o n procedures.

T a b l e VI E f f e c t o f S o n i c a t i o n Time on E x t r a c t i o n o f P r o t e i n From C o t t o n s e e d and Soybean Meal Sonication Time (Min) 1 2 4 8 12 16

Total % Kjeldahl Protein Extracted C o t t o n s e e d Meal Soybean Meal 70.0

+ 1.2

93.7

+ 4.2

73.2 61.3 71.6

+ 6.1 + 9.0 + 7.8

94.8

+ 2.7

Functionality of Extracted

Protein

P r o t e i n e x t r a c t e d from c o t t o n s e e d meal by t h e enzy m a t i c c h e m i c a l p r o c e s s i s q u i t e f u n c t i o n a l . The s o l u b i l i t y o f p r o t e i n c o n c e n t r a t e s produced by i s o e l e c t r i c p r e c i p i t a t i o n was t y p i c a l o f s o l u b i l i t y o f c o t t o n s e e d p r o t e i n as a f u n c t i o n o f pH (42). S o l u b i l i t y was minimal a t a c i d i c pH's and i n c r e a s e d i n b a s i c pH's (Crenwelge e t a l . , 1974). There were no marked d i f f e r ences i n s o l b u i l i t y i n 0.1 M NaCl v s w a t e r . T h i s sugge s t s l i t t l e o r no i n t e r a c t i o n between pH and i o n i c strength. There were no s i g n i f i c a n t d i f f e r e n c e s i n t h e w a t e r o r o i l - h o l d i n g c a p a c i t i e s o f t h e samples (Table V I I ) . However, t h e e m u l s i f y i n g c a p a c i t y o f t h e NaOH f r a c t i o n produced by t h e combined t e c h n i q u e was s i g n i f i c a n t l y g r e a t e r than t h a t o f o t h e r f r a c t i o n c o n c e n t r a t e s . It has o f t e n been s u g g e s t e d t h a t e m u l s i f y i n g c a p a c i t y i s a f u n c t i o n o f m o l e c u l a r r a d i u s (45) and t h e s e d a t a tend to c o n f i r m t h a t o b s e r v a t i o n s i n c e t h e h i g h e s t e m u l s i f i c a t i o n c a p a c i t y was n o t e d w i t h t h e c o n c e n t r a t e having the l o w e s t amount o f low m o l e c u l a r w e i g h t n i t r o g e n ( 4 2 ) .

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

ENZYMES IN FOOD AND BEVERAGE PROCESSING

314

Table V I I F u n c t i o n a l i t y o f c o t t o n s e e d meal p r o t e i n i s o l a t e s p r e p a r e d by the p r o t e l y t i c enzyme-chemical t e c h n i q u e (PC) and the u l t r a s o n i c - e n z y m a t i c (UE) t e c h nique. ( A l l d a t a a r e t h e mean o f t h r e e r e p l i c a t e s + standard deviation) Oilholding capacity (ml/g)

Sample Enzyme fraction (PC) NaOH fraction (PC) NaOH fraction (UE)

4.03

+ 1.10

Waterholding capacity (ml/g)

2.43

+ 0.86

Emulsifying capacity (ml/g)

