Enzymes in Food and Beverage Processing 9780841203754, 9780841204034, 0-8412-0375-X

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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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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

11

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.

20

ENZYMES

IN FOOD AND BEVERAGE

PROCESSING

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

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

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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.

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

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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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COGGON

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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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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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29

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* 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.

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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).

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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

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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AMORiM

AND AMORIM

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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.

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

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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.

AMORiM AND AMORIM

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51

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

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

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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.

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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.

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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

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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 ,

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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

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

62

ENZYMES

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

PROCESSING

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.

4.

SHiPE

63

Modification of Milk Fhvor

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.

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

64

E N Z Y M E S IN FOOD A N D

B E V E R A G E PROCESSING

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.

4.

SHiPE

Modification of Milk Flavor

65

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.

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

PROCESSING

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.

ENZYMES

68

IN FOOD A N D

BEVERAGE

PROCESSING

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.

5.

woYCHiK

AND

HOLSiNGER

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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

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

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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.

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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

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 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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W O Y C H I K A N D HOLSINGER

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

27.

Lactase in Dairy Manufacturing

79

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.

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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

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 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.

DAUSSANT

Amylases in Barley and Wheat

Figure 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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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^ 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 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) 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

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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)

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(10)

(11) (12) (13)

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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.

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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 -

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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

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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 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

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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

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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 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)

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

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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).

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 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.

256

ENZYMES IN FOOD AND BEVERAGE PROCESSING

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).

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

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257

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

B E V E R A G E PROCESSING

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).

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, 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).

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

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.

14.

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Soybean Processing and Quality Control

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 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 .

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

288

ENZYMES

IN FOOD A N D

BEVERAGE

PROCESSING

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.

16.

PALUMBO

A N D SMITH

% 100

Sausage Fermentation and Ripening

distribution

I

6

0

25

5 0 days Journal of Food Science

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)

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

289

290

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

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.

PALUMBO

AND

SMITH

Sdusdge Fermentation dnd Ripening

291

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,