Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors 9780841203303, 9780841202672, 0-8412-0330-X

Table of contents :
Title Page......Page 1
Copyright......Page 2
ACS Symposium Series......Page 3
FOREWORD......Page 4
PdftkEmptyString......Page 0
PREFACE......Page 5
1 Role of Flavones and Related Compounds in Retarding Lipid—Oxidative Flavor Changes in Foods......Page 6
Literature Cited......Page 16
What Is Tea?......Page 19
The Chemistry Of Tea Manufacture......Page 20
The Taste of Black Tea Derives Mainly From The Tea Polyphenols......Page 29
Summary And Conclusions......Page 45
Experimental......Page 47
Literature Cited......Page 49
3 Wine Flavor and Phenolic Substances......Page 52
Abstract......Page 71
Literature Cited......Page 72
Tastes and Odors in Water.......Page 76
Other Taste and Odor Sources.......Page 77
Analysis of Water for Taste and Odor Substances.......Page 78
Nitrogen Compounds.......Page 83
Treatment of Water for Taste and Odor Removal.......Page 86
Literature Cited......Page 89
5 Simultaneous Detection of Nitrogen and Sulfur Containing Flavor Volatiles......Page 90
Instrument Design and Modifications......Page 91
Application......Page 97
Literature Cited......Page 99
6 Flavor Precursors in Food Stuffs......Page 101
Flavor in Food Systems......Page 103
Meat Flavor Compounds......Page 105
Application of Meat Flavors......Page 111
Literature Cited......Page 117
Reaction products of aldehydes and hydrogensulfide......Page 119
Reaction products of α-dicarbonylcompounds, aldehydes, hydrogen-sulfide and ammonia......Page 120
Literature cited......Page 126
8 Non-Enzymic Transamination of Unsaturated Carbonyls: A General Source of Nitrogenous Flavor Compounds in Foods......Page 127
Experimental Section......Page 128
Reaction of PA with d-Carvone.......Page 129
2,4 Hexadienylamine.......Page 130
A. Reactions of Aldehydes.......Page 131
Β. Ketones.......Page 132
Discussion......Page 133
Literature Cited......Page 136
9 Identification and Flavor Properties of Some 3-Oxazolines and 3-Thiazolines Isolated from Cooked Beef......Page 138
Literature Cited......Page 149
10 Nonvolatile Nitrogen and Sulfur Compounds in Red Meats and Their Relation to Flavor and Taste......Page 151
Glycopeptides......Page 153
Amino Acids......Page 160
Peptides......Page 162
Effect Of Heating On Meat Flavor Precursors......Page 163
Furans in Meat......Page 179
Taste of Flavor Precursors......Page 182
Pharmacological Effects......Page 183
ABSTRACT......Page 184
Literature Cited......Page 185
11 Furans Substituted at the Three Position with Sulfur......Page 189
Experimental......Page 192
Literature Cited and Notes......Page 198
12 Cat Neural Taste Responses to Nitrogen Compounds......Page 199
Literature Cited......Page 210
Β......Page 212
F......Page 213
K......Page 214
Ο......Page 215
S......Page 216
V......Page 217
W......Page 218

Citation preview

Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors George Charalambous, EDITOR Anheuser-Busch, Inc.

Ira Katz, EDITOR International Flavors and Fragrances

A symposium sponsored by the Division of Agricultural and Food Chemistry at the 170th Meeting of the American Chemical Society, Chicago, Ill., August 2 5 - 2 6 ,

ACS SYMPOSIUM SERIES

1975

26

AMERICAN CHEMICAL SOCIETY WASHINGTON, D. C. 1976

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

Library of Congress QE Data Phenolic, sulfur, and nitrogen compounds in food flavors. (ACS symposium series; 26 ISSN 0097-6156) Includes bibliographical references and index. 1. Flavoring essences—Congresses. 2. Phenols—Congresses. 3. Flavonoids—Congresses. I. Charalambous, George, 1922. II. Katz, Ira, 1933. III. American Chemical Society. Division of Agricultural and Food Chemistry. IV. Series: American Chemical Society. ACS symposium series; 26. TP418.P46 ISBN 0-8412-0330-X

664'.06

76-16544 ACSMC8 26 1-215

Copyright © 1976 American Chemical Society All Rights Reserved. No 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. PRINTED I N THE UNITED STATES OF AMERICA

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

ACS Symposium Series Robert F. Gould, Editor

Advisory Board Kenneth B. Bischoff Jeremiah P. Freeman E. Desmond Goddard Jesse C. H. Hwa Philip C. Kearney Nina I. McClelland John B. Pfeiffer Joseph V. Rodricks Aaron Wold

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

FOREWORD The ACS SYMPOSIUM SERIES was founded in 1974

to provide

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 Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

PREFACE T o u r i n g the past decade there has been a growing interest in all aspects of our food supply. This concern has been precipitated by a variety of factors such as predicted world food shortages, interest in the development of new foods and food analogs, diet and health, consumer concern, etc. In order to solve these problems we must know more about the chemistry of the foods we consume. Implicit in this is a better understanding of flavor chemistry. In this respect, this volume is very timely since sulfur, nitrogen, and phenolic compounds ar recently have we begun to realize how ubiquitous these chemicals are in food systems. The papers in this book will give some insight into the complexity and breadth of this area of food chemistry. Hopefully, it will stimulate the flavor chemist to continue and expand his endeavors. Anheuser Busch, Inc. St. Louis, Mo.

GEORGE

International Flavors and Fragrances Union Beach, N.J. January 23, 1976

CHARALAMBOUS

IRA

KATZ

vii In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

1 R o l e of Flavones a n d Related C o m p o u n d s i n Retarding L i p i d — O x i d a t i v e Flavor Changes i n Foods DAN E. PRATT Department of Foods and Nutrition, Purdue University, West Lafayette, Ind. 47907

The term f l a v o n o i the group of p l a n t phenol skeleton C -C -C . The b a s i c s t r u c t u r e of these compounds c o n s i s t s of two aromatic r i n g s l i n k e d by a three carbon aliphatic chain which normally has been condensed t o form a pyran or l e s s commonly a furan ring. As the name i m p l i e s flavone may be considered the general type compound of the f l a v o n o i d group. Based chiefly on the o x i d a t i o n s t a t e of the a l i p h a t i c fragment, these compounds may be subdivided i n t o s e v e r a l groups (1, 2, 3). The widest and most inclusive classification (2) places the flavonoids i n t o three c l a s s e s : 1) The anthoxanthins include all flavonoids t h a t possess a carbonyl group in the 4 - p o s i t i o n . The center condensed r i n g may be e i t h e r the pyran or furan s t r u c t u r e ; or in one case (the chalcones) the a l p h a t i c fragment i s not condensed i n t o a ring. 2) The flavans i n c l u d e flavonoids t h a t do not possess a carbonyl in the 4 - p o s i t i o n . The center condensed ring is always i n t a c t and i s the pyran s t r u c t u r e . 3) The anthocyanins are f l a v y l i u m salts. These may be considered as flavans in the highest s t a t e of o x i d a t i o n . These three c l a s s e s may be further d i v i d e d as shown in Figures 1 and 2. Several phenolic compounds t h a t are not flavonoids, but are c l o s e l y r e l a t e d t o flavonoids b i o s y n t h e t i c a l l y and i n t h e i r d i s t r i b u t i o n , must a l s o be considered. These compounds are i n the cinnamic acids (3-phenyl propenoic a c i d d e r i v a t i v e s ) , e s t e r s of cinnamic acids and hydroxy and/or methoxy d e r i v a t i v e o f coumarin. In the p l a n t kingdom, the angiosperms account for 6

3

6

1

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

2

PHENOLIC, SULFUR, AND NITROGEN COMPOUNDS IN FOOD FLAVORS

A.

TYPE COMPOUND

ANTHOXANTHINS

DESCRIPTION OF CLASS H y d r o x y l a t e d and/or methoxylated derivatives of flavone The 3-hydroxyf1avones a r e commonly r e f e r r e d t o as flavonols.

2.

Flavanones lated derivatives of f l a v a none ( 2 , 3 - d i h y d r o f l a v o n e ) . The 3 - h y d r o x y f l a v a n o n e s a r e commonly r e f e r r e d t o as flavanonols.

3.

Isoflavones

OCM3 4.

Chalcones

Analogous t o t h e f l a v o n e s w i t h the aromatic r i n g l i n k e d t o carbon 3 i n s t e a d o f carbon 2.

H y d r o x y l a t e d and/or methoxyl a t e d d e r i v a t i v e s o f two a r o m a t i c r i n g s l i n k e d by a three carbon a l i p h a t i c fragment.

4' Figure I.

Classification of flavonoids

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

1.

PRATT

A.

ANTHOXANTHINS DESCRIPTION OF CLASS

TYPE COMPOUND 5.

3

Fhvones and Related Compounds

H y d r o x y l a t e d and/or methoxyl a t e d d e r i v a t i v e s of benzalcoumranone.

Aurones

a:>o B.

FLAVANS f

3,7,4 -Hydroxyflavans which may a l s o be h y d r o x y l a t e d a t the 5, 3 , and/or 5 p o s i ­ tions .

Catechins

f

HO

f

(χχο Leucoanthocyanins

H y d r o x y l a t e d and/or methoxyl a t e d d e r i v a t i v e s of 3,4-dihydroxyflavan.

OH

Figure 1.

Classification of flavonoids (continued)

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

PHENOLIC, SULFUR, AND NITROGEN COMPOUNDS IN FOOD FLAVORS

Cinnamic

acids

(3-phenylproponoic a c i d

derivatives)

CH-COOH

Quinic

acid

(1,3,4,5-tetrahydroxycyclohexanecarboxylic a c i d ) ,

Quinic

a c i d e s t e r s o f c i n n a m i c a c i d s , and coumarins

( h y d r o x y and/or methoxy d e r i v a t i v e s o f c o u m a r i n ) .

00°

Figure 2. Classification of related compounds

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

1.

PRATT

Ffovones and Related Compounds

5

between 250 and 300 thousand s p e c i e s . Of t h i s number l e s s t h a n 400 s p e c i e s a r e c u l t i v a t e d o r g a t h e r e d as human f o o d s . These 400 s p e c i e s i n c l u d e 33 o f t h e 51 o r d e r s and 89 o f t h e 279 f a m i l i e s . A l l p a r t s o f p l a n t s a r e e a t e n - r o o t s , stems, l e a v e s , f l o w e r s , f r u i t , and seeds - b u t i n most s p e c i e s t h e e d i b l e p o r t i o n s are r e s t r i c t e d t o one p a r t . F l a v o n o i d s and r e l a t e d com­ pounds have been i s o l a t e d f r o m , o r d e t e c t e d i n about o n e - h a l f o f t h e s e e d i b l e p l a n t s , b u t n o t always i n t h e edible portions. The same compound, o r group o f com­ pounds, are n o t always p r e s e n t t h r o u g h o u t t h e p l a n t . Flavonoids occur i n a l l types of higher p l a n t t i s s u e - wood, b a r k , stems, l e a v e s , f r u i t , r o o t s , f l o w e r , p o l l e n and s e e d s . T a b l e I shows t h e g e n e r a l d i s t r i b u t i o n o f f l a v o n o i d s and c i n n a m i c a c i d s i n tlie v a r i o u s p a r t s o f th n o i d s are more c h a r a c t e r i s t i c o f some t i s s u e s t h a n others. I n f r u i t b e a r i n g p l a n t s , however, t h e same groups o f f l a v o n o i d s t h a t o c c u r i n t h e l e a v e s a l s o o c c u r i n t h e f r u i t i n a l e s s e r amount. A n t h o c y a n i n s a r e t y p i c a l l y i n f r u i t s , f l o w e r s , and some l e a v e s . The g r e a t e s t n a t u r a l s o u r c e o f f l a v a n s - c a t e c h i n s and l e u c o a n t h o c y a n i n s - a r e from woods and b a r k s . How­ e v e r , t h e s e do o c c u r i n non-woody t i s s u e as t e a l e a v e s , c o c o a b e a n s , and f r u i t p u l p s . C h a l c o n e s and aurones a r e c h i e f l y found i n f l o w e r p e t a l s , and t o a l e s s e r e x t e n t i n l e a v e s and stems o f some s p e c i e s b u t a r e n o t as w i d e l y d i s t r i b u t e d as o t h e r groups o f f l a v o n o i d compounds. F l a v o n e s and f l a v o n o n e s a r e p r e s e n t in many p l a n t t i s s u e s and cannot be c o n s i d e r e d as com­ ponents o f any one t y p e o f t i s s u e . Perhaps t h e g r e a t e s t s t i m u l u s t o t h e s t u d y o f f l a v o n o i d s and r e l a t e d compounds came w i t h t h e d e v e l o p ­ ment o f paper chromatography and i t s e v o l u t i o n i n t o t h i n - l a y e r techniques. Paper chromatography p r o v i d e d the f i r s t s a t i s f a c t o r y procedures o f surveying p l a n t t i s s u e s f o r the presence of f l a v o n o i d s (4). These compounds p o s s e s s j u s t t h e r i g h t range 6Έ s o l u b i l i t y c h a r a c t e r i s t i c s f o r ease i n s e p a r a t i o n (5, 6) and most o f them p o s s e s s c h a r a c t e r i s t i c s p e c t r a i n u l t r a v i o l e t a n d / o r v i s i b l e r e g i o n s (6, 1). N e a r l y t h i r t y y e a r s ago paper chromatography was employed f o r t h e s e p a r a t i o n o f f l a v o n o i d s . There have been many e x c e l l e n t r e v i e w s on t h e s u b j e c t (5, (3, 8_, —* i £ ' ϋ . ) · The s e l e c t i o n o f a s p e c i f i c c h r o m a t o g r a p h i c p r o c e d u r e depends on t h e o b j e c t i v e s o f an i n v e s t i g a t i o n . The i s o l a t i o n and p u r i f i c a t i o n o f a f l a v o n o i d (or a c i n n a m i c a c i d ) can be a c h i e v e d b y v a r i o u s p r e p a r a t i o n techniques u s i n g e i t h e r one- or two-dimensional p r o ­ cedures. In p r e p a r a t i v e p r o c e d u r a l work i n our

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

6

PHENOLIC, SULFUR, A N D NITROGEN COMPOUNDS I N FOOD FLAVORS

l a b o r a t o r y has u s u a l l y been b y s e r i e s o f o n e - d i m e n s i o n a l techniques. In e i t h e r case, s e v e r a l separations a r e r e q u i r e d and s e v e r a l d e t e c t i o n t e c h n i q u e s must be used. Many o f t h e f l a v o n o i d s and r e l a t e d compounds have strong antioxidant c h a r a c t e r i s t i c s i n lipid-aqueous and l i p i d f o o d systems (Tables I I , H I , F i g u r e 3 ) . As may be seen c e r t a i n f l a v o n e s , f l a v o n o l s , f l a v o n o n e s , f l a v a n o n a l s , and c i n n a m i c a c i d d e r i v a t i v e s have c o n siderable antioxidant a c t i v i t y . The v e r y low s o l u b i l i t y o f t h e s e compounds i n l i p i d s i s o f t e n c o n s i d e r e d a d i s a d v a n t a g e and i s c o n s i d e r e d a s e r i o u s d i s a d v a n t a g e i f an aqueous phase i s a l s o p r e s e n t (12). However, f l a v o n o i d s suspended i n t h e aqueous phase o f a l i p i d aqueous system o f f e r a p p r e c i a b l e p r o t e c t i o n t o l i p i d o x i d a t i o n (13, 14, 15 Swoboda (12J7 n e a r l y twenty y e a r s ago, found t h a t f l a v o n o l s were e f f e c t i v e a n t i o x i d a n t s when suspended i n l i p i d systems. The a n t i o x i d a n t a c t i v i t y was measured u s i n g 20 mg. o f l i n o l e i c a c i d , 200 mg. o f Tween 40, and 1 m l . o f 0.02% B - c a r o t e n e i n c h l o r o f o r m . The c h l o r o f o r m was removed b y e v a p o r a t i o n on a w a t e r - b a t h a t 5 0 ° C . , u s i n g a rotary evaporator. 50 m l . o f oxygenated water was added, and 5 m l . a l i q u o t s o f t h i s e m u l s i o n were p l a c e d i n spectrometer tubes w i t h 2 m l . o f the a n t i o x i d a n t s o l u t i o n under t e s t . For the c o n t r o l , 2 m l . o f d e i o n i z e d , d i s t i l l e d w a t e r , o r e t h a n o l , as a p p r o p r i a t e , were added t o t h e e m u l s i o n . Readings a t 470 nm. were taken immediately. The t u b e s were s t o p p e r e d , and p l a c e d i n a w a t e r - b a t h a t 50 C . Readings o f t h e o p t i c a l d e n s i t y were t a k e n a t r e g u l a r i n t e r v a l s u n t i l t h e c o n t r o l was b l e a c h e d . The a n t i o x i d a n t i n d e x was c a l c u l a t e d by d i v i d i n g the l o s s o f o p t i c a l d e n s i t y o f the c o n t r o l a t t h e end o f t h e i n d u c t i o n p e r i o d , b y t h e l o s s o f o p t i c a l d e n s i t y o f the t e s t s o l u t i o n at t h a t time. Polyphenolic antioxidants, sparingly soluble i n l i p i d systems, have been c o n v e r t e d i n t o r e a d i l y f a t s o l u b l e form b y a l k y l a t i o n or e s t e r i f i c a t i o n w i t h l o n g chain f a t t y acids or a l c o h o l s . Such a p r o c e d u r e o f f e r s promising r e s u l t s with f l a v o n o i d s . The a c t i o n f l a v o n o l a n t i o x i d a t i o n i s b i - m o d a l . F l a v a n o l s are known t o form complexes w i t h m e t a l s . C h e l a t i o n occurs at the 3-hydroxy, 4-keto grouping a n d / o r a t t h e 5 - h y d r o x y , 4 - k e t o g r o u p , when t h e A r i n g i s k y d r o x y l a t e d i n t h e 5 p o s i t i o n . An o - q u i n o l g r o u p i n g on t h e B - r i n g can a l s o demonstrate m e t a l - c o m p l e x i n g a c t i v i t y (19, 20). However, t h e major v a l u e o f f l a v o n o i d s and c i n n a m i c a c i d s i s i n t h e i r p r i m a r y

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

1.

PRATT

7

Fhvones and Related Compounds

TABLE I .

The G e n e r a l D i s t r i b u t i o n o f F l a v o n o i d Compounds i n P l a n t T i s s u e s

Plant Tissue Wood

R e l a t i v e C o n c e n t r a t i o n o f Compounds Catechins ^ Leucoanthocyanins > f l a v o n o l s > cinnamic a c i d s A s wood b u t g r e a t e r t o t a l q u a n t i t y Flavonols c i n n a m i c a c i d s > c a t e c h i n s #w Leucoanthocyanins Cinnamic a c i d s > c a t e c h i n s ^ L e u c o a n t h o c y a n ­ ins > flavonols

Bark Leaf Fruit

TABLE I I ,

Antioxidant A c t i v i t y o f Flavones Antioxidant Compound (

Aglycones: Quercetin (3,5,7,3·,4·-Pentahydroxy) Fisetin (3,7,3 ,4 -Tetrahydroxy) Myricetin (3,5,7,3 ,4 ,5 -Hexahydroxy) Robinetin (3,7,3',4*,5'-Pentahydroxy) Rhammnetin ( 3 , 5 , 3 , 4 - T e t r a h y d r o x y 7-Methoxy) Glycosides: Quercetin ( Q u e r c e t i n 3-Rhamnoside) Rutin ( Q u e r c e t i n 3-Rhamnoglucoside) 1

TABLE I I I .