333

+

4.0

3.53 + 0.32

2.80 + 0.26

443 + 4.0

3.66 + 0.15

2.73 + 0.15

586 + 2.5

In summary, i n v e s t i g a t i o n s w i t h e x t r e m e l y h e a t de n a t u r e d s u b s t r a t e s (NSI 0.15) such as c o t t o n s e e d meal, a l f a l f a meal and soybean meal have shown t h a t t r y p s i n t r e a t m e n t w i l l a l l o w e f f i c i e n t p r o t e i n e x t r a c t i o n . The e f f i c i e n c y o f t h i s e x t r a c t i o n can be markedly i n c r e a s e d by i n t e r f a c i n g u l t r a s o n i c energy w i t h t h e system. Iso l a t e s produced from the e x t r a c t e d p r o t e i n s a r e f u n c t i o n al. REFERENCES 1. Koshiyama, I. 1972. Agri. Biol. Chem. 36:62. 2. Altschul, A.M., N . J . N e u c e r e , A.A. Woodham, and J.M. Dechary. 1964. N a t . 203:501. 3. Daussant, J., N . J . N e u c e r e , and L . Y . Y a t s u . 1969. P l a n t P h y s i o l . 44:471. 4. S t . A n g e l o , A.J., L . Y . Y a t s u , and A.M. Altschul. 1968. A r c h . Biochem. B i o p h y s . 124:199. 5. V a r n e r , J.E. and G. S c h i d l o v s k y . 1963. P l a n t P h y s i o l . 38:139. 6. Graham, Τ . A . and B . E . S . Gunning. 1970. N a t . 228:81. 7. M e t z g e r , Η . , M.B. S h a p i r o , J.E. Mosiman, and J.E. V i n t o n . N a t . 219:1166. 8. D i e c k e r t , J.W. and M.C. D i e c k e r t . 1976. J Fd Sci. 41:475. 9. Sugarman, N. 1956. U.S. P a t e n t 2, 762, 820.

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

18.

CHILDSETAL.

Solubilization of Seed and Leaf Proteins

315

10. Wang, L.C. 1975. J F d Sci. 40:549. 11. C h i l d s , E.A. 1975. J Fd Sci. 40:78. 12. M a r t i n e z , W . H . , Berardi, L.C. and L.A. G o l d b l a t t . 1970. J A g r F d Chem. 1 8 : 9 6 1 . 13. V i x , H.L.E. 1968. Oil Mill G a z . 72:53. 14. M a r t i n e z , W . H . 1969. P r o c . XVII C o t t o n P r o c Clin. ARS Report 72-69:18. 15. Circle, S.J. and A.K. S m i t h . 1972. "Soybeans:Chemistry and T e c h n o l o g y . " 638. AVI P u b l i s h i n g C o . 16. N e u r a t h , H. and K. Bailey. 1973. "The P r o t e i n s . " 542. Academic P r e s s . 17. Pirie, N.W. 1971. "Leaf P r o t e i n : I t s Agronomy, P r e p a r a t i o n , Q u a l i t y and U s e . " IBP Handbook #20. Blackwell Scientific. 18. K n u c k l e s , B.E., R.R. S p e n c e r , M.E. L a z a r , E.M. Bickoff. and G.O. K o h l e r 1970 J A g r F d Chem 18:1086 19. K o h l e r , G.O. an Congress o f Foo 20. Lazar,M.E., R.R. S p e n c e r , B.E. K n u c k l e s , and E.M.J. B i c k o f f . 1971. J A g r Fd Chem. 19:944. 21. Miller, R.E., R.H. Edwards, M.E. L a z a r , E.M. Bickoff, and G.O. K o h l e r . 1972. J Agr Fd Chem. 20:1151. 22. S p e n c e r , R.R., A.C. M o t t o l a , E.M. B i c k o f f , J.P. C l a r k , and G.O. K o h l e r . 1971. J A g r Fd Chem. 19:504. 23. Chayen, I.H., R.H. S m i t h , G.R. T r i s t r a m , D. Thirkell, and T . Webb. 1961. J S c i Fd A g r . 12:502. 24. Hartman, G.H., W.R. A k e s o n , and M.A. Stohmann. 1967. J A g r F d Chem. 1 5 : 7 4 . 25. Huang, K.T., M.C. T a o , M. B o u l e t , R.R. Riel, J.P. Julien, and G.J. G r i s s o m . 1971. Can I n s t Fd T e c h . 4:85. 26. W i l s o n , R.F., and J.M.A. Lilley. 1965. J Sci F d A g r . 16:173. 27. B y e r s , M. 1967. J S c i Fd A g r . 18:34. 28. Cowlishaw, S.J., D.E. E y l e s , W . F . Raymond, and J.M.A. Tilley. 1956. J S c i F d A g r . 7:775. 29. deFremery, D., E.M. B i c k o f f , and G.O. K o h l e r . 1973. J A g r F d Chem. 20:1155. 30. Henry, K.M. and J.E. F o r d . 1965. J Sci Fd Agric. 16: 425. 31. L e x a n d e r , K., R. C a r l s o n , V . S c h a l e n , A. Simonsson, and T . L u n d b o r g . 1970. A n n a l . A p p l . Biol. 66:193. 32. Subba-Rau, B.H., S. Mahadeviah, and N . S i n g h . 1969. J S c i F d A g r . 20:355. 33. Edwards, R.H., R.E. Miller, D. deFremery, B.E. K n u c k l e s , E.M. B i c k o f f , and G.O. K o h l e r . 1975. J Agr F d Chem. 23:620. 34. L u , P.S. and J.E. Kinsella. 1972. J Fd Sci. 37:94. 35. Hang, Y.D., W . F . W i l k e n s , A.S. Hill, K.H. S t e i n k r a u s , and L.R. H a c k l e r . 1970. J A g r Fd C h . 1 8 : 9 .

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

316

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

36. Abdo, K.M. and K.W. K i n g . 1967. J A g r Fd Chem. 15:83. 37. S r e e k a n t i a h , K.R., H. E b i n e , J. O h t a , and M . Nakans. 1969. F d T e c h . 23:1055. 38. A r z u , Α., H. Mayorga, J. G o n z a l e s , and C . R o l z . 1972. J A g r F d Chem. 20:805. 39. F u j i m a k i , M . H. K a t o , S. Arai, and E. Tamaki. 1968. Fd T e c h . 22:889. 40. M o l i n a , M . R . and P . A . L a C h a n c e . 1973. J Fd Sci. 38: 607. 41. C h i l d s , Ε . A . 1976. U n p u b l i s h e d d a t a . 42. C h i l d s , E.A. and J.L. Forte'. 1976. J F d Sci. 41: 652. 43. K u , Y . and E.A. Childs. 1976. Tenn Fm Hm Sci. 97:26. 44. C r e n w e l g e , D.D., C . W . Dill, P . T . T y b o r , and W . A . Landmann. 1974. J Fd Sci. 39:175 45. C a r p a n t e r , J.L. an

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

INDEX A Acetaldehyde 2 Acid phosphatase 104 Adenosine-5'-monophosphate (AMP) 114 5'-Adenylate deaminase 114 Alcalase 200 Alcohol 76 dehydrogenase 20,21 aldehyde-alcohol conversio liquid foods by 132 NAD oxidoreductase reaction 2 Aleurone 90 Alfalfa meal 306 Alkaline phosphatase 217 Alkylamine glass 105 Allopolyploids as bridge species 224 Alumina 105 Amberlite IR-45 118 Amine content of various sausages 292 Amino acid(s) enrichment 263 production of L 103, 111 in sausages, changes in the con centration of free 290 Aminoacylase 103, 111 Ammonia 266 Amylases in barley and wheat 80 immunochemical characterization of 80 α-Amylases 108,117 antigen characteristic for germinated seeds 94 bacterial 93 in cereals during germination 90 fungal 93 in wheat seeds 89 β-Amylases 117,206 Anaerobic metabolism 1 Anthocyanase 193 Antioxidants 233 Apohemoglobins 296 Appearance of fruits and vegetables .. 193 Apple(s) 159 calcium uptake in 203 development 173 Golden Delicious 201 juice 136 processing applications 206

Apple(s) (Continued) slices during processing, texture modification of softening varieties, Appalachian Arabica coffees, extractable lipids of .. Arachin

203 195 195 37 215

Asparagus fiber formation in postharvest Astringency in coffee Autoradiograms of apple pieces Auxin Avocado fruit polygalacturonase in