3.8 3.8

1

1

1

index

1

4.5

1

4.5 3.6

1

3.7 1.6

A n t i o x i d a n t A c t i v i t y o f Flavanones A n t i o x i d a n t Index Compound (5 χ 10 M)

Aglycones: Naringenin (5,7,3*-Trihydroxy) Dihydroquercetin (3,5,7,3 ' , 4 '-Pentahydroxy) Hesperitin (5,7,3*-Trihydroxy-4 -Methoxy) Glycosides : Hesperidin ( H e s p e r i t i n 7-Rhamnoglucoside) Neohesperidin (Hesperitin 7-glucoside)

1.6 3.8 1.2

1

1.2 1.3

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

—

8

PHENOLIC, SULFUR, AND NITROGEN COMPOUNDS IN FOOD FLAVORS

Compound

Structure

Antioxidant Index (5 χ 10~ M) %

Hesperidin Methyl Chalcone

1.3

*C ^ O H M

OH

R-%hamnoglucoside D-Catechin

3.5

Chlorogenic Acid

3.7 OH

G H ^

U

V ^ O O H ^-~CH=CH-C00

C a f f e i c Acid

3.6 OH

CH=CH-C00H

Quinic A c i d

1.5

Nu

7^ COOH

OH

Propyl G a l l a t e

2.1 OH HO ^

^-C00C H 3

7

OH

Figure 3. Antioxidant indices of some flavonoids and related compounds

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

1. PRATT

Flavones and Related Compounds

9

a n t i o x i d a n t a c t i v i t y ( i . e . , as f r e e r a d i c a l a c c e p t o r s and as c h a i n - b r e a k e r s ) . The major e v i d e n c e t h a t t h e s e compounds work m a i n l y as p r i m a r y a n t i o x i d a n t s i s t h e i r a b i l i t y t o work e q u a l l y w e l l i n m e t a l c a y a l y z e d and u n c a t a l y z e d systems. They a r e a l s o e f f i c i e n t a n t i o x i d a n t s i n systems c a t a l y z e d b y r e l a t i v e l y l a r g e m o l e c u l e s , such as heme and o t h e r p o r p h y i n compounds. They are a l s o e f f e c t i v e against lipoxygenase catalyzed r e a c t i o n s . These compounds cannot be e n f i s a g e d as f o r m i n g complex­ es w i t h f l a v o n o l s . In a d d i t i o n , h e s p e r i t i n ( 5 , 7 , 3 ' t r i h y d r o x y - 4 ' me t h o x y f l a v o n e ) which p o s s e s s e s an a c t i v e m e t a l - c o m p l e x i n g s i t e has demonstrated n e g l i b i b l e antioxidant a c t i v i t y . The p o s i t i o n and t h e degree o f h y d r o x y l a t i o n i s o f p r i m a r y importanc vity. There i s g e n e r a l agreement t h a t o r t h o - d i h y d r o x y l a t i o n o f the Β r i n g c o n t r i b u t e s markedly t o the a n t i ­ o x i d a n t a c t i v i t y o f f l a v o n o i d s (12, 13, 14, 21, 22, 23, 24). The p a r a - q u i n o l s t r u c t u r e οΈ t E ê Β r i n g T i a F l > e e n sïïbwn t o i m p a r t even g r e a t e r a c t i v i t y them t h e o r t h o q u i n o l s t r u c t u r e ; w h i l e t h e meta c o n f i g u r a t i o n has no e f f e c t on a n t i o x i d a n t a c t i v i t y (21). However, p a r a and meta h y d r o x y l a t i o n o f t h e Β r i n g a p p a r e n t l y does n o t o c c u r commonly i n n a t u r e . A l l f l a v o n o i d s with the 3 ' , 4 - d i h y d r o x y c o n f i g u r ­ a t i o n possess antioxidant a c t i v i t y . Two ( r o b i n e t i n and m y r i c e t i n ) have an a d d i t i o n a l h y d r o x y l group a t the 5 p o s i t i o n , which i n c r e a s e s the a n t i o x i d a n t a c t i ­ v i t i e s over those of the corresponding flavones w i t h t h e 5 - h y d r o x y l g r o u p , f i s e t i n and q u e r c e t i n . Two f l a v a n o n e s ( n a r i n g e n i n and h e s p a r i t i n ) h a v i n g a s i n g l e h y d r o x y l group on t h e Β r i n g p o s s e s s e s o n l y s l i g h t antioxidant a c t i v i t y . H y d r o x y l a t i o n o f the Β r i n g i s a major c o n s i d e r a t i o n f o r a n t i o x i d a n t a c t i v i t y . Meta 5 , 7 - h y d r o x y l a t i o n o f t h e A r i n g a p p a r e n t l y has l i t t l e , i f any, e f f e c t on a n t i o x i d a n t a c t i v i t y . T h i s i s e f i d e n c e d b y t h e f i n d i n g s t h a t q u e r c e t i n and f i s e t i n have r e l a t i v e l y t h e same a c t i v i t y and m y r i c e t i n p o s s e s s e s t h e same a c t i v i t y as r o b i n e t i n . Heimann and h i s a s s o c i a t e s (23, 24) r e p o r t e d t h a t meta 5 , 7 - h y d r o x y l ­ a t i o n lowered a n t ï b x ï c t a n t a c t i v i t y . To t h e c o n t r a r y , Mehta and S e s h a d r i (22) found q u e r c e t i n t o be a more e f f e c t i v e a n t i o x i d a n t than 3 , 3 , 4 - t r i h y d r o x y f l a v o n e . Data from our l a b o r a t o r y s u p p o r t t h e f i n d i n g o f Mehta and S e s h a d r i . The importance o f o t h e r s i t e s o f h y d r o x y l a t i o n were s t u d i e d b y L e a and Swoboda (12); Mehta and S e s h a d r i (22); Simpson and U r i (21); and U r T (25). The two former groups found q u e r c e t a g e t i n (3,¥75,7,3,4'-hexa1

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h y d r o x y f l a v o n e ) and g o s s y p e t i n ( 3 5 , 7 , 8 , 3 , 4 ' - h e x a h y d r o x y f l a v o n e ) t o be v e r y e f f e c t i v e a n t i o x i d a n t s . U r i (25) found t h a t t h e o r t h o - d i h y d r o x y g r o u p i n g on one r i n g and t h e p a r a d i h y d r o x y g r o u p i n g on the o t h e r ( i . e . , 3 , 5 , 8 , 3 * 4 ' - and 3 , 7 , 8 , 2 , 5 - p e n t a h y d r o x y vlavones) produced v e r y potent a n t i o x i d a n t s . These f o u r p o l y h y d r o x y f l a v o n e s are t h e most p o t e n t f l a v o n o i d s , as a n t i o x i d a n t s , y e t r e p o r t e d i n non-aqueous s y s t e m s . Simpson and U r i (21) found 7-n-butoxy-3,2 ,5 -trihyd r o x y f l a v o n e t o be t h e most e f f e c t i v e a n t i o x i d a n t o f 30 f l a v o n e s s t u d i e d i n aqueous e m u l s i o n s o f m e t h y l linoleate. The 3 g l y c o s i d e s p o s s e s s a p p r o x i m a t e l y t h e same a n t i o x i d a n t a c t i v i t y as t h e c o r r e s p o n d i n g a g l y c o n e when t h e g l y c o s y l s u b s t i t u t i o n i s w i t h m o n o s a c c h a r i d e . In t h e c a s e o f r u t i a disaccharide antioxidant a c t i v i t y i s reduced. The a n t i o x i d a n t c a p a c i t y o f a commercial p r e p a r a t i o n o f r u t i n i s c o n s i d e r a b l y lower t h a n t h e c o r r e s p o n d i n g aglycone, q u e r c e t i n . K e l l e y and Watts (19) s t u d i e d t h e a n t i o x i d a n t e f f e c t o f s e v e r a l f l a v o n ô T d s and found r u t i n womewhat i n f e r i o r t o q u e r c e t i n and q u e r c i t r i n b u t t h e d i f f e r e n c e s were n o t as g r e a t as we have f o u n d . Chromatographic p u r i f i c a t i o n and t h e use o f s e v e r a l c o m m e r c i a l l y a v a i l a b l e samples (to e l i m i n a t e t h e e f f e c t o f p o s s i b l e contamination) d i d not a l t e r the f i n d i n g . K e l l e y and Watts (19), u s i n g a c a r o t e n e - l a r d system a l s o found t h a t q u e r c i t r i n had a p p r o x i m a t e l y the same p r o t e c t i o n as q u e r c e t i n . C r a w f o r d e t a l , (26) found t h a t m e t h y l a t i o n o f t h e 3 - h y d r o x y l group o f q u e r c e t i n o n l y s l i g h t l y lowered a n t i o x i d a n t a c t i v i t y . However, c o n s i d e r a b l e importance has been a t t a c h e d t o t h e f r e e 3 - h y d r o x y l b y o t h e r s (12, 21, 22, 2 3 ) . Mehta and S e s h a d r i (22) p o s t u l a t e d t h a t t E e " ^ h y d r o x y 1 and t h e 2,3 double"T>ond a l l o w e d t h e m o l e c u l e t o undergo i s o m e r i c changes t o d i k e t o forms w h i c h would p o s s e s s a h i g h l y r e a c t i v e - C H group ( p o s i t i o n 2 ) . D i h y d r o q u e r c e t i n was found t o have t h e same a n t i o x i d a n t a c t i v i t y as q u e r e c t i n i n d i c a t i n g e i t h e r t h a t t h e 2 , 3 , d o u b l e bond i s n o t o f major importance t o antioxidant a c t i v i t y or t h a t conversion of d i h y d r o q u e r c e t i n t o q u e r c e t i n t o o k p l a c e w h i l e t h e compound was i n c o n t a c t w i t h t h e o x i d i z i n g f a t . Mehta and S e s h a d r i (22) s u g g e s t e d t h a t c o n v e r s i o n might a c c o u n t f o r t h e a n t i o x i d a n t a c t i v i t y o f d i h y d r o q u e r c e t i n . Howe v e r , c h r o m a t o g r a p h i c t e s t s demonstrated t h a t d i h y d r o q u e r c e t i n i s not converted t o q u e r c e t i n by the h y d r o l y s i s p r o c e d u r e , nor c o u l d q u e r c e t i n be c h r o m a t o g r a p h i c a l l y d e t e c t e d i n t h e c a r o t e n e - l a r d system i n which d i h y d r o q u e r c e t i n was u s e d as a n t i o x i d a n t . Dihydrof

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q u e r c e t i n was s t i l l p r e s e n t a f t e r 12 h o u r s i n t h e system. Perhaps t h e g r e a t e s t p o t e n t i a l s o u r c e o f f l a v o n o i d s f o r f o o d a n t i o x i d a n t s i s from wood as a b y - p r o d u c t o f lumber and p u l p i n g o p e r a t i o n s . Whole b a r k o f t h e D o u g l a s f i r c o n t a i n s about f i v e p e r c e n t d i h y d r o quercetin ( 3 , 4 , 7 , 3 ' , 4 pentohydroxyflavonone). The c o r k f r a c t i o n , r e a d i l y s e p a r a t e d from t h e b a r k , c o n t a i n up t o 22% d i h y d r o q u e r c e t i n ( 2 7 ) . K i r t h (28) r e p o r t e d t h a t a p p r o x i m a t e l y 150 m î T l i o n pounds o f d i h y d r o q u e r c e t i n are p o t e n t i a l l y a v a i l a b l e a n n u a l l y i n Oregon and Washington a l o n e . Q u e r c e t i n ( 3 , 5 , 7 , 3 *4' p e n t o h y d r o x y f l a v o n e ) has been p r o d u c e d c o m m e r c i a l l y as an a n t i o x i d a n t from wood s o u r c e s ( 2 9 ) . Quercetin i s p r e s e n t i n much i s dihydroquercetin q u a n t i t y by o x i d a t i o n o f d i h y d r o q u e r c e t i n . As m e n t i o n e d e a r l i e r o t h e r p l a n t c o n s t i t u e n t s w h i c h m i g h t be e x p e c t e d t o show a n t i o x i d a n t powers w o u l d be p r i m a r i l y p h e n o l i c compounds, e x p e c i a l l y o - and p - d i h y d r o x y p h e n o l s such as t h e h y d r o x y c i n n a m i c a c i d s , c a f f e i c and f e r u l i c a c i d s . While t h e s e a c i d s u s u a l l y o c c u r i n p l a n t t i s s u e as w a t e r s o l u b l e e s t e r s , commonly c h l o r o g e n i c a c i d o r d a f f e o y l q u i n i c a c i d , and s u g a r e s t e r s , t h e y have a l s o been i s o l a t e d as complex l i p o p h i l i c e s t e r s o f g l y c e r o l , l o n g - c h a i n d i o l s , and -w-hydroxy a c i d s . These l i p o p h i l i c e s t e r s have b e e n r e v e a l e d as a n t i o x i d a n t s i n a comprehensive i n v e s t i g a t i o n o f t h e a n t i o x i d a n t s i n o a t s (30, 3 1 , 32, 3 3 ) . The c a f f e o y l e s t e r s have c o n s i d e r a b l y more a n t i o x i d a n t a c t i v i t y t h a n do t h o s e o f f e r r u l i c a c i d . Other l i p i d - s o l u b l e e s t e r s o f f e r u l i c a c i d w i t h c y c l o a r t e n o l and o t h e r t r i t e r p e n o i d s have been shown b y Ohta e t a l . (34) t o o c c u r i n r i c e b r a n o i l , w h i l e a f e r u l a t e o f d i H y d r o x y - B - s i t o s t e r o l has been i s o l a t e d from m a i z e b y Tamura e t a l . ( 3 5 ) . Wheat has a l s o been shown t o c o n t a i n s i m i l a r s t e r o i d e s t e r s (36). The p r e s e n c e o f two i s o m e r s o f c h l o r o g e n i c a c i d , a l s o f e r u l i c a c i d , and s e v e r a l o t h e r p h e n o l i c a c i d s , has been c o n f i r m e d i n hexane d e f a t t e d s o y f l o u r b y A r a i et a l . (37). Literature Cited 1. 2. 3.

Geissman, T . A . and Hinreiner, E . Botan. Rev. (1952) 18:77. B a t e - S m i t h , E. C. Advances in Food R e s e a r c h . (1954) 5:261. Geissman, T . A . "The C h e m i s t r y of F l a v o n o i d Com―

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p o u n d s " , C h a p t e r 1. ( E d i t e d b y T . A . Geissman) The M a c m i l l a n Company, New Y o r k . (1962). 4 . B a t e - S m i t h , E . C . Chem. I n d . R . (1956) 32. 5 . Harborne, J. B. J. Chromatography. (19591) 2:581. 6 . M a b r y , T. J., Markham, K . R . and Thomas, M. B. "The Systematic Identification of Flavonoids". Springer-Verlag, Berlin (1970). 7 . J u r d , L. "The C h e m i s t r y o f F l a v o n o i d Compounds", Chapter 5. ( E d i t e d b y T . A . Geismann) The Mac­ millan Company, New Y o r k . (1962). 8. H a r b o r n e , J. B. J. Chromatography (1958) 1:473. 9 . Thompson, J. F., Honda, S. I., H u n t , G. Ε., K r u p k a , R. M., Morris, C. J., Powell, Jr., L. Ε., Silber­ stein, O. O., Towers, G. Η. Ν., and Z a c h a r i u s , R. M . B o t a n . Rev 1 0 . Seikel, M. K. "Th pounds", Chapter 3 ( E d i t e d b y T . A . Geissman) The M a c m i l l a n Company, New Y o r k . (1962). 1 1 . Seikel, M. K. " B i o c h e m i s t r y of P h e n o l i c Compounds", C h a p t e r 2 ( E d i t e d b y J. B. Harborne) Academic P r e s s , New Y o r k . (1964). 1 2 . L e a , C . H . and Swoboda, P . A . T . Chem. I n d . (1956) 1426. 1 3 . Pratt, D. E. and W a t t s , B. M. J. Food Sci. (1964) 29:27. 1 4 . Pratt, D. E. J. Food Sci. (1965) 30:737. 15. Cofer, A . Unpublished data. (1961) Florida State University. 16. C o f e r , A . Unpublished data. (1963) Florida State University. 17. Cofer, A . Ph.D. Thesis. (1965) Florida State University. 1 8 . Ramsey, M. B. and W a t t s , Β . M . Food T e c h n o l . (1963) 17:1056. 1 9 . Kelley, G . G . and W a t t s , Β . M . Food R e s e a r c h (1957) 22:308. 2 0 . D e W i t t , K . W. Chem. I n d . (1955) 1551. 21. S i m p s o n , T . H . and Uri, N . Chem. I n d . (1956) 2 2 . M e h t a , A . C . and S e s h a d r i , T. R. J. Sci. I n d . Research (1959) 18B: 2 4 . 2 3 . Heimann, W . , Heimann, Α . , Gremminger, M . and Holman, Η. H . F e t t e u. S o i f e n (1953) 55:394. 2 4 . Heimann, W. and Reiff, F. F e t t e u. S o i f e n (1953) 55:451. 2 5 . Uri, N. 1 9 6 1 . Mechanism of antioxidation. " A u t o x i d a t i o n a n d A n t i o x i d a n t s " , Chapter 4 ( E d i t e d b y W. O. L u n d b e r t ) Interscience Publishers, New Y o r k . 2 6 . C r a w f o r d , D. L., S i n n h u b e r , R. O. and Aft. H . J. Food Sci. (1962) 26:139.

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27. H e r g e r t , H. L. and K u r t h , E . F . T a p p i (1952) 35:59. 28. Kirth, E. F. I n d . E n g . Chem: (1953) 45:2096. 29. Anon. Chem. E n g . News (1958) 36 (No. 7 ) , 58. 30. D a n i e l s , D. G. H., K i n g , H . G . C . and M a r t i n , H . F . Sci. Food A g r . (1963) 14:385. 31. D a n i e l s , D . G . H . and M a r t i n , H. F. Chem. I n d . (1964) 2058. 32. D a n i e l s , D . G. H . and M a r t i n , H. F. J. Sci. Food Agr. (1967) 18:589. 33. D a n i e l s , D. G. H . and M a r t i n , H. F. J. Sci. Food Agr. (1968) 19:710. 34. Ohta, G. and Shimuzu, M . Pharm. Bull. (Tokyo) (1957) 5:40. 35. Tamura, T . , Sakaedani Nippon Kagaku 36. Tamura, T . , H i b i n o , T., Yokoyama, D. and Matsumoto, T. Nippon Kagaku Z a s s h i (1959) 80:215. 37. Arai, S., S u z u k i , H., F u j i m a k i , M . and Sakurai, Y. A g r . Biol. Chem. (Tokyo) (1966) 30:364.

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

2 C o n t r i b u t i o n of P o l y p h e n o l i c C o m p o u n d s to the Taste of T e a GARY W. SANDERSON, ARVINDS.RANADIVE, LARRYS.EISENBERG, FRANCIS J. FARRELL, ROBERT SIMONS, CHARLES H. MANLEY, and PHILIP COGGON ThomasJ.Lipton, Inc., 800 Sylvan Ave., Englewood Cliffs, N. J. 07632

What Is Tea? Tea i s a processed vegetable material used to prepare a stimulating, delicately flavored beverage that i s one of the most popular drinks i n the world. Tea is manufactured from the tender shoot tips ( i . e . the "flush") of the tea plant Camellia sinensis, (L.) O. Kuntze, cultivated in many t r o p i c a l and subtropical areas around the world. The tea manufacturing process (1,2,3) causes the fresh green tea leaf to be converted to commercial tea products such as green tea (not fermented), oolong tea ( p a r t i a l l y fermented), or black tea (fully fermented). Tea fermentation refers to an oxidation of the flavanols found in the tea leaf which is brought about by a catechol oxidase enzyme that i s endogenous to the leaves of tea plants (2,4): Control of this reaction is central to good tea manufacturing practices. The chemical composition of a tea beverage prepared from a commercial black tea blend, i.e. a Lipton tea bag, is shown i n Table 1. This set of analyses agrees closely with other analyses that have been published (5,6,7) indicating that the black tea studied in this investigation is representative of black teas i n general. As shown in Table 1, polyphenolic compounds are estimated to comprise about 48.5% of the t o t a l solids in a cup of tea. As will be shown l a t e r in this paper, these polyphenolic compounds make a most important contribution to the taste of tea, and the exact nature of this contribution is determined by the kind of polyphenolic compounds that are present i n the tea beverage. Accordingly, to understand the chemistry underlying the taste of tea, one must understand the chemistry of tea manufacture, esp e c i a l l y the tea fermentation process, since this determines the makeup of the polyphenolic compounds in tea products. Returning for a moment to our question; namely, "What i s tea?", we recognize that most people think of tea as the beverage that they obtain by steeping a tea bag containing black tea 14

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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Polyphenolic Compounds in the Taste of Tea

i n b o i l i n g h o t water, o r by d i s s o l v i n g i n s t a n t t e a i n c o l d water. In e i t h e r case, the average t e a beverage i n the U n i t e d States has a t e a s o l i d s c o n c e n t r a t i o n o f about 0.30% t e a s o l i d s obtained by s t e e p i n g a t e a bag (contains about 2.27g b l a c k t e a l e a f ) i n a cup w i t h about 6 oz. o f hot water ( i n i t i a l l y a t about 100°C) f o r about 1 min. ( t h i s produces about 5.2 oz. o f beverage a f t e r the tea bag i s removed), o r by d i s s o l v i n g a g e n t l y rounded teaspoonf u l o f i n s t a n t t e a (about 0.70g o f i n s t a n t t e a s o l i d s ) i n 8 oz. of c o l d water. Most of our s t u d i e s have been c a r r i e d out on an approximation o f t h i s standard American b l a c k t e a beverage p r e ­ pared from t e a bags ( a l s o c a l l e d a t e a i n f u s i o n ) s i n c e i t i s o f g r e a t e s t concern t o the authors. As w i l l be e x p l a i n e d l a t e r , the c a f f e i n e i n t e a has an im­ p o r t a n t modifying e f f e c t on the t a s t e o f t e a beverages. Accord­ i n g l y , i t i s noteworth b l a c k t e a beverage s t u d i e 0.026% (8.61% o f the t e a e x t r a c t s o l i d s themselves) which equates to about 40mg c a f f e i n e i n the "average cup o f t e a " . This v a l u e f o r the amount o f c a f f e i n e i n a cup o f t e a compares w i t h the value of 41 mg/cup reported by Burg (8) who i n v e s t i g a t e d t h i s matter. The Chemistry Of Tea Manufacture The chemistry o f t e a manufacture i s d e s c r i b e d i n some de­ t a i l elsewhere (2,9). I n b r i e f , one begins the b l a c k t e a manu­ f a c t u r i n g process by p l u c k i n g ( h a r v e s t i n g ) the f l u s h o f the r a p i d ­ l y growing t e a p l a n t s . The f l u s h i s p a r t i c u l a r l y r i c h i n p o l y ­ p h e n o l i c compounds C2,j)) and o f p a r t i c u l a r importance a r e the flavanols, i . e . (-)-epicatechin ( I ) , (-)-epicatechin-3-gallate ( I I ) , (-)-epigallocatechin ( I I I ) , (-)-epigallocatechin-3-gallate ( I V ) , (+)-catechin (V), and ( + ) - g a l l o c a t e c h i n ( V I ) . The t o t a l amount o f f l a v a n o l s present i n f r e s h t e a f l u s h w i l l vary from about 15 t o 25% (dry weight b a s i s ) : The exact amount o f these compounds present i n any p a r t i c u l a r l o t o f f r e s h l y harvested tea shoot t i p s i s determined by h o r t i c u l t u r a l f a c t o r s such as the clones of t e a p l a n t s from which the t e a shoot t i p s were har­ vested and the c l i m a t e that p r e v a i l e d w h i l e the t e a shoot t i p s were developing. The t e a manufacturing process begins w i t h i n a few hours a f t e r h a r v e s t i n g o f the f r e s h t e a f l u s h , and the fermentation step i s the most c h a r a c t e r i s t i c , and the most important, step i n the p r o ­ cess. Tea fermentation i s i n i t i a t e d by macerating the f r e s h t e a shoot t i p s causing the endogenous t e a c a t e c h o l oxidase t o come i n t o contact w i t h the f l a v a n o l s that a r e a l s o present i n these t i s s u e s . The consequence o f t h i s process i s an o x i d a t i o n o f the f l a v a n o l s ( I - V I ) , and g a l l i c a c i d ( V i l a ) by coupled o x i d a t i o n (10), which leads t o the formation o f the b i s - f l a v a n o l s A ( V I I I ) , Β ( I X ) , and C (X); t h e a f l a v i n ( X I ) ; t h e a f l a v i n g a l l a t e s A ( X I I ) and B ( X I I I ) ; t h e a f l a v i n d i g a l l a t e (XIV); e p i t h e a f l a v i c a c i d (XV); 3 - g a l l o y l - e p i t h e a f l a v i c a c i d (XVI); and t h e a r u b i g i n s which a r e 1

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

15

16

PHENOLIC, SULFUR, AND

Table 1:

NITROGEN COMPOUNDS IN FOOD FLAVORS

Proximate A n a l y s i s of a Black Tea I n f u s i o n . (The b l a c k tea used was L i p t o n tea bag blend and the tea e x t r a c t s o l i d s represented about 33% of the t o t a l tea l e a f dry weight. See the Experimental S e c t i o n f o r more d e t a i l s on a n a l y t i c a l procedures). Amount of Each C o n s t i t u e n t In the Tea In the Extract Beverage, Calcd (% χ 10 ) S o l i d s (%)

Chemical C o n s t i t u e n t

2

9969 Water 16.0 Polyphenols, t o t a l (-)-Epicatechin ( I ) (-)-Epicatechin-3-gallat (-)-Epigallocatechi (-)-Epigallocatechin-3-gallate (IV) F l a v o n o l g l y c o s i d e s and others B i s f l a v a n o l s (VIII-X) T h e a f l a v i n s (XI-XIV) E p i t h e a f l a v i c a c i d s (XV-XVI) Thearubigins (XVII-XX and other unknowns) Gallic acid (Vila) Chlorogenic a c i d Caffeine Theobromine Theophylline Carbohydrates Polysaccharides, t o t a l difference) Pectin Sugars, t o t a l Fructose Glucose meso-Inositol Sucrose Maltose Raffinose

(by

4, 75 48. 5 0.4 1.2

15 68

Trace Trace 0.8 Trace 11.4

Trace Trace 2.50 Trace 34.2

2.4 0.1 0.2

7.20 0.24 0.66

1.3

3.97 0.05

2.2

0.15 6.52

0.60 0.57 0.15 0.48 0.03 0.10

2.0 1.9 0.50 1.6 0.1 0.3

(continued on next page)

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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SANDERSON ET AL.