149 151 33 198 159 163 185

198

Β Baking industry 117 Banana fruit 147,159 Barley seeds, freeze-dried 92 Barley and wheat, a- and βamylases in 80 Beans 149 Beer 117,136 production 76, 80 Beet sugar industry I l l , 113 Berkeley process for enzymatic con version of cellulose-glucose 121 Biochemical changes in grapefruit 2 Black tea 12 aroma, formation of 18 Blanching 251 of peanuts 241 Blood in foods as combined protein and iron fortifier and coloring agent 295 Blood of slaughtered animals 295 Blue cheese 61 Bologna 281 Braunschweiger 291 Breadmaking 80,89,244 lipoxygenase in 246 Brewing industry 117 Brewing, malt in 80 Bromelain 309 Butter 61 Butylated hydroxyanisole (BHA) 233 Butylated hydroxytoluene (BHT) 233

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

318

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

c Caffeine 31 Calcium 81,149,175,184 carbonate 104 chloride 152 -firming plant tissue 198 tolerance 255 uptake in apples 203 Carbohydrate modification of soy products 255 Carbon dioxide 155,192 Carbonyls 288 Carboxyesterase 5 Carboxymethy cellulose 117 Catalytic activity of peroxidases 145 CDHP content of peanut butters 234 Cellulases 23,177,194,306 Cellulose(s) degradation 121 -glucose Berkeley, process fo enzymatic conversion of 121 substituted 103 Cereals during germination, α-amylases in 90 Cheddar cheese 61,73 Cheese manufacturing 120 Chemical components of green coffee 35 Chickpeas, nitrogen extraction from .. 306 Chitin 104,119 Chlorogenic acid 31 Chlorophyl 193 Chlorophyllase 193 Chorley wood bread process 247 α-Chymotrypsin 104 Citric acid 3 Citrus products, enzymes affecting flavor and appearance of 1 Cocoa 29 Coffea arabica L 27,31 canephora 27,31 dewevrei 31 liberica 27,31 Coffee(s) aroma and taste 48 bean, physical measurements of the whole 37 beans, mechanical injury to 48 chemical components of green 35 cherry 32 classifications 32 discoloration 46 drying process of 43 enzyme activities in 30 enzymes and quality 27 fermentation process of 41 fruit, harvesting and processing of 29 -handling processes 30

PROCESSING

Coffee(s) (Continued) oil, carbonyls in 46 physical and chemical changes of green 47 roasting process, enzyme activities of 48 spoiled 46 storage, role of enzymes during 44 washed 38 whiteners 61 Cold storage on peroxidation of fatty acids in raw peanuts, effect of 239 Collagen 106,119 Conjugated diene hydroperoxide (CDHP) 230 Copper 63,231 Corn starch 112 Corn syrup, high-dextrose 112 Cranberries, polygalacturonase in 186 Cucumbers, polygalacturonase in 186 Curdling of milk 120 Cured meat 279 Cyanide 236 Cytidine-5'-monophosphate (CMP) .. 114 Cytokinin 159 D Dairy products, lactase in the manufacture of Dates, polygalacturonase in DEAE-dextran 5-Dehydroshikimate reductase Dextran Diamine oxidase Diethylaminoethyl (DEAE) substituents Diffusional effects of surface-bound and entrapped enzymes Diphenol oxidase substrate specificity of orange Disease in plants Distilling industry Drying of meat

67 185 117 21 117 271 103 123 5 6 213 117 279

Ε Edible quality of tissue Edistin from hemp seed Elastin Electrophoretic analyses of extracts of germinated and developing wheat seeds Electrophoretic studies of isoenzymes in higher plants, esterases in