Polyphenolic Compounds in the Taste of Tea

Table 1, (continued)

Chemical C o n s t i t u e n t

Amount of Each C o n s t i t u e n t In the I n the Tea Beverage, Extract Calcd (% χ 10 ) S o l i d s (%)

Organic A c i d s , t o t a l (pH) ( T o t a l a c i d i t y as c i t r a t e ) Oxalic Malonic Succinic Malic trans-Aconitic Citric

0.8

Lipids, total

1.

M i n e r a l s (Ash) Potassium Sodium Calcium Magnesium Iron Maganese Aluminum

3.0

Peptides (6.25 χ N ) Amino a c i d s , t o t a l Aspartic acid Threonine Serine Glutamic a c i d Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Ammonia Lysine Histidine Arginine Glutamine Asparagine Tryptophane Theanine

2.52 (5,1) (2.36) 0.42 0.01 0.02 0.09 0.003 0.27

(7.85) 1.4 0.02 0.09 0.30 0.01 0.80

9.08 1.37 0.03 0.02 0.075 0.0013 0.015 0.014

a

4.6 0.11 0.08 0.25 0.005 0.05 0.05

1.9

5.71

2.1

6.29 Trace Trace Trace Trace Trace Trace Trace Trace Trace Trace Trace Trace Trace Trace Trace Trace Trace Trace Trace 1.1

0.39 0.07 0.24 0.42 0.02 0.12 0.20 0.02 0.18 0.19 0.15 0.16 0.13 0.04 0.002 0.03 0.19 0.24 0.09 3.40

T o t a l n o n - c a f f e i n e Ν (1.92%) χ 6.25 — amino a c i d s

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

17

18

PHENOLIC, SULFUR, AND NITROGEN COMPOUNDS IN FOOD FLAVORS

OH

I , (-)-Epicatechin; R^=H; R =H I I , ( - ) - E p i c a t e c h i n - 3 - g a l l a t e ; R =H; R =VIIb I I I , (-)-Epigallocatechin (-)-Epigallocatechin-3-gallate 2

I V >

V, (+)-Catechin; R=H V I , ( + ) - G a l l o c a t e c h i n ; R=OH

V i l a , G a l l i c acid

V l l b , G a l l o y l group

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

SANDERSON E T A L .

XI, XII, XIII, XIV,

Polyphenolic Compounds in the Taste of Tea

T h e a f l a v i n ; R]=H; R =H T h e a f l a v i n g a l l a t e A; Ri=H; R =VIIb T h e a f l a v i n g a l l a t e B; R ^ V I I b ; R =H T h e a f l a v i n d i g a l l a t e ; R =VIIb; R =VIIb 2

2

2

1

2

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

20

PHENOLIC,

SULFUR,

AND

NITROGEN

COMPOUNDS

IN

FOOD

FLAVORS

polymeric proanthocyanidins; i . e . p r o c y a n i d i n ( X V I I ) , p r o c y a n i d i n 3 - g a l l a t e ( X V I I I ) , p r o d e l p h i n i d i n (XIX), p r o d e l p h i n i d i n - 3 - g a l l a t e (XX). The chemistry of the t h e a r u b i g i n s i s only p o o r l y understood at the present time, but i t i s known t h a t they are a heterogeneous group of polymers formed by the o x i d a t i v e condensation of the simple f l a v a n o l s (I-VI) (11). F u r t h e r , the t h e a r u b i g i n s have been c h a r a c t e r i z e d as polymeric proanthocyanidins (12, 13, 14), w i t h molecular weights ranging from 700-40,000 ( 7 ) . F i n a l l y , the exa c t composition of the t h e a r u b i g i n s probably v a r i e s w i t h the cond i t i o n s of t h e i r formation; i . e . the c o n d i t i o n s of b l a c k t e a manuf a c t u r e , which has made t h e i r determination a most d i f f i c u l t matter (15). The r e a c t i o n s of the tea polyphenols during tea fermentation and f i r i n g are o u t l i n e d i n Figure 1. Fermentation and f i r i n g leads to the i n s o l u b i l i z a t i o n of Çhe t e a l e a f p r o t e i n s , some of the t e a p o l y p h e n o l i m a t e r i a l c o n s i d e r a b l e s o l i d matte Those b l a c k t e a substances t h a t are e x t r a c t e d i n the "normal" brewing of b l a c k t e a l e a f are l i s t e d i n Table I . In green tea manufacture, the harvested tea shoot t i p s are steamed p r i o r to maceration i n order to i n a c t i v a t e the endogenous c a t e c h o l oxidase enzyme. As a r e s u l t , the tea f l a v a n o l s undergo very l i t t l e change i n t h i s process and green tea i s r i c h i n uno x i d i z e d f l a v a n o l s ( I - V I ) . In b l a c k t e a manufacture, the t e a fermentation process i s allowed to proceed to near completion so there are u s u a l l y only t r a c e s of unoxidized f l a v a n o l s (I-VI) r e maining i n the f i n i s h e d product. However, the exact mix of f l a v a n o l o x i d a t i o n products (VIII-XX, and o t h e r s ) w i l l vary depending on the p r e c i s e c o n d i t i o n s under which the tea manufacturing process takes p l a c e . Oolong t e a (commonly c a l l e d Chinese tea i n the United S t a t e s ) i s produced by a p a r t i a l fermentation so i t cont a i n s an a p p r e c i a b l e r e s i d u e of unoxidized f l a v a n o l s (I-VI) as w e l l as the f l a v a n o l o x i d a t i o n products (VIII-XX and o t h e r s ) . In a d d i t i o n to the p o l y p h e n o l i c compounds, aroma i s most important i n determining the f l a v o r and q u a l i t y of tea products. Many chemical changes take p l a c e d u r i n g the tea manufacturing process, e s p e c i a l l y d u r i n g t e a fermentation and the subsequent f i r i n g (drying) s t e p , t h a t are e s s e n t i a l to the formation of the aroma c h a r a c t e r i s t i c of tea. I t has been shown (16) that the o x i d a t i o n of the tea f l a v a n o l s t h a t takes p l a c e d u r i n g t e a fermentation i s i t s e l f an e s s e n t i a l d r i v i n g f o r c e f o r r e a c t i o n s t h a t are r e q u i r e d to develop the aroma t h a t i s c h a r a c t e r i s t i c of tea products, and f i r i n g has been shown to be e s s e n t i a l f o r b l a c k tea aroma format i o n (17, 18). The chemistry of f l a v o r formation during the manufacture of b l a c k tea was r e c e n t l y reviewed (19, 20), and i t i s summarized i n F i g u r e 2. A t t e n t i o n should be drawn to c a f f e i n e (XXI) s i n c e c a f f e i n e does p l a y a p a r t i n determining the t a s t e of a cup of t e a . Caff e i n e i s b i o s y n t h e s i z e d i n the tea p l a n t (21, 22), and i t undergoes p r a c t i c a l l y no change during the b l a c k tea manufacturing process (2). Therefore, the amount of c a f f e i n e present i n tea prod-

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

2.

SANDERSON ET AL.

Polyphenolic Compounds in the Taste of Tea

IH HO

Τ^,ΟΗ

XV, E p i t h e a f l a v i d a c i d R=H XVI, 3 - G a l l o y l e p i t h e a f l a v i c a c i d ; R=VIIb f

OH

0R

Η

2

Η or Y XVII, P r o c y a n i d i n ; R}=H; R =H XVIII, Procyanidin g a l l a t e , R =H; R =VIIb 2

X

2

XIX, P r o d e l p h i n i d i n ; R,=0H; R XX, P r o d e l p h i n i d i n g a l l a t e ; R =OH; R =VIIb x

2

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

2

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

Figure I.

(V,VI)

(VIII-X)

Bisflavanols

(tea fermentation and f i r i n g )

(XVII-XVIII)

Procyanidins

BLACK TEA

(XVII-XVII)

P r o d e l p h i n i d i n s (XIX-XX)

Mixed Proanthocyanidins (XVII-XX)

->· I n c r e a s i n g l e v e l o f o x i d a t i o n and p o l y m e r i z a t i o n

(XI-XIV)

Theaflavins

• Unknowns

E p i t h e a f l a v i c a c i d s (XV-XVI)/-*- P r o c y a n i d i n s

Thearubigins

> Unknowns

Summary of changes undergone by the tea flavanols during tea fermentation and firing in black tea manufacture

FRESH GREEN TEA FLUSH

+ 0„

(+)-Catechins

(-)-Epigallocatechins (III,IV)

(-)-Epicatechins (1,11) + 0.

+Gallic acid (Vila)

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

(direct)

O x i d i z e d Tea Flavanols

Black Tea Aroma Constituents

(

j B l a c k Tea Polyphenols ^ (VIII-XX and other unknowns; Pigments, astringents, etc.)

Figure 2. Summary of reactions taking place during tea fermentation and firing in black tea manufacture. Tea flavanol oxidation has an essential role in causing various chemical changes that are important to the formation of black tea flavor

Black Tea F l a v o r Precursors

Tea F l a v a n o l s + (I-VI)

(Tea Catechol Oxidase)

24

PHENOLIC, SULFUR, AND

NITROGEN COMPOUNDS IN FOOD FLAVORS

ucts i s f i x e d by h o r t i c u l t u r a l p r a c t i c e s , and cannot be changed by c u r r e n t l y known tea manufacturing processes. The Taste of Black Tea Derives Mainly From The Tea

Polyphenols

The Taste of a Black Tea I n f u s i o n and Various F r a c t i o n s of This I n f u s i o n . A b l a c k t e a i n f u s i o n was c h e m i c a l l y analyzed (Table 1 ) and o r g a n o l e p t i c a l l y evaluated (Table 2 , F r a c t i o n 1 ) as a f i r s t step i n our program t o e l u c i d a t e the chemistry u n d e r l y i n g the t a s t e of b l a c k t e a . The beverage obtained was found to have a c h a r a c t e r i s t i c b l a c k t e a t a s t e that was described as being " f l o w e r y , p l e a s i n g , m i l d l y green and h a y - l i k e , and d i s t i n c t l y b l a c k tea l i k e " . When a t t e n t i o n was given s p e c i f i c a l l y to the a s t r i n g e n c y of the i n f u s i o n , i t was decided a f t e r lengthy d e l i b e r a t i o n by the p a n e l i s t t h a t th a s t r i n g e n c best described as having two components sharp and puckery w i t h (thi f i c u l t to d e s c r i b e type of a s t r i n g e n c y that i s c h a r a c t e r i s t i c of b l a c k t e a ) , and a non-tangy component t h a t was completely t a s t e l e s s , mouth-drying and mouth c o a t i n g , w i t h a l i n g e r i n g (more than 60 sec.) a f t e r t a s t e e f f e c t ( t h i s type of a s t r i n g e n c y i s t y p i c a l of unripe bananas). I t i s noteworthy that there i s v i r t u a l l y no b i t t e r n e s s i n t h i s whole b l a c k tea i n f u s i o n . Chemical a n a l y s i s of the "whole b l a c k t e a i n f u s i o n " showed that the s o l i d s were composed of about 4 8 % polyphenols, 7% c a f f e i n e and 4 4 . 3 % " o t h e r " m a t e r i a l s (Table 1 ) . Next, we f r a c t i o n a t e d the b l a c k t e a i n f u s i o n by a combination of s o l v e n t e x t r a c t i o n s and a d s o r p t i o n column treatments i n a n t i c i p a t i o n of being a b l e to i d e n t i f y the group of compounds r e s p o n s i b l e f o r each component of b l a c k tea t a s t e . And, of course, we were most i n t e r e s t e d i n i d e n t i f y i n g the c o n t r i b u t i o n of the p o l y phenols to the t a s t e of t h i s product. The t r i c h l o r o e t h y l e n e e x t r a c t (Table 2 , F r a c t i o n 2 ) contained mostly c a f f e i n e (XXI) and i t was b i t t e r w i t h no other n o t i c e a b l e taste attributes. The e t h y l acetate e x t r a c t (Table 2 , F r a c t i o n 4 ) contained mostly n e u t r a l b l a c k t e a polyphenols. These polyphenols were found by paper chromatography to be composed of the t r a c e s of uno x i d i z e d t e a f l a v a n o l s ( I - V I ) and the simple p o l y p h e n o l i c o x i d a t i o n products (VIII-XVI) and some of the t h e a r u b i g i n s : Roberts et a l . ( 2 3 ) named these the t h e a r u b i g i n s . This f r a c t i o n had a t r a c e of tangy a s t r i n g e n c y and a moderate l e v e l of non-tangy astringency. The aqueous phase remaining a f t e r removal of F r a c t i o n s 2 and 4 (Table 2 , F r a c t i o n 5 ) contained some complex p o l y p h e n o l i c compounds named the S and S J J t h e a r u b i g i n s by Roberts et a l ( 2 3 ) , and a l l the non-polyphenolic tea e x t r a c t s o l i d s (Table 1 ) except c a f f e i n e . This f r a c t i o n t a s t e d s i m i l a r to F r a c t i o n 4 i n t h a t i t had a f a i r l e v e l of non-tangy a s t r i n g e n c y w i t h none of the tangy astringency. I

A

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

2.

SANDERSON ET AL.

Polyphenolic Compounds in the Taste of Tea

Complete removal of p o l y p h e n o l i c compounds from the whole b l a c k tea e x t r a c t ( F r a c t i o n 1) was accomplished by passing t h i s e x t r a c t through a polyamide column (Table 2, F r a c t i o n 6 ) . The removal of the p o l y p h e n o l i c compounds from the t e a e x t r a c t was accompanied by a removal of a l l a s t r i n g e n c y from the whole tea e x t r a c t w i t h the concomitant appearance of b i t t e r n e s s t h a t was not present on the whole tea e x t r a c t . These r e s u l t s , together w i t h the r e s u l t s obtained f o r F r a c t i o n s 4 and 5, i n d i c a t e that the p o l y p h e n o l i c compounds i n a whole b l a c k t e a e x t r a c t are ast r i n g e n t and t h a t t h i s a s t r i n g e n c y i s expressed i n the whole b l a c k tea e x t r a c t . On the other hand, the c a f f e i n e i n a b l a c k tea e x t r a c t ( F r a c t i o n 2) i s present at a h i g h enough c o n c e n t r a t i o n to produce a b i t t e r t a s t e , but t h i s b i t t e r n e s s i s not expressed i n the whole b l a c k tea e x t r a c t ( F r a c t i o n 1 ) ; i t i s only expressed when the polyphenols ar Removal of the c a f f e i n (Fraction 6 F r a c t i o n 7) was e f f e c t i v e i n removing the b i t t e r ness from t h i s e x t r a c t showing again t h a t the c a f f e i n e i n a b l a c k tea e x t r a c t i s r e s p o n s i b l e f o r a b i t t e r t a s t e i n the absence of the b l a c k tea polyphenols. F r a c t i o n 8 (Table 2) was prepared by a d d i t i o n of pure c a f f e i n e i n the amount o r i g i n a l l y present i n the whole b l a c k tea i n f u s i o n ( F r a c t i o n 1) to the n e u t r a l b l a c k tea polyphenols ( F r a c t i o n 4 ) . This caused a modest i n c r e a s e i n the tangy a s t r i n g e n c y of F r a c t i o n 4 w i t h no change i n the non-tangy a s t r i n g e n c y . Most important, there was no n o t i c e a b l e b i t t e r n e s s i n F r a c t i o n 8. F r a c t i o n 9 (Table 2) was prepared by adding c a f f e i n e to Fract i o n 5 ( i . e . the a c i d i c b l a c k tea polyphenols and non-phenolic s o l i d s ) . This caused a s m a l l but s i g n i f i c a n t decrease i n the non-tangy a s t r i n g e n c y of F r a c t i o n 5, but no appearance of tangy a s t r i n g e n c y and no appearance of b i t t e r n e s s . F r a c t i o n 10 (Table 2) i s v i r t u a l l y a r e c o n s t i t u t i o n of the whole b l a c k t e a e x t r a c t ( F r a c t i o n 1 ) , and, as might be expected, i t was found to have t a s t e p r o p e r t i e s that were very s i m i l a r to the whole b l a c k t e a e x t r a c t . C o l l e c t i v e l y , these r e s u l t s (Table 2) i n d i c a t e d that the b l a c k tea polyphenols are a c e n t r a l e s s e n t i a l element i n d e t e r mining the t a s t e of black tea i n f u s i o n s . This i s i l l u s t r a t e d best by n o t i c i n g F r a c t i o n 6 which i s v i r t u a l l y the complete b l a c k t e a i n f u s i o n minus the black, t e a polyphenols and which has p r a c t i c a l l y no t a s t e other than some b i t t e r n e s s : The b i t t e r n e s s i s accounted f o r by the c a f f e i n e present i n t h i s f r a c t i o n . The primary c o n t r i b u t i o n of the b l a c k tea polyphenols to the " t a s t e " of b l a c k tea i n f u s i o n s appears to be a s t r i n g e n c y , and the "tangy" p o r t i o n of t h i s a s t r i n g e n c y was found to be most c h a r a c t e r i s t i c o f , and important t o , the t a s t e of b l a c k t e a . The r e s u l t s (Table 2) a l s o suggest that c a f f e i n e p l a y s a most important r o l e i n determining the l e v e l of tangy a s t r i n g e n c y i n a b l a c k tea i n f u s i o n . This was shown i n two ways. F i r s t , decaff e i n a t i o n of a b l a c k tea i n f u s i o n ( i . e . F r a c t i o n 1 -*· F r a c t i o n 3)

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

25

26

PHENOLIC, SULFUR, AND NITROGEN COMPOUNDS IN FOOD FLAVORS

Table 2.

Composition and Taste of F r a c t i o n s of Black Tea. (The c a r r i e d out as described i n the Experimental Section.) Composition of F r a c t i o n

Total Solids Present (%)

F r a c t i o n of Tea E x t r a c t (1) Whole b l a c k tea e x t r a c t ( D e t a i l e d composition shown i n Table 1)

100^

Amount of P o l y phenols (%) 48.5

(2) T r i c h l o r o e t h y l e n e solubles (Mainly caffeine)

8.1

0

(3) T r i c h l o r o e t h y l e n e e x t r a c t e d s o l i d s (Decaffeinated and dearomatized tea infusion)

92.3

48.5

(4) E t h y l acetate s o l u b l e s (Neutral black tea polyphenolsj i . e . f l a v a n o l s , t h e a f l a v i n s , S j t h e a r u b i g i n s , etc.)

17.0

17

(5) Aqueous phase a f t e r removal of Frac- 74.9 t i o n s 2 and 4 ( A c i d i c b l a c k tea p o l y phenols; i . e . S and S-J-J t h e a r u b i g i n s ; and non-polyphenolic s o l i d s )

31.5

(6) Polyamide column e f f l u e n t of Fract i o n 1 (Polyphenol f r e e tea s o l i d s )

51.5

0

(7) XAD-2 column e f f l u e n t of F r a c t i o n 5 (Polyphenol and c a f f e i n e f r e e tea extract)

43.4

0

(8) F r a c t i o n 4 (Neutral b l a c k tea p o l y phenols) + c a f f e i n e

24.2

17

(9) F r a c t i o n 5 (Tea e x t r a c t minus n e u t r a l b l a c k tea polyphenols) + caffeine

82.1

31.5

(10) F r a c t i o n 4 + F r a c t i o n 5 + C a f f e i n e + Aroma

99.1

48.5

I A

a

Astringency

b

The t o t a l s o l i d s e x t r a c t e d were about 33% of

r a t i n g s : 0 = none, 1 = t h r e s h o l d

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

2.

SANDERSON ET AL.