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

147 304 106 90 213

319

INDEX

Endocellulases 179 Endogenous enzymes in fruit and vegetables processing 192 Endosperm 90 Enzymatic conversion of cellulose-glucose, Berkeley process for the 121 hydrolysis of lactose 67 hydrolysis of ox-blood hemoglobin .. 295 modification of milk flavor 57 treatment by consumers 62 Enzyme(s) -active soy flours 245 activity (ies) in coffee 30 in coffee roasting process 48 in rabbit erythrocytes 211 affecting flavor and appearance of citrus products 1 as biological indicators, oilsee in black tea manufacture 1 books on food 100 in cell wall changes of ripening fruits 177 in citrus processing 7 in coffee fermentation 39 during coffee storage, role of 44 commercial application of immobilized Ill diffusional effects of surfacebound end entrapped 123 entrapment 109 in food processing 100 proposed uses of immobilized 115 in fruit and vegetable processing, endogenous 192 in fruit softening 172 home use of 62 immobilization of 140 immobilized 100,192 with impact on milk flavor 57,59 in instant tea manufacture 21 iron 144 in juice 4 methods to assess marine food quality 266 -modified soy products 255 multiplicity 210 and oxidative stability of peanut products 229 pectic 179 production and purification 101 saccharifying 112 in solubilization of seed and leaf proteins 304 in soybean processing 244 supports 102 inorganic 104 polysaccharide 103

Enzyme(s) (Continued) systems, immobilized starchhydrolyzing 114 systems related to fruit softening .... 195 Esterase(s) detecting 211 in electrophoretic studies of isoenzymes in higher plants .... 213 nonspecific 211 patterns 224 Ethanol 75 content in juice 2 Ethylene 155 wound 151 Extracted protein, functionality of .... 313 F Fat in coffee free(FFA) hydroperoxide oxidation, catalysts of peroxidation, linoleic acid in unsaturated Fermentation nitrate reduction in sausage process of coffee products in the fruit, accumulation of of sausages lactic acid production in substrate, whey as a Fiber formation in postharvest asparagus Fibrous proteins Fibrovascular tissue in vegetables .... Ficin Fish fresh freshness meter tester, intelectron Flavor of citrus juice of fruits and vegetables of hydrolyzed-lactose milk thresholds Floral primordium Flour enzyme-active soy Food by alcohol dehydrogenase, aldehyde-alcohol conversion in liquid application of soy products flavor ingredients lipids

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

31 288 230 231 233 132 283 41 1 281 282 74 151 106 149 308 266 267 267 4 193 70 3 172 90 245

132 257 133 245 229

320

ENZYMES

Food (Continued) preservation processing, proposed uses of immobilized enzymes in quality, enzyme methods to assess marine quality test, rapid marine technologist Fruit(s) accumulation of fermentation products in the cell wall structure of development of enzymes in cell wall changes of ripening intact pectinesterase in physical stress on respiration ripening and senescence, peroxidase in softening enzymes in enzymes systems related to polysaccharides in texture in tractor trailers -and vegetable(s) appearance of flavor of nutritional quality of peroxidase in postharvest processing, endogenous enzymes in products, lipase in Fudges Furanoterpenoid stress metabolites .... G Galactose utilization in yogurts α-Galactosidases enzyme source of β-Galactosidase Galacturonans Galacturonic acid Galacturonosylgalactose Galacturonosylxylose Gel electrophoresis for plant speciation entrapment filtration Genetic crossability Genoa salami Germination, α-amylases in cereals during Globulins from soybeans

279 115 266 274 144 1 174 173 177 1 180 1 1

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

PROCESSING

Glucoamylase 104,118 Glucosamine 104 Glucose 71,103,144,282 analyzers 120 isomerase 103,112 production from soybean whey 258 Glucuronic 176 Glutaraldehyde 104 Glycosidases 38 Gossypium 221 Grape 159 Grapefruit, biochemical changes in .... 2 Grapefruit bitterness 9 Green pea 132 "Greening" of sausage 283 Ground beef 292 Guanosine-5'-monophosphate (GMP) 114 H

15 187 172 195 195 195 1

coffee fruit Hematin Hemicellulases Hemicellulose degradation Hemin Hemoglobin, enzymatic hydrolysis of ox-blood 193 Hemp seed, edistin from 193 Hepatotoxins 193 n-Hexanal 143 Hexane-alcohol azeotrope extraction of defatted soy flakes 192 n-Hex-frans-2-enal 194 n-Hex-irarw-2-enol 62 High-dextrose corn syrup 155 High-fructose corn syrup High-maltose syrups Histamine content of sausages Hollow fibers 71 Horseradish 73 Hydrogels 38,113,244 Hydrogen donors I l l Hydrolyzed fats 187 Hydroperoxides 195 Hydroxylation reactions 175 Hydroxyproline 175 Hypoxanthine analysis 175 209 221 108 296 221 279 90 304