Polyphenolic Compounds in the Taste of Tea 2 7

f r a c t i o n a t i o n of the b l a c k t e a beverage, F r a c t i o n 1 , was

Taste D e s c r i p t i o n of F r a c t i o n Polyphenol and Amount of C a f f e i n e Free C a f f e i n e ( % ) S o l i d s (%) 7.2

7.2

0.4

Trace

Astringency NonTangy Tangy

Trace

Trace

Other Flowery, p l e a s i n g , m i l d green h a y - l i k e , black tea taste

44.3

43.4

a

Bitter

2

Very weak b l a c k t e a taste

2

Sweetish a f t e r taste

Trace

43.4

3

Chalky

7.2

44.3

0

Slightly bitter, green hay l i k e aroma

Trace

43.4

0

Malty

7.2

0

Plain

7.2

43.4

Chalky

7.2

43.4

3

S i m i l a r t o Fraction 1

l e v e l , 2 = weak, 3 = moderate, 4 = s t r o n g , the o r i g i n a l L i p t o n b l a c k tea bag b l e n d .

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

28

PHENOLIC,

SULFUR,

AND

NITROGEN

COMPOUNDS

IN

FOOD

FLAVORS

caused a marked r e d u c t i o n i n the tangy a s t r i n g e n c y and i n the b l a c k tea t a s t e of the i n f u s i o n . And second, w h i l e c a f f e i n e i t s e l f ( F r a c t i o n 2) i s b i t t e r and has no a s t r i n g e n c y , the presence of c a f f e i n e together w i t h the b l a c k t e a polyphenols, and e s p e c i a l l y w i t h the n e u t r a l b l a c k t e a polyphenols, was necessary f o r the expression of a reasonable amount of tangy a s t r i n g e n c y (compare F r a c t i o n 4 and F r a c t i o n 8 ) . The aroma i n the b l a c k tea i n f u s i o n was a l s o found to be important i n determining the f l a v o r of the beverages. None of the f r a c t i o n s ( i . e . Table 2, F r a c t i o n s 2-9 ) of the whole b l a c k tea i n f u s i o n ( F r a c t i o n 1) was n o t i c e a b l y b l a c k tea l i k e unless aroma was present w i t h the b l a c k t e a polyphenols and c a f f e i n e (compare F r a c t i o n 10 w i t h F r a c t i o n 1). I t i s noteworthy i n t h i s connection that aroma w i t h c a f f e i n e and a l l other b l a c k t e a s o l i d s except the polyphenols ( F r a c t i o 6) had weak s l i g h t l b i t t e r greenish t a s t e that was The E f f e c t Of Tea Fermentation And F i r i n g On The Taste Of Tea I n f u s i o n s . Samples of t e a were prepared t h a t had been f e r mented f o r v a r i o u s p e r i o d s of time and that had been e i t h e r f i r e d or not f i r e d ( i . e . f r o z e n immediately a f t e r fermentation and f r e e z e d r i e d ) f o r use i n determining the e f f e c t of fermentat i o n and f i r i n g on the tea polyphenols and on the t a s t e of the r e s u l t i n g tea products. These samples were prepared i n our l a b o r a t o r y using f r e s h tea f l u s h , and i t i s recognized that the r e s u l t s of these experiments s u f f e r from the l i m i t a t i o n s imposed by these non-optimal c o n d i t i o n s . In s p i t e of these l i m i t a t i o n s , we b e l i e v e that our r e s u l t s are i n d i c a t i v e of the r o l e of tea polyphenols i n determining the t a s t e of b l a c k t e a i n f u s i o n s . The r e s u l t s of the analyses and the o r g a n o l e p t i c e v a l u a t i o n of the i n f u s i o n s produced from these samples are summarized i n Table 3. These r e s u l t s may be b r i e f l y summarized as f o l l o w s : (a) The amount of s o l i d s e x t r a c t e d from the tea l e a f by the brewing process increases a p p r e c i a b l y d u r i n g the i n i t i a l stage of tea fermentation, i . e . during the l e a f maceration process and the very f i r s t minutes of the formal tea fermentation p e r i o d , a f t e r which the e x t r a c t a b l e s o l i d s decrease as the tea fermentation p e r i o d i n c r e a s e s . F i r i n g causes an a d d i t i o n a l app r e c i a b l e l o s s of e x t r a c t a b l e s o l i d s . Apparently, the very f i r s t products of t e a fermentation (Figure 1) are more e a s i l y e x t r a c t e d than the tea f l a v a n o l s themselves, although tea fermentation (and f i r i n g ) do l e a d to the formation of l e s s and l e s s s o l u b l e products as the length of the process i n c r e a s e s . (b) The amount of t o t a l f l a v a n o l s i n the i n f u s i o n decreases as t e a fermentation proceeds: This decrease i n f l a v a n o l s i s most r a p i d i n the e a r l y stages of tea fermentation. F i r i n g causes an a p p r e c i a b l e a d d i t i o n a l decrease i n f l a v a n o l s , e s p e c i a l l y i n the i n i t i a l stages of tea fermentation. I t i s noteworthy that the g a l l o - f l a v a n o l s ( I I I , I V ) are o x i d i z e d more r a p i d l y than the c a t e c h o l - f l a v a n o l s (1,11) (Figure 3).

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

2.

SANDERSON ET AL.

Polyphenols Compounds in the Taste of Tea

29

Figure 3 . The disappearance of teaflavanoUin macerated teaflushas a result of tea fermentation and firing. Fermented and freeze dried (i.e. not fired): 0, ί V; Δ , II; Ο , III; •, IV. Fermented and fired: •, I and V; A , II; ·, III; •, IV. These samples are described further in Table 3.

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

30

PHENOLIC, SULFUR, AND NITROGEN COMPOUNDS IN FOOD FLAVORS

Table 3.

E f f e c t of Tea Fermentation and F i r i n g on the Taste of Composition of Tea I n f u s i o n S o l i d s

Theaflavins (mg/cup)

Thea rubigins (mg/cup)

Fermented and then Freeze D r i e d (before maceration) 763 ( a f t e r maceration) 283 872

2.3 4.1

129 202

Length of Tea Fermentation P e r i o d (min.) A. 0 0

C

Total Total Solids Flavanols (mg/cup) (mg/cup)

15 30

804

171

12.7

232

60 90 120 180 240

824 825 781 732 746

111

17.3 16.3 13.1 13.6 12.2

274 278 276 283 294

B.

34 26

Fermented and F i r e d (before maceration) 763 ( a f t e r maceration) 851

173

2.3 7.4

129 242

15 30 60

828 786 758

154 107 92

10.1 12.9 14.7

256 271 285

90

735

15.4

293

120 180 240

706 680 630

13,3 11.9 11.5

290 276 276

0 0

C

33 29

a

A l l t e a i n f u s i o n s were prepared by e x t r a c t i n g 2.27g of dry tea produced about 5.2 oz. of beverage ( i . e . the i n f u s i o n ) . A n a l y s i s caffeine/cup. b

A s t r i n g e n c y r a t i n g s : 0 = none, 1 = t h r e s h o l d , 2 = weak,

c

This sample of f r e s h green tea l e a f was analyzed b e f o r e any macerated p r i o r to the formal tea fermentation p e r i o d . A c c o r d fermentation, would have taken p l a c e i n these samples p r i o r to

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

2.

SANDERSON E T A L .

Polyphenols Compounds in the Taste of Tea

Table 3 (cont.) Tea I n f u s i o n s Organoleptic E v a l u a t i o n o f I n f u s i o n s

Astringency b Tangy Non-Tangy

Black Tea Aroma

Black Tea Taste

None None

Bland, s l i g h t l y green Very green, h a r s h , bitter

Very green, h a r s h , slightly bitter Green, harsh Green, s l i g h t l y harsh Green, s l i g h t l y harsh Green, s l i g h t l y harsh Green, s l i g h t l y harsh

0 0

1 4

0 0

0

4

0

0

4

0

None

0 0 0 0 0

3 2 2 1 1

0 0 0 0 0

None None None None None

0 0

1 4

0 0

None None

0 0 1

4 3 2

0 0 1

None None Slight

1

2

1

Mild

1 1 1

2 1 1

1 2 2

Mild Weak Weak

Other t a s t e

Bland, s l i g h t l y green Green, h a r s h , s l i g h t l y bitter Green, s l i g h t l y harsh Green, s l i g h t l y harsh S l i g h t l y green, s l i g h t l y harsh S l i g h t l y green, s l i g h t l y harsh S l i g h t l y harsh None None

l e a f w i t h 6.0 oz. b o i l i n g water i n a t e a cup f o r 5 min. This showed that each i n f u s i o n contained between 38 and 40 mg.

3 = moderate, 4 = s t r o n g . treatments. A l l other samples of t e a l e a f were withered and i n g l y , some o x i d a t i o n of the t e a l e a f f l a v a n o l s , i . e . t e a the s t a r t of the formal t e a fermentation p e r i o d .

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

32

PHENOLIC, SULFUR, AND

NITROGEN COMPOUNDS IN FOOD FLAVORS

The reason f o r t h i s d i f f e r e n t i a l i n s u s c e p t i b i l i t y to o x i d a t i o n i n the t e a fermentation system i s d i f f i c u l t to e x p l a i n s i n c e a l l of the tea f l a v a n o l s have s i m i l a r a f f i n i t i e s f o r the tea c a t e c h o l oxidase enzyme (24), but the phenomena has been n o t i c e d before (18). C e r t a i n l y , the amount of each f l a v a n o l , and the r e l a t i v e r a t e of o x i d a t i o n of each f l a v a n o l , must be important determinants of the exact composition of t h e i r o x i d a t i o n products (Figure 1 ) , and c o n s e q u e n t i a l l y on the t a s t e of the f i n i s h e d b l a c k tea product. (c) T h e a f l a v i n s i n c r e a s e as the tea fermentation proceeds u n t i l a maximum i s reached ( a f t e r about 60 min. i n the u n f i r e d samples and 90 min. i n the f i r e d samples) a f t e r which they decrease i n amount. F i r i n g causes a l a r g e r amount of t h e a f l a v i n s to accumulate a f t e r short fermentation periods and s m a l l e r amounts to be present a f t e r longer fermentation p e r i o d s . (d) An a p p r e c i a b l f t h e a r u b i g i n i formed i th macerated tea l e a f p r i o t i o n p e r i o d : This must be due to the tea fermentatio t h a t takes p l a c e i n i n j u r e d c e l l s of the t e a f l u s h during w i t h e r i n g and, most important, the tea fermentation that takes p l a c e d u r i n g maceration of the tea f l u s h . The t h e a r u b i g i n s continue t o i n c r e a s e c o n t i n u ously as the tea fermentation p e r i o d increases (Table 3, p a r t A ) . F i r i n g causes an a d d i t i o n a l a p p r e c i a b l e i n c r e a s e i n the amount of t h e a r u b i g i n s e x t r a c t e d from samples w i t h l o n g , i . e . greater than about 120 min. i n these experiments, fermentation periods (Table 3, p a r t B). The decrease i n e x t r a c t a b l e t h e a r u b i g i n s a f t e r r e l a t i v e l y long tea fermentation periods and f i r i n g appears to be c l o s e l y r e l a t e d to the decrease i n t o t a l e x t r a c t a b l e s o l i d s that i s a s s o c i a t e d w i t h these treatments. (e) The tea fermentation t h a t takes p l a c e p r i o r to the f o r mal tea fermentation p e r i o d , i . e . during w i t h e r i n g and maceration of the f l u s h , causes a l a r g e i n c r e a s e i n the non-tangy a s t r i n g e n c y of tea i n f u s i o n s prepared from t h i s m a t e r i a l . Tea fermentation per se (Table 3, p a r t A) causes a decrease i n non-tangy astringency that i s present i n withered, macerated tea l e a f p r i o r to the f o r mal tea fermentation p e r i o d , but n e i t h e r tangy a s t r i n g e n c y nor b l a c k tea aroma develop, hence no b l a c k tea t a s t e develops unless the samples undergo t e a fermentation ( f o r at l e a s t about 90 min. i n these experiments) and are f i r e d (Table 3, p a r t B). I t i s noteworthy t h a t the t h e a f l a v i n s and t h e a r u b i g i n s content of the i n f u s i o n from the u n f i r e d sample fermented f o r 180 min. was almost i d e n t i c a l to the t h e a f l a v i n s and t h e a r u b i g i n s content of the i n f u s i o n from the f i r e d sample t h a t had been fermented f o r 120 min., yet these samples had e n t i r e l y d i f f e r e n t t a s t e p r o f i l e s ( i . e . the former was green and harsh w i t h no b l a c k tea c h a r a c t e r whereas the l a t t e r was midly a s t r i n g e n t and p l e a s a n t l y b l a c k tea l i k e ) : Very s i m i l a r r e s u l t s and conclusions were reported p r e v i o u s l y (18). This p o i n t s out the s e r i o u s f a i l i n g s of the Roberts method (15) f o r e v a l u a t i n g tea beverages by measurement of t h e a f l a v i n s and thearubigins i n s p i t e of much work to e s t a b l i s h the v a l i d i t y of t h i s t e s t (25, 26, 27, 28). 1

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

2.

SANDERSON ET A L .

Polyphenolic Compounds in the Taste of Tea

The Taste Of I n d i v i d u a l P o l y p h e n o l i c Compounds Present In Tea I n f u s i o n s . The work described above e s t a b l i s h e d the impor­ tance of the tea polyphenols i n determining the t a s t e of b l a c k tea i n f u s i o n s . Next we were i n t e r e s t e d i n determining the t a s t e p r o p e r t i e s of each i n d i v i d u a l b l a c k t e a polyphenol as a step towards d e f i n i n g t h e i r separate c o n t r i b u t i o n s to the whole. Ac­ c o r d i n g l y , the p o l y p h e n o l i c compounds present i n tea beverages were p u r i f i e d and then they were o r g a n o l e p t i c a l l y evaluated f o r t h e i r t a s t e p r o p e r t i e s and t h e i r i n d i v i d u a l t a s t e t h r e s h o l d values. The r e s u l t s (Table 4) i n d i c a t e d that the only t a s t e p r o p e r t i e s as­ s o c i a t e d w i t h the t e a polyphenols are astringency and b i t t e r n e s s . The simple, non-gallated tea f l a v a n o l s ( I , I I I , V) are not as­ t r i n g e n t , although they do have a b i t t e r t a s t e . On the other hand, the simple, g a l l a t e d tea f l a v a n o l s ( I I , IV, VI) and the con­ densed t e a f l a v a n o l s (XI-XIX) a s t r i n g e n t i a d d i t i o t havin a b i t t e r t a s t e . Of p a r t i c u l a no case was the a s t r i n g e n c y y purifie polyphenol of the tangy type. These r e s u l t s c l e a r l y show the importance of the g a l l o y l groups ( V l l b ) on the tea f l a v a n o l s f o r the expression of a s t r i n ­ gency and b i t t e r n e s s . R e s u l t s (Table 4) obtained w i t h the v a r i o u s t h e a f l a v i n s (XI-XIV) a l s o i n d i c a t e s the importance of the g a l l o y l groups ( V l l b ) i n determining the a s t r i n g e n c y of condensed ( o x i ­ d i z e d ) p o l y p h e n o l i c compounds i n t e a . T h e a f l a v i n (XI) i s formed by o x i d a t i v e condensation of ( - ) - e p i c a t e c h i n ( I ) and ( - ) - e p i g a l l o c a t e c h i n ( I I I ) (which are not a s t r i n g e n t ) , y e t t h e a f l a v i n (XI) has some a s t r i n g e n c y even though i t has no g a l l o y l groups ( V l l b ) : This i s presumably due to the r e l a t i v e l y l a r g e molecular s i z e and the l a r g e number of p h e n o l i c groups of t h i s molecule as com­ pared to the simple n o n - g a l l a t e d t e a f l a v a n o l s ( I , I I I , V). How­ ever, there i s a p r o g r e s s i v e i n c r e a s e i n the i n t e n s i t y , i . e . de­ crease i n the t h r e s h o l d l e v e l , of the a s t r i n g e n c y of the thea­ f l a v i n s as the number of g a l l o y l groups ( V l l b ) per molecule i n ­ creases. That i s , t h e a f l a v i n (XI) i s l e s s a s t r i n g e n t t h a t the t h e a f l a v i n monogallates A and Β ( X I I - X I I I ) which are l e s s a s t r i n ­ gent than t h e a f l a v i n d i g a l l a t e (XIV). The t o t a l of the unoxidized f l a v a n o l s and the t h e a f l a v i n s i n b l a c k tea i s h a r d l y enough to reach t h e i r t a s t e t h r e s h o l d l e v e l . This leaves the t h e a r u b i g i n s , which u s u a l l y comprise over 30% of a l l the b l a c k tea s o l i d s e x t r a c t e d i n t o a cup of t e a , to ac­ count f o r most of the " t a s t e " of t e a . U n f o r t u n a t e l y , i t was not p o s s i b l e to p u r i f y the t h e a r u b i g i n s s u f f i c i e n t l y to determine t h e i r t h r e s h o l d v a l u e , but i t was determined that they are as­ t r i n g e n t . The chemistry of the t h e a r u b i g i n s i s only p o o r l y under­ stood at the present time, but i t i s known that they are a h e t e r ­ ogeneous group of condensed f l a v a n s (11). I t i s noteworthy that the astringency of tea beverages increases a p p r e c i a b l y d u r i n g the very e a r l y stages of tea fermentation, i . e . before the beginning of the formal t e a fermentation p e r i o d . The a s t r i n g e n c y of the tea i n f u s i o n s then decreases s t e a d i l y even though the l e v e l of

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

33

34

PHENOLIC,

Table 4:

SULFUR,

AND

NITROGEN

COMPOUNDS

IN

FOOD

FLAVORS

Threshold Levels For Astringency and B i t t e r n e s s Of Polyphenols

Threshold L e v e l (mg/100ml) Astringency B i t t e r n e s s 60 Not a s t r i n g e n t 50 20

Tea

Approximate L e v e l i n a Cup of Black T e a (mg/100ml) of beverage) Trace Trace a

Polyphenol (-)-Epicatechin ( I ) (-)-Epicatechin gallate (II) Trace 35 ( - ) - E p i g a l l o c a t e c h i n Not a s t r i n g e n t (III) 16-18 60 30 (-)-Epigallocatechin g a l l a t e (IV) (+)-Catechin (V) 5-11 60 70 Crude T h e a f l a v i n s , a n a t u r a l mixture (XI-XIV) 1.2 0.6 75- 100 80 T h e a f l a v i n (XI) 3.7 1.8 50 3036 T h e a f l a v i n monogallates A and B, a n a t u r a l mixture ( X I I - X I I I ) Not determined 2.4 - 4.8 12.5 Theaflavin d i g a l l a t e (XIV) c 3- 5 bitter G a l l i c acid (Vila) Not a s t r i n g e n t ^ Not 120 95determined^ Thearubigins (XVIINot determined" Not XX, others) 20 80 Tannic a c i d u

b

a

c

Based on amount of s o l i d s e x t r a c t e d from a standard American tea bag (2.27g. tea) brewed w i t h 6 oz. b o i l i n g tap water i n a cup f o r 3 min. Polymeric proanthocyanidins (12, 13). T e s t e d a t up to 1000 mg/100ml: Taste at t h i s l e v e l was sour w i t h sweet l i n g e r i n g a f t e r - t a s t e . I t was not p o s s i b l e to prepare samples of t h e a r u b i g i n s of suff i c i e n t p u r i t y o r g a n o l e p t i c a l l y to evaluate.

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

2.

SANDERSON ET A L .

Polyphenolic Compounds in the Taste of Tea

thearubigins continues to i n c r e a s e . I t can be surmised that the astringency and the b i t t e r n e s s of the thearubigins d e r i v e s both from the number of g a l l o y l groups per i n d i v i d u a l t h e a r u b i g i n molecule and from the degree of condensation ( s i z e ) of each i n d i v i d u a l t h e a r u b i g i n molecule which changes continuously d u r i n g tea fermentation and f i r i n g , but these f a c t o r s have yet to be s t u d i e d . The l a c k of tangy astringency i n any of the p u r i f i e d b l a c k tea polyphenols i s thought to be r e l a t e d to the absence of c a f f e i n e i n any of these p u r i f i e d p r e p a r a t i o n s : Evidence f o r the importance of c a f f e i n e i n determining b l a c k tea t a s t e i s given i n other p a r t s of t h i s r e p o r t ( c f . Tables 2, 4 and 5). The E f f e c t Of E x t r a c t i o n Time On The Taste Of Black Tea I n f u s i o n s . Tea bags were i n f u s e d f o r v a r y i n g lengths of time (1, 3, or 5 minutes) and th the amount of s o l i d s e x t r a c t e The r e s u l t s (Table 5, p a r t A) suggest t h a t tea aroma i s e x t r a c t e d f a s t e r than the a s t r i n g e n t p r i n c i p l e s ( i . e . the polyphenols), and that the tangy p o r t i o n of the astringency i s not e x t r a c t e d as f a s t as the non-tangy p o r t i o n of the a s t r i n g e n c y . However, the o v e r a l l "tea t a s t e " of the i n f u s i o n s appears to be determined by a comb i n a t i o n of the aroma and the a s t r i n g e n t p r i n c i p l e s . These i n d i c a t i o n s were v e r i f i e d i n the f o l l o w i n g f u r t h e r experiments:The E f f e c t Of Tea Aroma On The Taste Of Black Tea I n f u s i o n s . F i r s t , the aroma was removed from the tea i n f u s i o n s (Table 5, p a r t A) by s t r i p p i n g o f f the v o l a t i l e m a t e r i a l s present i n the tea i n f u s i o n s under reduced pressure. Removal of the aroma from the tea i n f u s i o n s was found (Table 5, p a r t B) to reduce the o v e r a l l t e a - l i k e q u a l i t y of the t e a i n f u s i o n s , but i t had v i r t u a l l y no e f f e c t on the l e v e l of astringency i n the i n f u s i o n s . The e f f e c t of removing aroma from the tea i n f u s i o n could be reversed by addi n g the aroma back t o the s t r i p p e d i n f u s i o n s . The E f f e c t Of D e g a l l a t i n g The Tea Polyphenols On The Taste Of Black Tea I n f u s i o n s . The importance of g a l l o y l groups ( V I l b ) on the t e a polyphenols i n determining the amount of astringency of these polyphenols was c l e a r l y i n d i c a t e d by r e s u l t s obtained by t a s t i n g i n d i v i d u a l tea polyphenols (see d i s c u s s i o n above of r e s u l t s summarized i n Table 3). The importance of g a l l o y l groups on tea polyphenols to the t a s t e of whole b l a c k tea i n f u s i o n s was t e s t e d by t r e a t i n g the whole b l a c k tea i n f u s i o n w i t h a p u r i f i e d preparat i o n of the enzyme tannase (EC. 3.1.1.20). This enzyme i s an esterase that a c t s s p e c i f i c a l l y on the e s t e r bond between g a l l o y l groups ( V l l b ) and g a l l a t e d tea polyphenols ( I I , IV, V I I I , IX, X I I XIV, XIX, XX, and others) (29, 30, 31). D e g a l l a t i n g a 3-minute i n f u s i o n of b l a c k tea l e a f (Table 5, p a r t C) completely e l i m i n a t e d the tangy p o r t i o n of the astringency of the i n f u s i o n but had no e f f e c t on the non-tangy p o r t i o n of the astringency. Dearomatizing the d e g a l l a t e d tea i n f u s i o n had the

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

35

PHENOLIC, SULFUR, AND NITROGEN COMPOUNDS IN FOOD FLAVORS

Table 5:

The E f f e c t of Various Treatments on the Taste of

T o t a l Amount of Tea S o l i d s i n I n f u s i o n (mg)

D e s c r i p t i o n of the Tea I n f u s i o n A.

E f f e c t of length of i n f u s i o n p e r i o d : Tea l e a f i n f u s i o - 5 minutes

B.

670

E f f e c t of aroma removal ( s t r i p p i n g ) : Tea l e a f i n f u s i o n - 3 minutes Stripped i n f u s i o n (aroma removed) Aroma R e c o n s t i t u t e d s t r i p p e d i n f u s i o n + aroma

590 590 0 590

C.

E f f e c t o f d e g a l l a t i n g t e a polyphenols ( i . e . , Tea l e a f i n f u s i o n - 3 minutes Infusion after degallating Stripped i n f u s i o n a f t e r d e g a l l a t i n g D e g a l l a t e d s t r i p p e d i n f u s i o n + aroma

treating 590 590 590 590

D.

E f f e c t of d e c a f f e i n a t i o n ( i . e . b l a c k t e a l e a f I n f u s i o n (3 minutes) of d e c a f f e i n a t e d (and dearomatized) t e a l e a f Decaffeinated i n f u s i o n + c a f f e i n e Decaffeinated i n f u s i o n + aroma Decaffeinated i n f u s i o n + c a f f e i n e + aroma f

decaffein535 580 535 580

E.

E f f e c t o f m i l k ( i . e . , adding 1 teaspoon m i l k to t e a i n f u 1 minute i n f u s i o n + m i l k 490 3 minutes i n f u s i o n + m i l k 590 5 minutes i n f u s i o n + m i l k 670 F. E f f e c t of adding lemon j u i c e ( i . e . , j u i c e squeezed from a Tea l e a f i n f u s i o n - 3 minutes (pH 4.8) 590 I n f u s i o n + 3 ml lemon j u i c e (pH 3.2) 590 I n f u s i o n + HC1 (pH 3.2) 590 2.27g b l a c k t e a l e a f was i n f u s e d i n a t e a cup w i t h 6 oz. Key t o r a t i n g s : 0 = none; 1 = t h r e s h o l d ; 2 = weak; 3 «

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

2.

SANDERSON E T A L .

Polyphenolic Compounds in the Taste of Tea

Table 5 (eont.)

Black Tea I n f u s i o n s

Organoleptic E v a l u a t i o n Amount o f Caffeine i n I n f u s i o n (mg)

Astringency Tangy Non-Tangy

41 48 54

48 48 0 48 the t e a i n f u s i o n 48 48 48 48

Aroma

Black Tea Taste

Very s t r o n g

4

3 3 0 3

Strong Flat F l a v o r y , pungent Strong

w i t h tannase enzyme): 3 0 0 0

Strong Mild Weak Mild

ated, and consequently

dearomatized, by s o l v e n t e x t r a c t i o n ) : Weak Mild Mild Strong

3 48 3 48 sions from A above): 41 0 48 0 54 1

2 3 3

lemon wedge) : 48 3 3 48 1 2 48 1 2 f r e s h l y b o i l e d tap water producing moderate; 4 = s t r o n g .

3 3 3

Weak, m i l k y M i l d , milky Strong, m i l k y

3 3 3

Strong M i l d , lemon M i l d , sour on average 5.2 oz. of beverage.

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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PHENOLIC, SULFUR, AND

NITROGEN COMPOUNDS IN FOOD FLAVORS

e f f e c t of reducing both the tangy and the non-tangy p o r t i o n s of the a s t r i n g e n c y and reducing the o v e r a l l t e a - l i k e q u a l i t y of the i n f u s i o n to a b a r e l y p e r c e p t i b l e l e v e l . The e f f e c t of dearomatizi n g the i n f u s i o n s was e n t i r e l y reversed by r e s t o r a t i o n of the aroma removed. The E f f e c t Of C a f f e i n e (XXI) On The Taste Of Black Tea I n f u s i o n s . Samples of d e c a f f e i n a t e d black tea l e a f and r e g u l a r b l a c k tea l e a f were brewed s i d e by s i d e and the i n f u s i o n s obtained were c h e m i c a l l y analyzed and o r g a n o l e p t i c a l l y evaluated. The r e s u l t s (Table 5, p a r t D) i n d i c a t e d that removal of c a f f e i n e from the t e a i n f u s i o n has a s i g n i f i c a n t e f f e c t on the t a s t e of the i n f u s i o n . S p e c i f i c a l l y , d e c a f f e i n a t i o n causes the b i t t e r n e s s of a b l a c k tea i n f u s i o n s l i g h t l y to i n c r e a s e and d e c a f f e i n a t i o n changes the nature of the a s t r i n g e n c i th i n f u s i o fro th tang type which i s c h a r a c t e r i s t i c t h e r , the r e s u l t s (Tabl , par degallatio tea polyphenols has only a r e l a t i v e l y s m a l l e f f e c t on the t a s t e of d e c a f f e i n a t e d tea beverages ( t h i s e f f e c t i s a s m a l l general decrease i n a l l t a s t e p r o p e r t i e s ) , whereas d e g a l l a t i o n of the tea polyphenols i n a r e g u l a r tea i n f u s i o n causes a marked r e d u c t i o n i n the astringency of the i n f u s i o n . I t has long been known that c a f f e i n e (the predominant xant h i n e compound i n t e a ; see Table 1) complexes w i t h t e a polyphenols In f a c t , the complexation of b l a c k t e a polyphenols and c a f f e i n e i s r e s p o n s i b l e f o r much, but not a l l , of the tea cream formation ( i . e . the p r e c i p i t a t i o n of tea s o l i d s ) that occurs when b l a c k tea i n f u s i o n s c o o l down (32, 33, 34). A more d e t a i l e d i n v e s t i g a t i o n by C o l l i e r et a l . (35) showed that condensation of the tea f l a v a n o l s (ex. I + I I I + 02** X) and the presence of g a l l o y l groups ( V l l b ) on the tea polyphenols, decreases the s o l u b i l i t y of c a f f e i n e / t e a polyphenol complexes. Our r e s u l t s (Table 5, p a r t D) suggest that c a f f e i n e complexes w i t h the b l a c k tea polyphenols i n a way that prevents these polyphenols from complexing w i t h themselves to form l a r g e r p o l y p h e n o l i c molecules. These c a f f e i n e / b l a c k tea polyphenol complexes are l e s s s o l u b l e i n c o l d water than the i n t e r n a l b l a c k tea polyphenol/polyphenol complexes, and the c a f f e i n e / p o l y p h e n o l complexes have a more sharp tangy a s t r i n g e n c y than the polyphenol/ polyphenol complexes which have a l i n g e r i n g mouth-drying, mouthc o a t i n g e f f e c t ( i . e . non-tangy a s t r i n g e n c y ) . The a b i l i t y p a r t i a l l y to transform the a s t r i n g e n c y i n r e g u l a r b l a c k tea i n f u s i o n s ( c o n t a i n i n g c a f f e i n e ) to something very c l o s e to the a s t r i n g e n c y of the d e c a f f e i n a t e d tea i n f u s i o n s by d e g a l l a t i n g the tea polyphenols (Table 5, p a r t s C and D) suggests t h a t the g a l l o y l groups on the b l a c k tea polyphenols are the s p e c i f i c s i t e s i n v o l v e d i n the complexation w i t h c a f f e i n e , or other b l a c k tea polyphenols. I f the above i s t r u e , then i t i s a l s o true t h a t the g a l l o y l groups on the t e a polyphenols are c r i t i c a l determinants of the type of a s t r i n g e n c y that e x i s t s i n tea beverages. I t

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

2.

SANDERSON ET A L .

Polyphenolic Compounds in the Taste of Tea

i s noteworthy t h a t i t i s now known (36, _37, 38) t h a t d e g a l l a t i o n of b l a c k tea polyphenols w i l l e l i m i n a t e most of the b l a c k tea cream f u r n i s h i n g another p i e c e of evidence f o r the importance of the s p e c i f i c nature of the complexation between c a f f e i n e (XXI) and the g a l l o y l groups ( V l l b ) of b l a c k tea polyphenols ( I I , IV, V I I I , IX, XII-XIV, XVI, XIX, XX, and o t h e r s ) . The E f f e c t Of M i l k On The Taste Of Tea I n f u s i o n s . M i l k i s o f t e n added to b l a c k tea i n f u s i o n s to ameliorate the t a s t e of the beverage. The e f f e c t of t h i s p r a c t i c e was s t u d i e d by adding m i l k to b l a c k tea i n f u s i o n s and determining the o r g a n o l e p t i c p r o p e r t i e s of the r e s u l t i n g beverage. The r e s u l t s (Table 5, p a r t E) showed that the a d d i t i o n of m i l k to b l a c k t e a i n f u s i o n s caused a marked lowering of the a s t r i n g e n c y of the i n f u s i o n s . The r e d u c t i o n of a s t r i n g e n c y i n these experiment and 3-min. i n f u s i o n s an Of course, the e f f e c t noted here w i l l be h i g h l y dependent on the amount of tea s o l i d s i n the cup and the amount of m i l k added. I t i s noteworthy that the a d d i t i o n of m i l k to these t e a i n fusions had p r a c t i c a l l y no e f f e c t on e i t h e r the o v e r a l l t e a - l i k e q u a l i t y or the aroma. The a d d i t i o n of m i l k to b l a c k tea i n f u sions d i d cause other e f f e c t s on the t a s t e of the t e a i n f u s i o n s (not noted i n Table 5 ) ; such as an i n c r e a s e i n the body of the beverage, c o n t r i b u t i o n of a smoothness to the t a s t e , and c o n t r i b u t i o n of a m i l k y t a s t e per se; but these e f f e c t s are f o r e i g n to the subject of t h i s d i s c u s s i o n . Since the a s t r i n g e n c y of b l a c k t e a i n f u s i o n s i s e s t a b l i s h e d to be due to the p h e n o l i c compounds present, i t can be s a f e l y assumed that the m i l k has i t s e f f e c t by t y i n g up the tea polyphenols i n such a way that they no longer have a s t r i n g e n t p r o p e r t i e s : The m i l k p r o t e i n s are prime candidates f o r the agents i n m i l k t h a t cause t h i s t y i n g up of the tea polyphenols. Brown and Wright (39) i s o l a t e d m i l k p r o t e i n / b l a c k tea polyphenol complexes and s t u d i e d t h e i r e l e c t r o p h l o r e i c p r o p e r t i e s . The i n t e r a c t i o n of p r o t e i n s w i t h p o l y p h e n o l i c compounds i s a w e l l known, f r e q u e n t l y observed phenomena (40), but i t i s important to t h i s d i s c u s s i o n to note two consequences of the r e a c t i o n between tea polyphenols and m i l k p r o t e i n s : F i r s t , the complexes do not p r e c i p i t a t e as do v i r t u a l l y a l l other p r o t e i n / t e a polyphenol complexes. Presumably, the m i l k p r o t e i n - t e a polyphenol complexes form s t a b l e c o l l o i d a l suspensions. Second, these complexes have the e f f e c t of a p p r e c i a b l y reducing the a s t r i n g e n c y of b l a c k tea i n f u s i o n s which i s d e s i r a b l e to most consumers (G.W. Sanderson, unpublished d a t a ) . The E f f e c t Of Lemon J u i c e On The Taste Of Black Tea Infusions. Lemon j u i c e added to a b l a c k tea i n f u s i o n was found (Table 5, p a r t F) to cause a marked r e d u c t i o n i n the astringency of the beverage. F u r t h e r , the tangy p a r t of the tea a s t r i n g e n c y was more a f f e c t e d than the non-tangy p a r t . The o v e r a l l e f f e c t of t h i s r e d u c t i o n i n astringency was to reduce the t e a t a s t e im-

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p r e s s i o n of the beverage from " s t r o n g " to " m i l d " . The pH of a second b l a c k t e a i n f u s i o n was adjusted down w i t h h y d r o c h l o r i c a c i d from an i n i t i a l pH 4.8 to pH 3.2 (the same pH change e f f e c t e d by a d d i t i o n of the lemon j u i c e described above). This t e a beverage w i t h pH adjusted by means of an i n o r g a n i c a c i d was found (Table 5, p a r t F) to have v i r t u a l l y the same t a s t e p r o p e r t i e s as the tea beverage w i t h lemon j u i c e , e s p e c i a l l y as regards the a s t r i n g e n c y and the s t r e n g t h of the tea t a s t e . These r e s u l t s i n d i c a t e that s l i g h t l y reducing the pH of a tea beverage (such as from pH 4.8 to about 3.2) w i l l reduce the a s t r i n g e n c y of the beverage and that t h i s change i s most p l e a s a n t l y accomplished by adding a l i t t l e lemon j u i c e : T h i s , of course, has the added advantage of c o n t r i b u t i n g a touch of lemon f l a v o r which complements the t e a f l a v o r . The f a c t t h a t the tangy p a r t of the tea astringenc the c a f f e i n e / p o l y p h e n o l change. The chemistry u n d e r l y i n g t h i s phenomena i s not understood, but the phenomena, i . e . the r e d u c t i o n i n a s t r i n g e n c y caused by lowering pH, has been described more than once before (40). Summary And

Conclusions

The r e s u l t s of our i n v e s t i g a t i o n s confirm and extend e a r l i e r research (9, 15, 41, 42, 43) t h a t i n d i c a t e s the prime importance of the tea polyphenols i n determining the t a s t e of b l a c k tea i n f u s i o n s (beverages). Through the process of tea fermentation (Figures 1 and 2) the green tea f l a v a n o l s (I-VI) are o x i d i z e d and condense to form the t h e a f l a v i n s (XI-XIV), the t h e a r u b i g i n s (VII-XX, and other unknowns), and other minor products ( V I I I - X , XV, XVI, and other unknowns). These changes are accompanied by changes i n the t a s t e of i n f u s i o n s from green, grassy, harsh, b i t t e r , w i t h s l i g h t non-tangy a s t r i n g e n c y ( f r e s h green tea f l u s h ) , to green, very harsh, w i t h s t r o n g non-tangy a s t r i n g e n c y (fermented but not f i r e d ) , to f l o w e r y , s l i g h t l y green, w i t h pleasant tangy a s t r i n g e n c y , and m i l d b l a c k tea f l a v o r (fermented and f i r e d ) . These changes were found to be d e f i n i t e l y a s s o c i a t e d w i t h the o x i d a t i o n of the t e a f l a v a n o l s i n that e l i m i n a t i o n of both the tea fermentation and the f i r i n g processes prevented the development of a c h a r a c t e r i s t i c b l a c k t e a t a s t e (Table 3) and removal of the polyphenols from a b l a c k t e a i n f u s i o n e f f e c t i v e l y removed a l l r e c o g n i z a b l e b l a c k tea c h a r a c t e r (Table 2 ) . F i r i n g of the fermented tea f l u s h m a t e r i a l was shown i n our i n v e s t i g a t i o n (Table 3) to be e s s e n t i a l to the development of b l a c k t e a f l a v o r . This f i n d i n g has been reported p r e v i o u s l y by Bokuchava et a l . (17) and by B h a t i a and U l l a h (18). The r o l e of f i r i n g i n the development of b l a c k tea f l a v o r i s not w e l l understood but the a v a i l a b l e evidence suggests that the f o l l o w i n g changes are brought about by f i r i n g that are important i n t h i s context: (a) F i r i n g f o l l o w i n g tea fermentation causes some

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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f u r t h e r changes i n the polyphenols t h a t resemble the changes tak­ ing p l a c e d u r i n g t e a fermentation, i . e . changes i n the amounts of t h e a f l a v i n s and t h e a r u b i g i n s . However, these changes which take p l a c e at higher temperatures and i n a more concentrated e n v i r o n ­ ment may be q u a l i t a t i v e l y d i f f e r e n t from those o c c u r i n g during tea fermentation i t s e l f . (b) F i r i n g causes c o n s i d e r a b l e i n s o l u b i l i z a t i o n of tea s o l i d s and the m a t e r i a l s i n s o l u b i l i z e d i n c l u d e both polyphenols and non-ρ οlyphenolie compounds. The p o l y p h e n o l i c m a t e r i a l s l o s t to the i n f u s i o n in t h i s way are mostly t h e a r u b i g i n s and the non-polyphenolic m a t e r i a l s l o s t are probably s m a l l amounts of peptides (44) and p o l y s a c c h a r i d e s (45). These l o s s e s may be most important e s p e c i a l l y i f i t were found that the compounds w i t h the harshest, strongest non-tangy a s t r i n g e n c y were p e r f e r e n t i a l l y l o s t i n t h i s process. ( c ) . The formation of b l a c k tea aroma i s e n t i r e l y dependent on tea fermentation and f i r i n g (16 17 18 Table 3 ) , and b l a c k te ment to the b l a c k t e a s o l i d f l a v o r (Table 5 ) . I t i s a l s o known that f i r i n g d r i v e s o f f ap­ p r e c i a b l e amounts of aroma c o n s t i t u e n t s (17, 46), and t h i s may lead to an improved balance of aroma c o n s t i t u e n t s as f a r as b l a c k tea aroma i s concerned. The above 3 p o i n t s c e r t a i n l y deserve f u r ­ ther i n v e s t i g a t i o n . The r e l a t i o n s h i p of g a l l o y l groups ( V I l b ) and c a f f e i n e (XXI) to the tangy a s t r i n g e n c y of tea i n f u s i o n s i s most important (Tables 2 and 5 ) . Tangy a s t r i n g e n c y i s p o s s i b l y what some other researchers (9, 25, 43), and the tea trade (47) c a l l b r i s k n e s s . In any case, tangy a s t r i n g e n c y i s d i f f i c u l t to d e f i n e , a f a c t recognized long ago by Bate-Smith (48) i n h i s review of a s t r i n ­ gency i n food products, yet i t i s a most important c h a r a c t e r i s t i c p a r t of the t a s t e of b l a c k t e a i n f u s i o n s . Roberts (9^, 49) had found t h a t " b r i s k n e s s " i n b l a c k tea i n f u s i o n s was c o r r e l a t e d to some extent w i t h the t h e a f l a v i n s and the c a f f e i n e content of these i n f u s i o n s and Wood and Roberts (25) provided some a d d i t i o n a l evidence i n support of t h i s c o n t e n t i o n . However, we can now say that i t i s the g a l l o y l groups on the t h e a f l a v i n g a l l a t e s ( X I I XIV) and other g a l l a t e d b l a c k tea polyphenols ( V I I I , IX, XVI, XX, and other g a l l a t e d unknowns) that r e a c t w i t h c a f f e i n e (XXI) to produce the tangy a s t r i n g e n c y a s s o c i a t e d w i t h " b r i s k n e s s " i n b l a c k tea i n f u s i o n s . I t i s noteworthy t h a t s t u d i e s of consumer p r a c t i c e s and p r e ­ ferences i n the United States (G.W. Sanderson, unpublished) i n ­ d i c a t e t h a t tea bags are u s u a l l y brewed f o r only about 1 min. F u r t h e r , the c r i t i c i s m of tea beverages that i s obtained more o f t e n than any other i s that the beverage i s too b i t t e r (consum­ ers appear to confuse b i t t e r n e s s w i t h a s t r i n g e n c y i n the case of tea beverages). Apparently, consumers i n the United States con­ t r o l the l e v e l of a s t r i n g e n c y i n t h e i r cup of t e a by using a r a t h e r s h o r t e x t r a c t i o n time, thereby l i m i t i n g the amount of tea s o l i d s e x t r a c t e d . As shown i n Table 5, p a r t A, t h i s i s an e f ­ f e c t i v e means of m i n i m i z i n g the a s t r i n g e n c y of the i n f u s i o n w h i l e

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at the same time p r o v i d i n g f o r a reasonable amount of tea f l a v o r to be e x t r a c t e d . Of course, reducing the pH of the b l a c k tea i n f u s i o n by adding lemon j u i c e i s a l s o a sound means of reducing the a s t r i n g e n c y of the i n f u s i o n (Table 5, p a r t F) and t h i s too i s a common p r a c t i c e i n the United S t a t e s . In many places o u t s i d e the United S t a t e s , i t i s customary to brew t e a much stronger than w i t h i n the United States (by brewing f o r 3 t o 5 min. r a t h e r than f o r 1 min. and/or by u s i n g more tea to prepare a s e r v i n g ) . However, i t i s a l s o customary to add m i l k to tea i n f u s i o n s i n these p l a c e s . The United Kingdom, I n d i a , and S r i Lanka are good examples of c o u n t r i e s where such p r a c t i c e s are almost u n i v e r s a l . The r e s u l t s shown i n Table 5, p a r t D, i n d i c a t e that the h i g h l e v e l of a s t r i n g e n c y n a t u r a l l y a s s o c i a t e d w i t h the stronger t e a i n f u s i o n s p r e f e r r e d i n many c o u n t r i e s outs i d e the United States i s n e u t r a l i z e d w i t h m i l k r a t h e r than be l i k e d or t o l e r a t e d , b On The Chemistry Of The Taste Of Green Tea A d e t a i l e d d i s c u s s i o n of green tea i s o u t s i d e the scope of t h i s paper. However, a t t e n t i o n should be drawn to a recent paper by Nakagawa (50) that provides much u s e f u l i n f o r m a t i o n on the chemistry u n d e r l y i n g the t a s t e of green tea i n f u s i o n s . Nakagawa s (50) r e s u l t s i n d i c a t e that the major components of t a s t e i n green tea are b i t t e r n e s s , a s t r i n g e n c y , brothy, and sweetness. The b i t t e r n e s s and astringency was shown to be due to the green tea polyphenols: The tea f l a v a n o l s ( I - V I ) , e s p e c i a l l y the g a l l a t e d f l a v a n o l s ( I I , I V ) , and leucoanthocyanins were considered to be most important i n determining these t a s t e c h a r a c t e r i s t i c s , but some u n i d e n t i f i e d phenol-type m a t e r i a l s were a l s o thought to make a s i g n i f i c a n t and d e s i r a b l e c o n t r i b u t i o n to green tea b i t t e r n e s s and a s t r i n g e n c y . The brothy t a s t e of green tea was shown to be due to amino a c i d s , and the sweetness to sugars. C a f f e i n e was reported to p l a y no s i g n i f i c a n t r o l e i n determining the t a s t e of green tea. The c o n t r a s t between Nakagawa s (50) r e s u l t s f o r green tea and the r e s u l t s discussed above f o r b l a c k tea d e r i v e i n p a r t from the d i f f e r e n t clones of tea p l a n t s c u l t i v a t e d f o r green tea manufacture, but mostly from the tea fermentation process (Figure 2) which i s p a r t of the b l a c k tea manufacturing process but which i s purposely prevented i n the green tea manuf a c t u r i n g process (51, 52). 1

1

Experimental The beverage s t r e n g t h e x t r a c t was prepared by brewing b l a c k tea l e a f i n 75 times i t s weight of d i s t i l l e d d e i o n i z e d water f o r 5 min. The e x t r a c t was f r e e z e - d r i e d from about 2% s o l u t i o n f o r use as r e q u i r e d . Aroma Recovery.