29 296 194 121 299 295 304 155 138 252 135 135 112 113 117 291 109 145 108 145 61 146 146 176 267

I IAA oxidase Immobilization of enzymes Immobilized enzyme(s) activity in food processing, proposed uses of half-life of

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

157 140 100,192 122 115 125

321

INDEX

Immobilized (Continued) -starch hydrolyzing enzyme systems xanthine oxidase Immunoabsorption Immunochemical characterization of a- and β-amylases Immunoelectroabsorption Immunoelectrophoretic analysis of extracts of germinated and developing wheat seeds Inorganic enzyme supports Inosine-5'-monophosphate (IMP) Insect problems Instant tea Iron enzymes fortifier and coloring agent, blood in foods as combined protein and -porphyrin group present in hemoglobin Isoenzymes in higher plants, esterases in electrophoretic studies of as multiple enzyme forms, identifying of peroxidase Italian sausages

114 271 82 80 86 88 90 104 114 221 12 231 144 295 29

M

213 210 146 281

Κ

Keratin Kidney toxins

Linoleic acid in fatty acid peroxidation 233 Linoleic acid hydroperoxide lyase 237 Lipase action in milk 60 Lipase in fruit and vegetable products 194 Lipid(s) of Arabica coffees, extractable 37 binding, effect of soy lipoxy genases on 249 oxidation 136 peroxidation on protein solubility, effect of 237 peroxides 229 of various sausages 287 Lipolysis 20,285 in milk 60 Lipoxygenase 231, 244,245 in raw peanuts 233 stability 235

107 155

L Lactase(s) (/?-D-galactosidase) .62,67,104 hydrolysis, modification of whey by 73 purity of 69 sources and properties 68 Lacti-aid 62 Lactic acid bacteria 280 Lactic acid production in sausage fermentation 282 Lactose 59 -derived sirups 74 enzymatic hydrolysis of 67 intolerance 69 low sweetness level of 74 milk, flavor of hydrolyzed70 in whole or skim milk, hydrolysis of 69 Lard, fresh 285 Leaves, protein from 305 Legumin from pea seeds, vicilin and 304 Leucine aminopeptidases 215,219 Lignin degradation 121 Lignin synthesis 147,159 Limonoate dehydrogenase 10

Malate:NAD oxidoreductase reaction 3 Malic enzyme 2 Malonaldehyde 230 Malt 80 Maltose 117 Mango 159 Manufacturing process of black tea ... 13 Margarines 61 Marine food quality enzyme methods to assess 266 index 267 rapid 274 Marine food spoilage 271 Mass transfer coefficient (k ) 123 Metalloproteins 231 Maturation and ripening, activation of pectinesterase during 206 Meat substitutes, cheap proteins as .... 295 Methanol 199 L-Methionine Ill N N'-Methylene bis(acrylamide) 108 Methyl esters 180 Microbial pellets 110 Microcompartmentation 147 Microfibrils 174 Microencapsulation 141 Milk 136,145 beverage use 69 chocolates 61 curdling of 120 fat, emulsions of 61 flavor enzymatic modification of 57 enzymes with impact on 57,59 c