Tea aroma was

recovered by c o l l e c t i n g about

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25% d i s t i l l a t e from a f r e s h l y prepared tea beverage. Distillat i o n was c a r r i e d out i n a r o t o r y evaporator under vacuum a t 40°C and w i t h a condenser temperature a t -5°C F r a c t i o n a t i o n . The beverage s t r e n g t h t e a e x t r a c t was concent r a t e d t o about 2% (w/w) s o l i d s p r i o r t o f r a c t i o n a t i o n by s o l v e n t e x t r a c t i o n . C a f f e i n e and other p u r i n e s o f tea were removed by e x t r a c t i n g the t e a concentrate w i t h t r i c h l o r o e t h y l e n e (TCE) f o r 48 h r . u s i n g a l i q u i d - l i q u i d e x t r a c t o r . A f t e r the e x t r a c t i o n was completed TCE was d i s t i l l e d under vacuum t o recover c a f f e i n e . Traces o f TCE were removed from the aqueous t e a e x t r a c t under vacuum u s i n g a r o t o r y evaporator. Next, t e a polyphenols were recovered from the c a f f e i n e f r e e t e a e x t r a c t by e t h y l a c e t a t e (EtAc) e x t r a c t i o n f o r 48 h r . u s i n g a l i q u i d - l i q u i d e x t r a c t o r . Polyphenols were recovered from the EtAc f r a c t i o n by removing EtAc under vacuum a f t e d i s t i l l e d wate added t t h a t f r a c t i o n . Added water by f r e e z e - d r y i n g . The polypheno aqueou f r a c t i o n o f the tea was then f r e e z e - d r i e d a f t e r removal o f t r a c e s of EtAc under vacuum. A t e a e x t r a c t f r e e o f a l l tea polyphenols was obtained by passing a f r e s h l y brewed t e a s o l u t i o n through a column packed w i t h hydrated polyamide CC6 (Brinkmann). The column was then washed 3 times w i t h hot d i s t i l l e d water. The e l u a t e s obtained were completely f r e e of polyphenols as judged by paper chromatography. A t o t a l polyphenol and c a f f e i n e f r e e t e a e x t r a c t was obtained by passing the above polyphenol f r e e e x t r a c t through a p r e s o l v a t e d XAD-2 column (53). P u r i f i e d t e a f l a v a n o l s were obt a i n e d or prepared as d e s c r i b e d p r e v i o u s l y (54). A n a l y t i c a l Methods. O r g a n o l e p t i c e v a l u a t i o n s were done using a panel c o n s i s t i n g of l a b o r a t o r y personnel. The panel was t r a i n e d f o r t h i s work and a l l the t e s t i n g was c a r r i e d out under standard c o n d i t i o n s . M i n e r a l s were determined by atomic a b s o r p t i o n and flame emission spectroscopy. P e c t i n s were determined by the method o f McComb and McCready (55). Sugars were determined by a m o d i f i c a t i o n (R. Simons, unpublished) o f a procedure by which sugars are p u r i f i e d by i o n exchange chromatography (56) and determined by q u a n t i t a t i v e gas chromatography (57). Organic a c i d s were separated by a m o d i f i c a t i o n (R. Simons, unpublished) of a procedure by F u j i m a k i e t a l . (58), and determined by gas chromatography (59). Amino a c i d s were determined by automatic amino a c i d a n a l y z e r . C a f f e i n e was determined by g . l . c . a f t e r c h l o r o f o r m e x t r a c t i o n (P.D. C o l l i e r , unpublished method). I n d i v i d u a l f l a v a n o l s were estimated using the method o f C o l l i e r and Mallows (60). T h e a f l a v i n and t h e a r u b i g i n s a n a l y s i s o f t e a s o l u t i o n s was

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carried out using the method of Roberts and Smith (15). A l l other a n a l y t i c a l methods were o f f i c i a l A.O.A.C. methods (61). Black Tea Manufacture: Freshly harvested green tea flush was air-freighted so as to reach our laboratory i n the evening of the day of harvesting (54). Black tea manufacture (62, 63) was carried out according to the following laboratory scale pro­ cedure : The flush was spread out on a bench top to wither overnight to a moisture content of about 65%. The withered flush was macerated by passing i t 3 times through a r o l l m i l l . The macerated tea flush was spread about 3 cm. deep i n trays covered with damp cheesecloth and allowed to undergo tea fermentation for the times specified. At the end of the desired tea fermentation period, one sample of fermente with crushed dry ice an sampl by forcing a i r at 97°C through the sample for about 25 min. A l l samples were dried to a moisture content of about 5% which ren­ dered them stable and ready for chemical analysis and organolep­ t i c evaluation. Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

Hainsworth, E. "Encyclopedia of Chemical Technology," 2nd ed., Standen, Α., E d . , pp. 743-755, Wiley (Interscience), New York, 1969. Sanderson, G.W. "Structural and Functional Aspects of Phytochemistry," Runeckles, V . C . , Tso, T . C . , E d . , pp. 247-316 Academic Press, New York, N . Y . , 1972. Peterson, M.S. "Encyclopedia of Food Technology," Johnson, A . H . , Peterson, M . S . , ed., pp.889-891, AVI Publishing Com­ pany, Westport, Connecticut, 1974. Sreerangachar, H.B. Biochem J. (1943) 37, 661. Kursanov, A.L. Kulturpflanze Beiheft (1956) 1, 29. Vuataz, L., Brandenberger, H. J. Chromatog. (1961) 5, 17. M i l l i n , D.J., Rustidge, D.W. Process Biochem. (1967) 2, 9. Burg, A.W., Tea and Coffee Trade Journal (1975) 147, January, 40. Roberts, E.A.H. "Chemistry of Flavonoid Compounds," Geissman, T . A . , E d . , pp.468-512, Pergamon Press, London, 1962. Berkowitz, J.E., Coggon, P., Sanderson, G.W. Phytochem. (1971) 10, 2271. Sanderson, G.W., Berkowitz, J.E., Co, Η., Graham, N.H. J. Food Sci. (1972) 37, 399. Brown, A . G . , Eyton, W.B., Holmes, Α., Ollis, W.D. Nature (London) (1969a) 221, 742. Brown, A . G . , Eyton, W.B., Holmes, Α., Ollis, W.D. Phytochem. (1969b) 8, 2333. Weinges, Κ., Muller, O. Chemiker Zeitung (1972) 96, 612.

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SANDERSON ET AL.

15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43.

Polyphenolic Compounds in the Taste of Tea

Roberts, E.A.H., Smith, R.F. J. S c i . Food Agric. (1963) 14, 689. Saijo, R . , Kuwabara, Y. Agr. B i o l . Chem. (1967) 31, 389. Bokuchava, M.A., Skobeleva, N . K . , Dmitriev, A . F . Dokl. Akad. Nauk SSSR (1958) 115, 183. Bhatia , I . S . , U l l a h , M.R. J. S c i . Food Agric. (1965) 16, 408. Sanderson, G.W., Graham, H.N. J. Agric. Food Chem. (1973) 21, 576. Sanderson, G.W. "International Symposium: Odour and Flavour Substances," Drawert, W., Ed., pp 65-97, Haarmen and Reimer, GmbH, Holzminden, W. Germany, 1975. Suzuki, T., Takahashi, T. Biochem. J. (1975a) 146, 79. Suzuki, T., Takahashi, T. Biochem. J. (1975b) 146, 87. Roberts, E . A . H . , Cartwright R . A . Oldschool M. J. S c i Food Agric. (1957 Coggon, P . , Moss, , , Phytochem (1973) 12, 1947. Wood, D.J., Roberts, E.A.H. J . S c i . Food Agric. (1964) 15, 19. Biswas, A . K . , Biswas, A . K . , Sarkar, A.R. J. S c i . Food Agric. (1971) 22, 196. Biswas, A . K . , Sarkar, A . R . , Biswas, A.K. J. S c i . Food Agric. (1973) 24, 1457. H i l t o n , P.J., Ellis, R.T. J. Sci. Food Agric. (1972) 23, 227. Dykerhoff, H . , Armbruster, R. Hoppe Seyler's Zeitschrift fur Physiologische Chemie (1933) 219, 38. Haslam, E . , Strangroom, J . E . Biochem. J. (1966) 99, 28. Iibuchi, S., Minoda, Y., Yamada, K. Agr. Biol. Chem. (1972) 36, 1553. Roberts, E.A.H. J . S c i . Food Agric. (1963) 14, 700. Wickremasinghe, R . L . , Perera, K.P.W.C. Tea Quart. (1966) 37, 131. Smith, R.F. J. Sci. Food Agric. (1968) 19, 530. C o l l i e r , P . D . , Mallows, R., Thomas, P.E. Phytochem. (1972) 11, 867. Tenco Brooke Bond Ltd. B r i t i s h Patent 1, 249,932 (1971). Takino, Y. New Zealand Patent 160, 729 (1971). Takino, Y. Canadian Patent 905205 (1972). Brown, P.J., Wright, W.B. J. Chromatog .(1963) 11, 504. Joslyn, M.A., Goldstein, J . L . Advan. Food Res. (1964) 13, 179. Bokuchava, M.A., Novozhilov, N.P. Biokhim. Chainogo P r o i z vodstva, Akad. Nauk SSSR (1946) 5, 190. Bokuchava, M . A . , Skobeleva, N . I . Advan. Food Res. (1969) 17, 215. M i l l i n , D.J., Crispin, D.J., Swaine, D. J. Agr. Food Chem. (1969) 17, 717.

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

45

46

44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63.

PHENOLIC, SULFUR, AND NITROGEN COMPOUNDS IN FOOD FLAVORS Wood, D.J., Bhatia, I . S . , Chakraborty, S., Chondhury, M.N.D., Deb, S.B., Roberts, E . A . H . , Ullah, M.R. J. S c i . Food Agric. (1964) 15, 14. Sanderson, G.W., Perera, B.P.M. Tea Quart. (1965) 36, 6. Yamanishi, T., Kobayashi, Α., Sato, Η., Nakamura, Η., Uchida, Α., Mori, S., Saijo, R. Agr. Biol. Chem. (1966) 30, 784. Palmer, D.H. J. S c i . Food Agric. (1974) 25, 153. Bate-Smith, E.D. Food (1954) April, 124. Roberts, E.A.H. J . S c i . Food Agric. (1958) 9, 381. Nakagawa, M. Nippon Shokuhin Kogyo Gakkai-Shi (1975) 22, 59. Ukers, W.H. "All About Tea", 2 vol., Tea and Coffee Trade Journal Co., New York, N . Y . , 1935. Ukers, W.H., Prescott, S.C. "Chemistry and Technology of Food and Food Products" 2nd ed. V o l 2 Jacobs M . B . Ed. pp. 1656-1705, Interscienc Gustafson, R . L . , Albright, , , J., Lirio, J.A., Reid, O.T., Jr. Ind. Eng. Chem. Prod. Res. Develop. (1968) 7, 107. Co, Η., Sanderson, G.W. J. Food S c i . (1970) 35, 160. McComb, E . A . , McCready, R.M. Ana1. Chem. (1952) 24, 1630. Cartwright, R . A . , Roberts, E.A.H. J. S c i . Food Agric. (1954) 5, 600. Larson, P.Α., Hobbs, W.E., Honold, G.R. Instrum. Food Beverage Ind. (1972) January, 1. Fujimaki, Μ., Kim, Κ., Kurata, T. Agric. B i o l . Chem. (1974) 38, 45. Fernandez-Florez, Ε., Johnson, A . R . , Fitelson, J . J . Assoc. Off. Anal. Chemists (1970) 53, 1193. C o l l i e r , P . D . , Mallows, R. J . Chromatog (1971) 57, 29. Horwitz, W. " O f f i c i a l Methods of Analysis," 12th ed., Assoc. Off. Anal. Chemists, Washington, D . C . , 1975. Eden, T. "Tea", 2nd E d . , Longmans, Green and Co., Ltd., London, England, 1965. Harler, C.R. "Tea Manufacture", Oxford Univ. Press, London, England, 1963.

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

3 W i n e F l a v o r a n d P h e n o l i c Substances V.L.SINGLETON andA.C.NOBLE Department of Viticulture and Enology, University of California, Davis, Calif. 95616

Phenolic substance odor of wines. They make direct contributions to flavor which may be very large and overriding as in some astringent red wines, but can be very subdued compared to the effects of other compounds in wines of other types. In dry red wines they usually are the most p l e n t i f u l constituents after alcohol, t a r t a r i c acid, and unfermentable sugars. Indirect effects of phenols on flavor can be large and both psychological and chemical. For example, sensory quality of wine is d i r e c t l y influenced to a considerable degree by color and the influence of color on judgement of flavor is difficult to avoid. The colors of wines are largely the result of anthocyanins, other phenols, and their reaction products whether the wines are pink, red, purplish, red-orange, yellow, golden, or amber. Reactions associated with the oxidation of the wine's phenols normally produce taste and odor changes as w e l l as color changes. Therefore, wine that appears oxidized from its color is likely to be judged as oxidized in flavor even if it is not. In most wines, polyphenols are the main pool of substances capable of autoxidation under normal wine-aging conditions (ambient or lower temperature, about pH 3.3, a i r access often restricted and at ambient pressure, etc.). Thus, processing and aging of wine produce direct taste or odor changes by modifying the phenols themselves, but also produce indirect flavor effects through associated reactions. The ideal situation has not been reached wherein we know the complete specific Qualitative and quantitative phenolic composition of wines and these substance's individual and collective role in flavor. Considerable progress is currently being made to t h i s goal. The phenolic substances of wine and their direct and indirect effects i n wine have been reviewed (1). Further effects associated with coloration and discoloration reactions have also been reviewed (2). In t h i s paper the focus i s on the s p e c i f i c , direct effects of wine

47 In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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PHENOLIC, SULFUR, AND NITROGEN COMPOUNDS IN FOOD FLAVORS

phenols on odor and t a s t e of wine emphasizing the present s t a t u s of our knowledge w i t h onlv a few o b s e r v a t i o n s on the r e a c t i o n s of phenols of i n d i r e c t but great importance t o wine f l a v o r . Color as such i s ignored. Tn an e f f o r t to be com­ p l e t e and to encourage f u r t h e r work, we have i n c l u d e d p e r t i n e n t f i n d i n g s not d i r e c t l y i n v o l v i n g wines and have d e l i b e r a t e l y included some observations and o p i n i o n s not s o l i d l y based on proven f a c t , Data drawn from the. l i t e r a t u r e are documented, but not e x h a u s t i v e l y . Facts considered common knowledge i n enology can be checked i n general t e x t s (e.g., 3)· !· Phenols as F l a v o r a n t s . What e f f e c t s on wine t a s t e and odor might be expected from the g e n e r a l knowledge of phenols and f l a v o r ? C e r t a i n phenols are known to have s e v e r a l d i f ­ ferent types of d i r e c pungents, sweet substances Odorants must, of course, be reasonably v o l a t i l e . To i n t e r a c t at the odor sensing s i t e and cause an odor s e n s a t i o n , ap­ p r e c i a b l e l i p i d s o l u b i l i t y and some water s o l u b i l i t y are considered r e q u i s i t e s ( 4 ) . Manv s m a l l o r simple phenols can be seen to f i l l these requirements w e l l . Odorous phenols of some i n t e r e s t i n foods range from phenol, c r e s o l s , and g u a i a c o l w i t h " m e d i c i n a l , " " p h e n o l i c , " o r p o s s i b l y smoky odors to more c h a r a c t e r i s t i c a l l y p l e a s a n t odorants such as v a n i l l i n and methyl s a l i c y l a t e . Flavonoids and o t h e r s i z e a b l e phenols have no s i g n i f i c a n t odor i n the pure s t a t e nor do h i g h l y p o l a r phenol d e r i v a t i v e s , g l u c o s i d e s or g a l l i c a c i d f o r example, which have h i g h water s o l u b i l i t y o r low vapor pressure. I n wine, the odorous phenols would be sought i n the v o l a t i l e , s o l v e n t e x t r a c t a b l e , n o n f l a v o n o i d f r a c t i o n . Pungency i s considered a "hot," p e n e t r a t i n g , burning sensation i n the mouth which at lower l e v e l s may be "warm," s p i c v , sharp or harsh. I t i s a l s o an odor d e s c r i p t o r and p a r t of the b u r n i n g , p e n e t r a t i n g , h a r s h , s p i c y " p h e n o l i c " odors o f some phenols i s b e l i e v e d another e x p r e s s i o n of pungency i n v o l a t i l e compounds. Pungency i s not confined t o phenols; c o n s i d e r , f o r example, a c r o l e i n , cinnamaldehyde and even h i g h concentrations of e t h a n o l which g i v e b u r n i n g " t a s t e " sensa­ t i o n s . Many phenols have a pungency component i n t h e i r f l a v o r and n o t a b l e pungents i n c l u d e eugenol from c l o v e s , g i n g e r o l s from g i n g e r , and c a p s a i c i n from c h i l e peppers. These l a t t e r compounds and some pungent s y n t h e t i c analogs a l l are οrtho-methoxypheno1s w i t h a nonpolar para s i d e c h a i n . Pungent phenols are a l s o expected t o be i n the n o n f l a v o n o i d f r a c t i o n e x t r a c t a b l e w i t h i m m i s c i b l e s o l v e n t s such as e t h y l a c e t a t e , but may (eugenol) o r may not ( g i n g e r o l s ) be v o l a t i l e even w i t h steam ( 5 ) . Astringency i s a c o n t r a c t i n g ( p u c k e r i n g ) , d r y i n g , mouth f e e l i n g i n v o l v i n g p r e c i p i t a t i o n of the p r o t e i n s of s a l i v a and the mucous surfaces ( 5 , 6). A s t r i n g e n c y among food c o n s t i t -

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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Wine Flavor and Phenolic Substances

49

uents i s almost e n t i r e l y from " t a n n i n s , " the l a r g e r n a t u r a l polyphenols. Based p a r t l y on t h e i r a b i l i t y to p r e c i p i t a t e p r o t e i n s , polyhydroxyphenols w i t h a molecular weight of the order of 500 are considered the minimum f o r a p p r e c i a b l e astringency and astringency g e n e r a l l y i n c r e a s e s w i t h increased degree of p o l y m e r i z a t i o n up t o some p o i n t o r the s o l u b i l i t y becomes l i m i t i n g (1, 6). None of the e f f e c t s mentioned have i n v o l v e d the primary t a s t e s : s a l t , sour, sweet, and b i t t e r . The s a l t y t a s t e i s r a r e l y of anv s i g n i f i c a n c e i n wine, but the sour t a s t e i s q u i t e important. Substances g i v i n g s a l t y o r sour t a s t e s are i o n i z a b l e (7) and phenols without carboxy groups (which do not i o n i z e a p p r e c i a b l y under wine c o n d i t i o n s ) are not d i r e c t l y i n ­ volved i n these t a s t e s . Sweetness can be and b i t t e r n e s s o f t e n i s a property of p h e n o l i l i k e p h l o r o g l u c i n o l ar sweetness i s weak compared t o sugars and accompanied by " m e d i c i n a l " or other f l a v o r s . A few phenols d e r i v e d from n a t u r a l f l a v o n o i d s are i n t e n s e l v sweet, n a r i n g i n d i h y d r o chalcone, f o r example. There i s , however, no evidence of a s i g n i f i c a n t sweetness c o n t r i b u t i o n by any phenol found i n grapes or wine. I t i s not uncommon t h a t sweet, b i t t e r , and t a s t e l e s s members occur w i t h i n the same s e r i e s of compounds and t h i s i s t r u e of phenols. N a r i n g i n i s the i n t e n s e l y b i t t e r 7-βneohesperidoside of n a r i n g e n i n ; 4 , 9 , 7 - t r i h y d r o x v f l a v a n o n e . I t i s the predominant b i t t e r substance i n g r a p e f r u i t and can be converted to the i n t e n s e l y sweet dihvdrochalcone. Naringenin 7-P-rutinoside i s t a s t e l e s s (8, 9 ) . Current t h e o r i e s i n d i c a t e that sweetness r e q u i r e s two e l e c t r o n e g a t i v e atoms such as oxygen about 3 angstroms a p a r t , one w i t h an a v a i l a b l e proton and the other as a p o t e n t i a l proton acceptor f o r i n t e r m o l e c u l a r hydrogen bonding w i t h the receptor s i t e ( 7 ) . B i t t e r substances appear to have a s i m i l a r arrangement except t h a t the proton to negative center d i s t a n c e i s reduced about h a l f , producing i n t r a m o l e c u l a r hydrogen bonding and r e l a t i v e h y d r o p h o b i c i t y (7). The s p a c i a l arrangement of the key groups i s a l s o important i n whether a given compound w i l l be sweet or b i t t e r and how i n t e n s e l y so. I t seems p e r t i n e n t t h a t i n the i n o s i t o l s e r i e s substances w i t h more than 4 hydroxv rroups are o n l y sweet whereas those w i t h l e s s than 4 were b i t t e r and one w i t h 4 hydroxvIs was t a s t e l e s s (10). The p i c t u r e of a water s o l u b l e hydroxylated r i n g w i t h some hydrophobic character f i t s n a t u r a l phenols w e l l and many phenols have some b i t t e r c h a r a c t e r i f they have f l a v o r at a l l . A few examples i n c l u d e p i c r i c a c i d , m a t a i r e s i n o l g l u c o s i d e i n s a f f l o w e r (11), an isocoumarin i n c a r r o t s , o l e u r o p e i n i n o l i v e s , and 2,4,5-trimethoxybenzaldehyde i n c a r r o t seed (12)» Of course, manv terpene, t r o p o l o n e , g l y c o s i d e , a l k a l o i d , and peptide d e r i v a t i v e s are a l s o b i t t e r , 1

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but no b i t t e r s other than p h e n o l i c have, to our knowledge, been demonstrated i n wines unless they were d e l i b e r a t e l y f l a v o r e d as w i t h vermouths. 1 1

· Sources of Phenols i n Wines. The major source o f phenols i n wine i s , of course, the grapes used t o make the wine. The n a t u r a l phenols p r e e x i s t i n g i n the grape may be modified by the enzymes and exposure i n c i d e n t t o c r u s h i n g and p r e p a r a t i o n for fermentation. The yeasts i n c r e a s e phenol content by conv e r s i o n of nonphenolic s u b s t r a t e s t o p h e n o l i c d e r i v a t i v e s and w i l l modify the p h e n o l i c mixture by i n f l u e n c i n g e x t r a c t i o n or s o l u b i l i t y through the a l c o h o l produced, by adsorption and p r e c i p i t a t i o n w i t h the yeast c e l l , and by metabolism i n t o new phenols. Other microorganisms, p a r t i c u l a r l y the m a l i c a c i d fermenting l a c t i organisms causing i n c i p i e n modify wine's phenols. P r o c e s s i n g , p a r t i c u l a r l y operations such as o x i d a t i o n (as i n sherry making) or p a s t e u r i z a t i o n , m o d i f i e s nhenols i n wine. Aging produces c m a l i t a t i v e and Q u a n t i t a t i v e changes i n the p h e n o l i c makeup of wine and p a r t i c u l a r l y f i n e wines are o f t e n aged f o r s e v e r a l y e a r s . The f i r s t stage of aging i s o f t e n i n wooden c o n t a i n e r s . Depending on p r i o r c o n t a i n e r useage, s u r f a c e of wood per u n i t of wine, and time o f c o n t a c t , a d d i t i o n a l phenols are c o n t r i b u t e d from the c o n t a i n e r wood t o the wine (13b). F i n a l l y , phenols might be i n wine as i n a d v e r t a n t contaminants or as p a r t of f l a v o r s added t o such wines as vermouths. I I I . T o t a l Phenol Contents of Wines. The t o t a l c o n c e n t r a t i o n of a l l phenols can be determined a u i t e s a t i s f a c t o r i l y i n substances such as wine by molybdotungstophosphoric c o l o r i m e t r y (14). The r e s u l t s are expressed as mg of g a l l i c a c i d e q u i v a l e n t (CAE) per l i t e r of wine. By comparison w i t h s u i t a b l e known phenols the c o n t r i b u t i o n of any other nhenol t o the t o t a l mg OAE/1 of the wine can be p r e d i c t e d s a t i s f a c t o r i l y (13a). For example, (+)-catechin behaves as an equimolar mixture of p h l o r o g l u c i n o l and c a t e c h o l and gives 1.5 times the molar c o l o r y i e l d of g a l l i c a c i d i n t h i s assay. I f the phenols of grape b e r r i e s are e x h a u s t i v e l y e x t r a c t e d w i t h aqueous e t h a n o l , the t o t a l i s c o n s i d e r a b l y dependent upon the grape v a r i e t y as shown i n t a b l e 1 (15).

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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SINGLETON AND NOBLE

Wine Flavor and Phenolic Substances

Table I T o t a l E x t r a c t a b l e Phenols I n Ripe Grape B e r r i e s Berry c o l o r White II II It tl II II

Red It If If II

V a-jr i e t y• Aligote Emerald R i e s l i n g French Colombard Muscat A l e x a n d r i a Pinot blanc Sauvignon b l a n c Sémi l i o n Calζin Catawba Grenach Petite Sira Delaware

°Brix 20.7 20.6 18.7 18.8 20.1 21.8 19.8 21.0 22.1

20.6

T o t a l phenol mg. GAE/kg. f r e s h wt. 3240 5470 2530 2840 6060 2090 2430 4980 4060

4360

I t f o l l o w s from these f i g u r e s t h a t i f a l l t h e phenols present i n the grape b e r r y were f r e e l y e x t r a c t e d i n t o t h e wine t h e phenol content would be about 2000-6000 mg/1. These v a l u e s may be somewhat low s i n c e grapes grown i n c o o l e r r e g i o n s have more t o t a l phenol and these grapes were from a warm area. Wines w i t h 6500 mg GAE/1 have been prepared when we d e l i b e r a t e l y attempted t o r a i s e the t o t a l phenol as much as p o s s i b l e . Such wine i s u n d r i n k a b l y a s t r i n g e n t . I f c l u s t e r stems were e x t r a c t e d , a d d i t i o n a l t o t a l phenol content could be c o n t r i b u t e d up t o about 2000 mg GAE/1. Stems g i v e t o wine a somewhat hot or peppery c h a r a c t e r t h e chemistry of which has not been c l a r i f i e d ( 1 ) . The data a v a i l a b l e from s e v e r a l sources (1) suggest about 5500 mg GAE/kp t o t a l phenol i n t h e average r e d wine grape and about 4000 mg GAE/kg i n t h e average w h i t e wine grape. The d i f f e r e n c e i s p a r t l y t h a t t h e w h i t e grapes do n o t make anthocyanins and t h i s p o r t i o n of the t o t a l phenol appears t o be l o s t r a t h e r than d i v e r t e d t o o t h e r p h e n o l i c d e r i v a t i v e s . Furthermore, the r e d b e r r i e s tend t o be s m a l l e r , thus have more seeds and s k i n s f o r a g i v e n weight o f f r u i t (16). The t o t a l phenol was d i s t r i b u t e d about 3.3% i n the s k i n s , 0.7% i n the pressed f l e s h , 3.4% i n the j u i c e and 62.6% i n the seeds f o r a s e r i e s of r e d grapes and 23.2, 0.9, 4.5, and 71.4% r e s p e c t i v e l y i n a s e r i e s o f w h i t e wine grapes ( 1 ) . Seedless grapes' t o t a l i s reduced about 2/3 by t h e absence o f the seeds. I n i t i a l winemaking p r a c t i c e s g r e a t l y i n f l u e n c e the amount of phenols which t r a n s f e r from t h e grape t o the wine. L i g h t white wines of h i g h q u a l i t y are u s u a l l y made by separ­ a t i n g the j u i c e immediately upon c r u s h i n g t h e grapes. The contact w i t h pomace ( s k i n s and seeds) i s thus minimal and

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the p r e s s i n g i s r e s t r a i n e d i n j u i c e r e c o v e r y . The j u i c e i s f r e q u e n t l y c l a r i f i e d p r i o r to f e r m e n t a t i o n . The phenols o f such wines are those of the j u i c e w i t h minimal t r a n s f e r from the s k i n s or seeds. The t o t a l phenol content i s u s u a l l y about 50 t o 350 mg GAE/1 i n these wines, averaging about 250 mg GAE/1 ( 1 ) . I f the grapes are o v e r r i p e , s h r i v e l e d , heated, frozen or otherwise damaged so as t o cause e x t r a c t i o n o f the phenols from the s o l i d p a r t s , the t o t a l phenol content w i l l r i s e . The same i s t r u e of prolonged pomace contact before j u i c e s e p a r a t i o n or e x c e s s i v e p r e s s i n g f o r j u i c e r e c o v e r y . Good white t a b l e wine may be d e l i b e r a t e l y made w i t h some pomace contact t o i n c r e a s e f l a v o r and p a r t o f the i n c r e a s e i s p h e n o l i c . Lower q u a l i t y w h i t e wines may have t r i p l e the u s u a l phenol content. White d e s s e r t wines and s h e r r i e s are normally made from r i p e c o n d i t i o n s such that th h i g h e r , 300 to perhaps 800 mg GAE/1. Pink wines are o r d i n a r i l y made by fermenting the mixed whole mass of destemmed, crushed r e d grapes f o r about one day to e x t r a c t some anthocyanins from the s k i n s . The time b e f o r e f l u i d s e p a r a t i o n from the fermenting mixture i s extended f o r red wines; perhaps 3-4 days i s t y p i c a l a t present. Maximum red c o l o r r e l e a s e i n t o the wine i s reached w e l l b e f o r e maximum phenol e x t r a c t i o n . T h i s i s p a r t l y because of slower d i f f u s i o n and e x t r a c t i o n of m o l e c u l a r l y l a r g e r t a n n i n s e s p e c i a l l y from the seeds. About h a l f of the s e e d s phenol content should be e x t r a c t e d i n the u s u a l c o n d i t i o n s of r e d wine making (17). Carignane rosé made by fermentation a t 25°C had o n l y 1.65% of the anthocyanin and 7.6% of the b e r r y t o t a l phenols and red wine made by fermenting some o f the same grapes 10 days at 25°C had 26.8% of the b e r r y anthocyanin and 31.2% of the t o t a l phenol (18). I f f e r m e n t a t i o n on the pomace i s continued long enough the t o t a l phenol content o f the wine decreases, e v i d e n t l y due t o p o l y m e r i z a t i o n and p r e c i p i t a t i o n . Bourzeix e t a l . (19) found the maximum p h e n o l i c content occurred at about 11 days of pomace f e r m e n t a t i o n . Considerable data on the t y p i c a l phenol content of p i n k and red wines was reviewed by S i n g l e t o n and Esau ( 1 ) . Although wines from i n d i v i d u a l grape v a r i e t i e s d i f f e r c o n s i d e r a b l y , rosé wines fermented one day on the s k i n s had about 500 mg/1 of t o t a l phenol and about 10% of the red c o l o r o f wines made from the same grapes and fermented 3-5 days on the s k i n s to reach about 1900 mg/1 of t o t a l phenol. S e l e c t i v e h e a t i n g of grape s k i n s t o produce presumably complete r e l e a s e o f anthocyanins and o t h e r phenols from the s k i n s w i t h o u t c o n t r i b u t i o n from the seeds produced about 1200 mg GAE/1 t o t a l phenol. The lower content of t o t a l phenol i n wine compared to the content i n grapes r e f l e c t s mainly incomplete e x t r a c t i o n from the grape's seeds and s k i n s . However, p r e c i p i t a t i o n , p a r t i c u l a r l y of the t a n n i n s w i t h the grape p r o t e i n s o r yeast 1

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c e l l s a l s o i s a p p r e c i a b l e i n t h e course o f wine making and c l a r i f i c a t i o n . F u r t h e r p r e c i p i t a t i o n and l o s s occurs d u r i n g aging ( 1 ) . Berg and A k i y o s h i (20) found t h a t a wine prepared without fermentation by a l c o h o l a d d i t i o n t o r e d j u i c e had 1240 mg/1 t o t a l phenol. Wine made by fermenting another p o r t i o n of the same j u i c e was s i m i l a r i n c o l o r but the phenol content f e l l t o 1020 mg/1. Other s t u d i e s show even g r e a t e r l o s s e s of t a n n i n added b e f o r e f e r m e n t a t i o n . There appears t o be a sudden t r a n s i t i o n i n t o t a l phenol contents i n wines from those under about 500 mg/1 t o those above about 1000 mg/1. T h i s seems t o represent t h e b u f f e r i n g e f f e c t of yeast and grape p r o t e i n t a n n i n p r e c i p i t a t i o n c a p a c i t y , f o l l o w e d by a r a p i d r i s e t o h i g h e r l e v e l s as t h i s c a p a c i t y i s exceeded ( 1 ) White wines tend t o have excess p r o t e i n and no t r u e t a n n i n t a n n i n content and undetectabl on l o w - c o l o r , low-sugar grapes a t c r u s h i n g and a t d a i l y i n t e r v a l s of fermentation on t h e pomace t h e r e a f t e r the phenol contents of the samples were 240, 600, 640, 660, 700, 1220 and 1490 mg/1 (22). The r i s e would be f a s t e r w i t h more p h e n o l - r i c h v a r i e t i e s and a l s o w i t h h i g h e r a l c o h o l p r o d u c t i o n or warmer fermentation temperature. I V

T

o

t

a

l

* Phenol Content and Wine F l a v o r and Q u a l i t y . I t has long been known that i n c r e a s e d t o t a l phenol content r e s u l t i n g from i n c r e a s e d e x t r a c t i o n o f grape s o l i d s d u r i n g wine p r e p a r a t i o n produces i n c r e a s e d f l a v o r , p a r t i c u l a r l y i n c r e a s e d a s t r i n g e n c y i n the wine ( I ) . I n l i p h t white t a b l e wines i n creased p h e n o l i c content has u s u a l l y been a s s o c i a t e d w i t h reduced q u a l i t y of the wine, although s t r i p p i n g of such wines w i t h e f f i c i e n t phenol adsorbing agents ( c h a r c o a l , polyamides, p o l y v i n y l p y r r o l i d i n o n e ) i s a l s o b e l i e v e d t o produce i n s i p i d , lower q u a l i t y wines. On t h e other hand, very r i p e grapes and some pomace contact are a s s o c i a t e d w i t h h i g h e r phenol content and h i g h q u a l i t y i n more robust white t a b l e and dessert wines. Examples of both types are provided by a study o f 554 German wines (23). The average phenol content i n c r e a s e d i n the order Mosel, Rheingau, Rheinhessen, R h e i n p f a l z and Baden and t h i s i s a l s o the s e r i e s considered t o be decreasing i n l i g h t n e s s , elegance, and q u a l i t y toward h e a v i e r wines. However, the phenol (leucoanthocyanidin) content f o r wines o f i n c r e a s i n g q u a l i t y and made from i n c r e a s i n g l y " r i p e " grapes averaged 10.6 mg./l. f o r K a b i n e t t , 12.6 f o r S p a t l e s e , 17.9 f o r A u s l e s e , 18.0 f o r Beerenauslese, and was between 24 and 133 mg./l. f o r Trockenbeerenauslese wines, i n s p i t e o f t h e f a c t t h a t B o t r y t i s c i n e r e a , a mold i n c r e a s i n g l y i n v o l v e d w i t h grapes f o r t h e l a t t e r t y p e s , commonly lowers the leucoanthocyanin content (24). Increased phenol (10 times normal leucoanthocyanidin) content i s a l s o considered r e s p o n s i b l e f o r decreased q u a l i t y and i n c r e a s e d inharmonious, b i t t e r , heavy o r coarse t a s t e i n white wines from f r o s t e d grapes (25). Over 35 mg/1 l e u c o -

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anthocyanidin i s r a r e among German wines except i n the l a t e or b o t r y t i z e d s p e c i a l i t y harvests (23). T h i s 35 mg/1 would he i n c l u d e d i n a t o t a l phenol content of about 250 mg GAE/1 (26). White wines fermented i n the normal f a s h i o n from o n l y j u i c e may not have s i g n i f i c a n t l y d i f f e r e n t q u a l i t y than those made w i t h up t o 12 hours pomace contact a f t e r c r u s h i n g (27), but longer contact lowered q u a l i t y . Phenol content and astringency r a t i n g i n c r e a s e w i t h time o f fermentation w i t h the pomace of white o r red grapes as shown by the average values f o r 7 s e t s i n v o l v i n g 6 white grape v a r i e t i e s , t a b l e 2 (28). Ratings f o r a s t r i n g e n c y and b i t t e r n e s s are on a 0-low to 5-high s c a l e and maximum q u a l i t y r a t i n g i s 20.

Phenol Content an

T o t a l phenol, mg GAE/1 A s t r i n g e n c y , mean r a t i n p B i t t e r n e s s , mean r a t i n g Q u a l i t y , mean r a t i n g

Pomace Contact During Fermentation 2 days 5 days 1 day 0 405 312 261 207 4.5 3.8 3.8 3.4 3.5 3.7 3.6 3.3 11.0 11.7 11.8 14.1

A l l of the q u a l i t y r a t i n g s ( t a b l e 2) and a l l but the 1and 2-day a s t r i n g e n c y r a t i n g s were s i g n i f i c a n t l y d i f f e r e n t (95% c o n f i d e n c e ) , but the b i t t e r n e s s r a t i n g s were not. From these data and comparisons w i t h i n each i n d i v i d u a l set of wines i t was concluded t h a t pomace contact s u f f i c i e n t t o g i v e about 100 mg GAE/1 a d d i t i o n a l phenol would g i v e a r e c o g n i z a b l e i n crease i n a s t r i n g e n c y whereas l e s s would not. That i s , the difference threshold f o r recognizable astringency increase i n white wine i s about 100 mg GAE/1 of t o t a l grape phenols. P a i r e d t e s t i n g r a t h e r than sample r a t i n g f o r a s t r i n g e n c y or b i t t e r n e s s would probably show a lower t h r e s h o l d . In t h i s study the h i g h e s t q u a l i t y r a t i n g f o r j u i c e - o n l y wine ( t a b l e 2) was mainly f o r nonphenolic reasons, but the a s s o c i a t i o n of high phenol content w i t h decreased q u a l i t y i n dry white wine agrees w i t h other r e p o r t s (e.g., 1, 23, 27). Since astringency i n c r e a s e d but b i t t e r n e s s r a t i n g s i n c r e a s e d only to a p o i n t w i t h i n c r e a s i n g phenol, i t appears p e r c e p t i o n of b i t t e r n e s s was suppressed, presumably by i n t e r f e r e n c e by h i g h astringency (28). Red wines cover a much w i d e r range of phenol content and astringency than whites ( 1 ) . Astringency i s a very s i g n i f i c a n t p a r t of a red wine's c h a r a c t e r . M i l d l y a s t r i n g e n t red wines o r d i n a r i l y have a t o t a l phenol content of the order of 1300 mg GAE/1 or l e s s , more robust wines about 1400 mg GAE/1, and over 2000 mg GAE/1 i n d i c a t e s a wine e x c e s s i v e l y a s t r i n g e n t f o r most people's preference today. The t y p i c a l

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phenol l e v e l i n red wines has been moving downward a p p a r e n t l y i n response to the p u b l i c ' s d e s i r e f o r l i g h t e r l e s s a s t r i n g e n t red wine. The t r e n d appears t o be w o r l d wide a t l e a s t i n wine we import. About 500 mg/1 of t o t a l phenol f o r w h i t e s and 2250 mg/1 f o r r e d C a l i f o r n i a wines were considered p r e f e r a b l e i n 1935 ( 1 ) . Commercial C a l i f o r n i a c l a r e t had about 2000, 1600, and 1500 mg GAE/1 t o t a l phenol i n 1935, 1940, and 1946, r e s p e c t i v e l y . C a l i f o r n i a port samples were l e s s than h a l f these v a l u e s and burgundy about 20% h i g h e r , but showed s i m i l a r t r e n d s . Wines made e x p e r i m e n t a l l y by recommended p r a c t i c e s averaged about 1300 mg/1 p r i o r t o 1941 and about 1150 mg GAE/1 s i n c e 1946. Much of t h i s downward s h i f t was accomplished by shortening the f e r m e n t a t i o n time on the s k i n s which probably has now reached the l i m i t of f u r t h e r r e d u c t i o n f o r most p r o ducers. A s e r i e s of 5 commercial C a l i f o r n i a 1186 mg GAE/1 t o t a l phenol. T h i s i n d i c a t e s a very low astringency l e v e l i n such wines and the t a s t e e f f e c t of the phenols i s lowered s t i l l f u r t h e r by the masking presence o f a s l i g h t sweetness from r e s i d u a l sugar. Premium dry r e d t a b l e wines, p a r t i c u l a r l y those intended f o r a p p r e c i a b l e a g i n g , are s t i l l g e n e r a l l y 1400 mg GAE/1 or more. On the b a s i s of the data quoted f o r w h i t e wines and cons i d e r i n g the t y p i c a l Weber-Fechner r e l a t i o n s h i p s the d i f f e r e n c e t h r e s h o l d f o r a s t r i n g e n c y i n the normal red wine would be expected to be about 250 mg GAE/1 of the m i x t u r e o f phenols e x t r a c t e d from pomace. Chis compares r a t h e r x*ell w i t h v a l u e s estimated by t a n n i n a d d i t i o n ( 1 ) . A s t r i n g e n c y r a t i n g s by an expert panel rose s i g n i f i c a n t l y i n r e d wines averaging 1500 mg GAE/1 when the f l a v o n o i d content i n c r e a s e d 280 mg GAE/1, but d i f f e r e n c e s were not s i g n i f i c a n t when the i n c r e a s e was only 150 mg GAE/1 (16). V. Nonflavonoids of Wine and Wine F l a v o r . The content o f phenols which are not f l a v o n o i d s i s r e l a t i v e l y constant i n a l l young wines at about 200 mg GAE/1 (29). T h i s group of compounds accounts f o r n e a r l y a l l of the phenols i n c l a r i f i e d , unheated, w h i t e grape j u i c e whereas the f l a v o n o i d s are e s s e n t i a l l y confined to the s k i n s and seeds. The only a p p r e c i a b l e source of n o n f l a v o n o i d s known from the s o l i d p a r t s of the grape i s the hydroxycinnamic a c y l groups which e a s i l y h y d r o l y z e from anthocyanins. Heating or m i c r o b i a l a c t i o n can a l s o lead t o some conversion of f l a v o n o i d t o n o n f l a v o n o i d phenols ( 1 , 30)· Wines aged i n wooden c o n t a i n e r s e x t r a c t phenols from the wood that are n e a r l y a l l n o n f l a v o n o i d i n nature (31). A. Odorous Phenols of Wine. The content of phenols d i s t i l l a b l e from wines i s normally low. Young w h i t e wines had about 1.5-3.2 mg/1 c a l c u l a t e d as phenol (32). With o l d e r wine t h i s increased t o 11.5 mg/1 i n one 9 years o l d . Red wines, part i c u l a r l y Georgian s t y l e which are s t o r e d on the grape pomace