f

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

322

ENZYMES

Milk (Continued) free fatty acids in 61 of hydrolyzed-lactose 70 oxidative stability of 64 powder 71 products 69 cultured 71 proteases 62 Mineral salts 106 5'-Mononucleotide flavor enhancers .. 114 Monosodium glutamate 114 Mucopolysaccharides 106 Ν NAD oxidoreductase 2 NADH 4 Naringinase 9 Natural coffee(s) 30,32 Nickel oxide 10 Nickel screens 10 Nicotinamide adenine dinucleotide (NAD) 20,132 Nicotinamide adenine dinucleotide phosphate (NADP) 20 Nitrate-reducing micrococci 280 Nitrate reduction in sausage fermentation 283 Nitrite 279 Nitrogen 192 compounds during ripening of Belgian salami, nonprotein 290 extraction from soybeans, chick peas, and sesame 306 Solubility Index (NSI) 250 Nitrosohemochrome 279 Nutritional value of foods, improved 229 of fruits and vegetables 193 of low-quality proteins, plastein reaction to improve the 263 Ο

Odor analysis detection concentration of alde hydes and alcohols development notes Oilseed enzymes as biological indicators Oilseeds, protein from Oleinase Orange Organoleptic evaluation of catfish Ox-blood hemoglobin, enzymatic hydrolysis of Ox blood, ultrafiltration of

133 133 194 136 209 304 57 133 267 295 300

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

Oxidatic reactions 146 Oxidative stability of peanut products, enzymes and 229 Oxygen 192 Ρ

Papain 102,308 Peach (es) 173 pectin solubility in 179 polygalacturonase in 184 Peanut(s) 213 arachin from 304 blanching of 241 butter(s) 231 CDHP content of 234 effect of cold storage on peroxida tion of fatty acids in raw 239 isoenzyme-cyanide experiments 236 stability of 229 Pear(s) 149,159 polygalacturonase in 186 Pea seeds, vicilin and legumin from .... 304 Pectic enzymes 179 Pectin 7,201 lyases 197 methyl esterase 20 solubilization 179 structure of 175 Pectinases 23,194 Pectinesterase 7,193 during maturation and ripening, activation of 206 in fruits 180 Pectolytic enzymes 182 cis-cw-l,4-Pentadiene 245 systems 233,234 Pepperoni 280 Peptidase 21 Pericarp 90,172 Peroxidase ( s ) 4,19,23,213,231,244 activity in maturing soybeans 253 catalytic activity of 145 cellular localization of 147 in fruit ripening and senescence 159 isoenzymes of 146 in postharvest fruits and vegetables 143 Peroxidatic reactions 145 Peroxidation, causes of 230 Peroxidation of fatty acids in raw peanuts, effect of cold storage on 239 Peroxide value (PV) determination .. 230 Phenolic resins 107 Phenolic substances 149 L-Phenylalanine Ill Phenylalanine ammonia lyase 21 5'-Phosphodiesterase 114

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

323

INDEX

Phosphogluconate dehydrogenase 217 Phospholipase C and D 63 Phosphotidylcholine 63 Physical stress on fruit 1 Plant pathogens 221 Plant speciation, gel electrophoresis for 221 Plastein reaction to improve the nutritional value of low-quality proteins 263 Plastein soy products 257 Pollution, problems brought on by 221 Polyacrylamide gels 118 Polygalacturonase ( s ) 8,197 in avocado 185 in cucumbers 186 in cranberries 186 in dates 185 in peaches 184 in pears 18 in tomatoes 182 Polyphenol oxidase 22,29, 31, 43,51 Polysaccharide enzyme supports 103 Polysaccharides in fruit softening 195 Poly (vinyl alcohol) gels 118 Poly (vinyl pyrrolidone) gels 118 Porous ceramic carriers 105 Porous glass 104 Potato 133 chips, shelf life of 233 Production of L-amino acids 103, 111 Production and purification, enzyme .. 101 Proteases 306 milk 62 Protein(s) Dispersibility Index (PDI) 250 fibrous 106 functionality of extracted 313 genetically independent 210 from heat-denatured substrates, extraction of 307 and iron fortifier and coloring agent, blood in foods as combined .... 295 from leaves 305 as meat substitutes, cheap 295 from oilseeds 304 plastein reaction to improve the nutritional value of low-quality 263 in sausages 288 soy 244 stability, effect of lipid peroxidation on 237 Proteolytic enzymes 120 Proteolytic modification of soy products 255 Purification, enzyme production and .. 101 Pyruvic decarboxylase 5

Q

Quinones

51 R

Rabbits Raffinose-sucrose hydrolysis Rancidity Red blood cells Rennets Rennin Resazurin Rhamnogalacturonan Ribonuclease RIN mutant Ripening, activation of pectinesterase during maturation and RNA, messenger

92 Ill 230 296 120 102 274 177 114 169

Saccharifying enzymes Salami nonprotein nitrogen compounds during ripening of Belgian Salting of meat Sand as a silica support Sausages amine content of various changes in the concentration of free amino acids in fat in fermentation of lactic acid production in nitrate reduction in and ripening "greening" of histamine content of lipids of various pH of commercial protein in semi-dry tyramine content in Sclereid development, excessive Seafood quality Seed deterioration, fungal-caused Semi-dry sausages Senescence, peroxidase in fruit ripening and Sephadex Sesame, nitrogen extraction from Shelf-life stability Shortening Silica Sirups, lactose-derived Smoking of meat S0 Solubilization of seed and leaf proteins, enzymes in

112 279

2

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

206 169

290 279 105 292 290 285 281 282 283 279 283 291 287 282 288 281 291 149 266 217 281 159 103 306 229 247 104 74 279 192 304

324

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

Sonication Soy flakes, hexane-alcohol azeotrope extraction of defatted globulins from lipoxygenases in lipid binding, effect of meal, hexane-defatted nitrogen extraction from peroxidase activity in maturing processing, enzymes in products enzyme-modified food applications of plastein whey, glucose production from Speciation Spoilage, marine food Starch preparation of sugar syrups fro Stress metabolites, furanoterpenoi Substrate flux Sucrose Sugar solutions, sweetness of Sugar syrups from starch, prepara tion of Sulfhydryl groups Sulfhydryl oxidase Sulfur-containing amino acids Superoxide anion Superoxide dismutase Sweetness of sugar solutions Sweet potato roots

313 252 304 249 306 306 253 244 255 257 257 258 221 271 80,90 123 203 74 116 57 58 107 58 58 74 155

Τ Tannase Tannins Tea black catechol oxidase cream fermentation flavanols flush instant metalloprotein Terpenes as fungistatic agents Texture modification of apple slices slices during processing Thiele modulus Thiobarbituric acid (TBA) Tobacco Tomato (es) cellulase in fruit, pericarp of pectinesterase in polygalacturonase in Transaminase

21 241 133 12 12 21 16 16 12 12 20 155 203 124 230 178 163 178 173 181 182 21

Triglycerides Trigonelline Trimethylamine dehydrogenase Triticale Trypsin L-Tryptophane Tyramine content in sausages Tyrosinase

PROCESSING

288 31 266 277 90 61,108,308 Ill 291 231

U

Ultrafiltration 296 of ox-blood 300 Ultrasonic energy techniques 312 Ultrazim 100 42 Urease 244,250 Uridine-5'-monophosphate (UMP) .. 114 V

L-Valine Vegetable(s) appearance of fruits and fibrovascular tissue in flavor of fruits and nutritional quality of fruits and oils, shelf life of peroxidase in postharvest fruits and processing, endogenous enzymes in fruit and products, lipase in fruit and Vicilin and legumin from pea seeds ....

Ill 193 149 193 193 233 143 192 194 304

W

Washed coffee Water supplies, problems brought on by dwindling Wheat a- and β-amylases in barley and flour seeds α-amylase in electrophoretic analyses of extracts of germinated and developing freeze-dried immunoelectrophoretic analyses of germinated and developing Whey as a fermentation substrate by lactase hydrolysis, modifica tion of Wine and beer production

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

30 221 80 247 89 90 92 90 73 74 73 75

INDEX

Χ

Xanthine oxidase colorimetry Xylan Xylanase Xylitol sweetness Xyloglucan

57 267 122 122 122 177

Yeast Yogurt galactose utilization Yuzuhada disorder

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