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and had 2400-2800 mg GAE/1 t o t a l phenol, gave c o n s i d e r a b l y higher d i s t i l l a b l e phenol i n i t i a l l y 22-36 mg/1, and a l s o i n creased w i t h aging to 42 mg/1 or so. The s m a l l phenols s o l v e n t - e x t r a c t a b l e without d i s t i l l a t i o n from commercial red wines were m-cresol, 4-ethylphenol, 4 - v i n y l p h e n o l , 4 - e t h y l g u a i a c o l , and t y r o s o l (33). A l s o present i n s m a l l e r amount were p - e r e s o l , g u a i a c o l , 4 - v i n y l g u a i a c o l , i s o e u g e n o l , v a n i l l i n , and ( i n one of those wines) 2,6-dimethoxyphenol. S e v e r a l of these had been reported p r e v i o u s l y i n wine or wine d i s t i l l a t e s along w i t h phenol, s a l i c y l i c a c i d and i t s methyl and e t h y l e s t e r s , and v a n i l l i c a c i d (34). A l l of these p l u s a d d i t i o n a l phenols of s i m i l a r types have been found i n d i s t i l l e d beverages aged i n oak c o n t a i n e r s or i n the oak i t s e l f i n c l u d i n g £c r e s o l , 2'-hydroxyacetophenone, 2 - e t h y l phenol, 4-methylg u a i a c o l , 2'-hydroxy-5 s y r i n g i c a c i d , 2-isopropylphenol c o n i f e r a l d e h y d e , eugenol, e t h y l v a n i l l a t e , and sinapaldehyde (34). These compounds may enter wine as e x t r a c t a n t s from wood, but some of them e v i d e n t l y can be generated i n wine by other means. Thermal degradation of f e r u l i c a c i d produces g u a i a c o l and i t s 4-methyl, 4 - e t h y l , and 4 - v i n y l d e r i v a t i v e s , v a n i l l i n , a c e t o v a n i H o n e , and v a n i l l i c a c i d (35). C a f f e i c and £coumaric a c i d s would be expected t o produce analogous d e r i v a t i v e s . These along w i t h a t o t a l of 32 phenols are p r i n c i p a l components of smoke and smoky f l a v o r s (36). However, these compounds are produced under c o n d i t i o n s much s h o r t of p y r o l y s i s such as mashing of g r a i n f o r whiskey (37) or h e a t i n g of apple j u i c e (38). C e r t a i n s t r a i n s of l a c t o b a c i l l i have been shown to convert c h l o r o g e n i c o r c a f f e i c and £-coumaric a c i d i n c i d e r v i a r e d u c t i o n and d e c a r b o x y l a t i o n t o 4 - e t h y l c a t e c h o l and 4ethylphenol (39). I n f a c t , the same organism can convert shikimate or quinate t o c a t e c h o l thus generating a new odorous phenol from nonphenolic precursors (40)· The e f f e c t of most of these phenols i n grape wine f l a v o r has not been determined, but i n f o r m a t i o n can be drawn from other s t u d i e s . In aqueous ethanol s o l u t i o n 4 - e t h y l g u a i a c o l has a sensory t h r e s h o l d of 0.05 m*/l w i t h a warm, sweet, s p i c y , burnt t o f f e e , p h e n o l i c c h a r a c t e r . S i m i l a r l y 4-ethylphenol had a t h r e s h o l d of 1.0 mg/1 and was d e s c r i b e d as woody, p h e n o l i c , m e d i c i n a l , and heavy c i d e r i n odor (41). These odorants are considered p a r t of the c h a r a c t e r i s t i c odor of b i t t e r - s w e e t E n g l i s h c i d e r s s i n c e t h e i r content i s about double the t h r e s h o l d for 4-ethylphenol and 2 0 - f o l d t h a t of 4 - e t h y l g u a i a c o l . At high l e v e l s they a l s o c o n t r i b u t e d to o f f - f l a v o r . Four-vinylphenol and 4-vinylguaiaco1 are r e p o r t e d to have t h r e s h o l d s i n water of 0.02 and 0.01 m/1 r e s p e c t i v e l y (38). I n beer the t h r e s h o l d of 4-ethylphenol was 0.3 mg/1 ( p h e n o l i c , a s t r i n g e n t ) ; 4 - e t h y l e u a i a c o l was 0.13 mg/1 ( p h e n o l i c , b i t t e r ) ; and 4 - v i n y l g u a i a c o l was 0.30 mg/1 ( a s t r i n g e n t , b i t t e r ) ( 4 2 ) . The t a s t e t h r e s h o l d s i n water of three major smoke c o n s t i t u e n t s g u a i a c o l , 4-methyl-

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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g u a i a c o l , and 2,6-dimethoxyphenol were 0.013, 0.065, and 1.65 mg/1 and the odor t h r e s h o l d s were un t o 60% h i g h e r (43). The combined r e c o g n i t i o n t h r e s h o l d of the phenol complex from smoke vapor i n water was £ 6.2 mg/1 o f phenol by t a s t e and < 21 mg/1 by odor w h i l e the most d e s i r a b l e c o n c e n t r a t i o n was 27 mg/1 by t a s t e or 42 mg/1 by odor o r l e s s depending on the method of smoke generation (44). The sum of the i d e n t i f i e d v o l a t i l e phenols e x t r a c t e d by pentane from Jamaica rum was 1.52 ppm w i t h 4 - e t h y l p h e n o l , g u a i a c o l and v a n i l l i n each at 0.25 pom ( 4 5 ) . The content i n Scotch whiskey of a l l i d e n t i f i e d v o l a t i l e phenols was 0.12 ppm, about 1/3 was eugenol (46). Phenols and c r e s o l s g i v e d e t e c t a b l e o f f f l a v o r s when added t o beer at 0.03 mg/1 ( 4 7 ) . I t i s obvious t h a t stud i needed d i r e c t l wines, but i t seems v e r group of v o l a t i l e phenol , especially age oak cooperage, can c o n t r i b u t e d e t e c t a b l e f l a v o r t o wines. While the content i s low and may not reach t h r e s h o l d f o r any one component i n wine, the f l a v o r s of many are s i m i l a r l y described as s p i c y , smoky, p h e n o l i c , m e d i c i n a l , e t c . and should be a d d i t i v e i n f l a v o r (42). An a b s o r p t i o n of o n l y 0.2 mg/1 of phenol and c r e s o l s i n t o wine from a t m o s p h e r i c a l l y contaminated grapes grown near a f a c t o r y which emitted these phenols produced o b j e c t i o n a b l e o f f - t a s t e i n the wine (48). I t a l s o appears that a s l i g h t warm, sharp, pungent f l a v o r may be c o n t r i b u t e d i n p a r t by these compounds. The content of pungent phenols i s much l e s s than i n such v e r y hot p r o ducts as c l o v e o i l or g i n g e r o l e o r e s i n , but e t h a n o l demonstrably p o t e n t i a t e s such "hotness." V a n i l l i n and the r e l a t e d c o n i f e r a l d e h y d e , s y r i n g a l d e h y d e , sinapaldehyde, v a n i l l i c a c i d , and e t h y l v a n i l l a t e are bel i e v e d t o be i n wine p r i m a r i l y as a l c o h o l e x t r a c t a n t s and a l c o h o l y s i s products from wood ( 1 ) . I n a few very o l d wines and brandies v a n i l l i n odor becomes r e c o g n i z a b l e . The recogn i t i o n t h r e s h o l d of v a n i l l i n i n water i s v a r i o u s l y r e p o r t e d to be 0.5-4 mg/1 w i t h the d e t e c t i o n t h r e s h o l d about 0.1 mg/1 (49) . The t h r e s h o l d o f v a n i l l i c a c i d i n beer i s about 10 mg/1 (50) . I n brandy aged i n American oak b a r r e l s the content of v a n i l l i n i s about 11 mg/1, syringaldehyde about 16 mg/1, and a l l aromatic aldehydes about 55 mg/1 c a l c u l a t e d as v a n i l l i n (51) . Up t o 0.25 mg/1 of v a n i l l i n has been r e p o r t e d i n wines aged i n wood, but over 0.5 mg/1 was considered as presumptive evidence of v a n i l l i n a d d i t i o n (52). A d d i t i o n of o n l y 0.05 t o 0.5 mg/1 of v a n i l l i n o r jv-hydroxybenzaldehyde t o s y n t h e t i c sake improved the q u a l i t y (53). I t appears and c e r t a i n l y i s the b e l i e f of wine t a s t e r s t h a t v a n i l l i n and r e l a t e d f l a v o r s can be part of the mellowing e f f e c t of b a r r e l aging on wine, but they are not u s u a l l y present at i n d i v i d u a l l y suprathreshold l e v e l s .

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

58

PHENOLIC, SULFUR, AND NITROGEN COMPOUNDS I N FOOD FLAVORS

B. F l a v o r E f f e c t s of Nonflavonoids o f Low V o l a t i l i t y . The odorous, v o l a t i l e phenols of wine apparently account f o r only a few ppm of the t y p i c a l 200 mg GAE/Ï o f nonflavonoids. The remainder i s l a r g e l y accounted f o r by cinnamic and benzoic a c i d d e r i v a t i v e s p l u s some t y r o s o l and r e l a t e d compounds. T y r o s o l i s the only phenol known to be produced i n s i g n i f i c a n t amounts from nonphenolic p r e c u r s o r s by y e a s t fermentation. I t , l i k e other " f u s e l o i l " a l c o h o l s , i s produced by fermenting yeasts from carbohydrate by d e c a r b o x y l a t i o n and r e d u c t i o n o f the o-|

50-1a HO-j 3

0-1 1

ao-f a ι ;

/

1

Figure 3. Preparative thin layer chromatogram of PGPC-BFP, MN 300 cellulose plate, 500 μ thickness, developing solvent: propanolr-water, 7:3 (v/v), silver nitrate visualiza­ tion

Table I. Comparison of PTLC Fractions of PGPC of Beef Flavor Precursors

Fraction No.

Nitrogen Content I

1

4.42

2 3

9.87 13.13

4 5

7.03 4.28

6 7

3.52 2.88

PGPC-BFP Ultrafiltrate

Yield % of PGPC Odor Description ) )29 ) ) )45 ) 10

BFP, Very Sweet. Strong BFP, Very Sweet. Weak BFP,Oily,Not Sweet. Weak BFP, Sweet. Heated Protein,Fishy,Musty, Not Sweet. Wet Cardboard. Musty, Hay.

7.92 9.20

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

PHENOLIC,

SULFUR, AND NITROGEN

COMPOUNDS

I N FOOD FLAVORS

Table II. Amino Acids Present in PTLC Fractions of PGPC-Beef Flavor Precursors.

Amino Acids 1 Histidine Glutamic B-alanine Aspartic Glycine Alanine Arginine Serine Tyrosine Leucine 1-methylhistidine Tryptophan Threonine Lysine Valine Isoleucine Ammonia Ornithine Phenylalanine a-amino-n-butyric acid Proline Methionine ) Methionine ) Sulfoxide) Cysteic Acid

μΜ/mg. of Fraction No. 2 3 4 5

0.]26 0.098 0.110 0.052 0.059 0.040 0.017 0.022 0.009 0.010

0.023 0.003 0.023 0.281 0.078 0.014 0.171 0.001 0.003

0.008 0.005 0.008 0.006 0.006 0.004 0.024 0.001 0.001

0.004 0.023 0.020 0.003 0.006 0.002 0.212 0.002 0.001

6

0.001 . . . . .

7 .....

0.004 0.001 0.003 0.001 0.0004 0.020 0.003 0.003 0.012 0.0001 0.503 0.207 0.008 0.006 0.0001 0.023 0.003 0.008 0.006 0.0004 0.003 0.004 0.051 0.427 0.041 0.017 0.001 0.066 0.011 0.004 0.001 0.006 0.014 0.413 0.002 0.002 0.047 0.091 0.019 0.020 0.001 0.001 0.006

0.002 0.0001 0.009 0.223 0.021 0.005 0.0001 0.003

0.001 0.003 0.010 0.048 0.005 0.002 0.002 0. 001 0.041 0.001

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

10.

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Nonvolatile Nitrogen and Sulfur Compounds in Red Meats 153

Table III. The Major Amino Acids i n PTLC Fractions

Amino Acids 1

Histidine Glutamic B-alanine Aspartic Glycine a-alanine Arginine Serine Ammonia Tryptophan Proline Valine Leucine Isoleucine Methionine + Methionine Sulfoxide Threonine

yiM/mg. of Fraction No. 3 2 4

0.126 0.098 0.110 0.052 0.059

5

6

0.413 0.051 0.047 0.041

0.427 0.223

0.299

0.281

0.171 0.091 0.041 0.048

0.066

Figure 4. Preparative thin layer chromatogram of PTLC, fraction #2, MN 300 cellulose plate, 500 μ thickness, developing solvent: propanolr-water, 7:3 (v/v), ninhydrin visualization

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

PHENOLIC,

154

SULFUR,

A N DN I T R O G E N

COMPOUNDS

I N FOOD

FLAVORS

Table IV. Weight of PTLC Subfractions of PTLC Fraction 2

Subfraction No. 1 2 3 4 5 6 7 8

% of Recovered Material

Weight g.

12.7 19.6 13.9 7.4 10.2 16.8 10.5 9.0

0.31091 0.47833 0.33921 0.18154 0.24900 0.41012 0.25748 0.22028

Weight of PTLC Fraction #2 used = 1.62 g.

Figure 5. Gel permeation chromatography of "contaminants," 28 X 0.7 cm column, Sephadex G-JO

Figure 6. Gel permeation chromatography of PTLC subfraction #3, 28 X 0.7 cm column, Sephadex G-10

15

25

35

45

55

65

TUBE NUMBER

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

75

10.

MABROUK

Nonvolatile Nitrogen and Sulfur Compounds in Red Meats 155

s u b f r a o t l o n s from the c o n t a m i n a n t s . The homogeneity of each s u b f r a c t i o n needs t o be evaluated before subject­ ing t o sensory e v a l u a t i o n and a n a l y s i s *

Amino A o l d s Of 27 amino compounds i n aqueous beef e x t r a c t , 23 were i d e n t i f i e d and q u a n t i f i e d (mg./lOQ g . f r e s h weight meat): p h o s p h o s e r i n e , 1 . 8 4 ; t a u r i n e , 0 . 0 9 ; a s p a r t i c ,

0.95 ; t h r e o n i n e , 3 . 1 ? » s e r i n e , 3 . 6 0 ; g l u t a m i c , 1 1 · 6 0 ; p r o ­ line,0.9^5 g l y c i n e , 3 * 1 0 ; c9,10) on the p r e s e n c e o f c a r n o s i n e , a n s e r i n e , and g l u t a t h i o n e , no p u b l i s h e d work e x i s t s i n l i t e r a t u r e on t h e p r e s e n c e o f o t h e r p e p t i d e s I n r e d meats. As the t o t a l c o n t e n t o f the amino a c i d s i d e n t i f i e d I n aqueous meat e x t r a c t s account f o r a s m a l l p o r t i o n o f t h e t o t a l n i t r o g e n c o n t e n t , the b a l a n c e o f the n i t r o g e n o u s compounds s h o u l d account f o r p e p t i d e s , g l y o o p e p t i d e s , n u c l e o t i d e s , n u c l e o t i d e d e r i v a t i v e s , and a m i n e s . The i n c r e a s e i n the number o f the components o b t a i n e d b y f r a c t i o n a t i o n o f meat f l a v o r p r e c u r s o r s b y PGPC, P T L C , and a n a l y t i c a l GPC , i n d l o a t e s t h e p r e s e n c e o f p e p t i d e s a n d / o r g l y o o p e p t i d e s depending upon the p r e s e n c e o f c a r b o h y d r a t e m o i e t i e s ( F i g u r e s 3 and k) and

(Tables I I ,

1 1 1 and I V ) ·

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

158

P H E N O L I C ,

S U L F U R ,

A N D

N u c l e o t i d e s and Nucleotide

N I T R O G E N

C O M P O U N D S

I N

F O O D

F L A V O R S

Derivatives

Nucleotides present i n red meats vary according not only t o species, breed, age of the animal, and feed but a l s o t o the freshness of the meat* 5 - I n o s l n e mono1

phosphate

(IMP) and 5 ' - g u a n o s l n e

monophosphate(GMP)

have f l a v o r enhancing e f f e c t s , and these e f f e c t s are considered t o be e s s e n t i a l l y the same. The f l a v o r e f f e c t of GMP i s s a i d t o be b r o a d e r than that of IMP and g e n e r a l l y produces a more harmonizing e f f e c t * GMP i s about 4 times s t o n g e r t h a n IMP i n aqueous solutions» Meats c o n t a i n a very small q u a n t i t y o f GMP i n a d d i t i o n to IMP, b u t the l e v e l i s so small that i t cannot be considered t o have any e f f e c t on t a s t e ( 2 2 ) . Eight f r a c t i o n s of p e p t i d e - b o u n d nucleotide reported i beef, one f r a c t i o n e x h i b i t e l e d meats c o n t a i n n u c l e o t i d sugar y r e l a t e d t o the metabolism and b i o s y n t h e s i s o f sugars. Nucleotide c h o l i n e i s i n v o l v e d i n l i p i d m e t a b o l i s m . 5 - c y t l d l n e monophosphate, 5 ' - a d e n o s i n e monophosphate, and 5 u r i d i n e monophosphate a r e a l s o p r e s e n t i n meat i n s m a l l c o n o e n t r a t i o n s . N u c l e o t i d e content i n r e d meats i s t a b u l a t e d i n T a b l e V I I . f

1

Effect

Of H e a t i n g On Meat F l a v o r P r e c u r s o r s

D u r i n g heat p r o c e s s i n g , the types o f r e a c t i o n s which can o c c u r t o the non-aqueous f l a v o r p r e c u r s o r s a r e : a u t o x l d a t i o n , h y d r o l y s i s , d e h y d r a t i o n and d e c a r b o x y l a t i o n of f a t s g i v i n g r i s e to aldehydes, f a t t y acids, lactones, ketones, hydrocarbons, a l c o h o l s , . . e t c . The aqueous f l a v o r p r e c u r s o r s may be s u b j e c t e d t o g l y c o s i d e s p l i t t i n g , o x i d a t i o n , p y r o l y s i s ( t h e r m a l decomp o s i t i o n ) t o y i e l d v o l a t i l e and n o n - v o l a t i l e compounds t h a t i n f l u e n c e f l a v o r . P r o d u c t s o f amino a c i d s p y r o l y s i s a r e v e r y complex. The most Important f l a v o r p r o d u c i n g c h e m i c a l r e a c t i o n i s t h e non-enzymic browning r e a c t i o n o f the amino a c i d s . T h i s r e a c t i o n i n v o l v e s o x i d a t i v e d e a m i n a t i o n o f a n amino a c i d m o l e c u l e w i t h the f o r m a t i o n o f an aldehyde w i t h one c a r b o n atom l e s s t h a n the o r i g i n a l a c i d . a- Nucleotides. I n a s t u d y on the browning r e a c t i o n o f 5 ' - r i b o n u c l e o t i d e s w i t h D - g l u o o s e , F u j i m a k i et al. (26) c o n c l u d e d t h a t phosphate p l a y s an important r o l e I n the browning r e a c t i o n of a l d o s e s w i t h n u c l e o t i d e s , and t h a t the phosphate e s t e r at the p r i m a r y a l c o h o l of r l b o s e r e s i d u e , as w e l l a s , the i n o r g a n i c orthophosphate w h i c h i s l i b e r a t e d due t o the h y d r o l y s i s o f t h e e s t e r causes the development of the r e a c t i o n . At

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

10.

MABROUK

Nonvolatile

Table VI.

Nitrogen and Sulfur Compounds

in Red Meats

159

Classification of Free Amino Compounds in Red Meats.

Percent Amino Compound

Neutral Amino Acids Hydroxy Amino Acids Acidic Amino Acids Sulfur - Containing Amino Acids Basic Amino Acids Aromatic Amino Acids Dipeptides "Anserine + Carnosine"

Beef

Lamb

Pork

14.4 5.5 3.4

18.1 6.2 5.9

9.4 3.4 2.9

9.8 2.0 55.8

20.2 2.9 19.7

7.1 1.0 61.1

Table VII. Nucleotide Content in Red Meats

Meat CMP Beef Beef Pork Mutton

12.0 1.0 1.9 1.9

Nucleotide Content, mg/100g. IMP IMP GMP AMP 13.0 1.6 1.6 0.6

150.0 107.0 123.0 83.5

8.0 2.1 2.5 5.1

17.0 6.6 7.6 6.8

Reference

24 22 22 25

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

160

P H E N O L I C ,

S U L F U R ,

A N D

N I T R O G E N

C O M P O U N D S

I N

F O O D

F L A V O R S

higher temperature ( 1 2 0 ° C ) , glucose a c c e l e r a t e the deg r a d a t i o n of IMP, which may be a t t r i b u t e d t o i n t e r a c t i o n o f the n u c l e o t i d e w i t h some r e a c t i v e compounds such as osones and others which are p o s s i b l y formed through 1 , 2 - and/or 2 , 3 - e n o l i z a t i o n of aldoses and f u r t h e r degradation, condensation, and p o l y m e r i z a t i o n * a l though IMP i s more s t a b l e than GMP, I M P - g l u c o s e s o l u t i o n produced more 3-deoxy-D-gluoosone and developed more intense brown c o l o r than OMP-glucose s o l u t i o n d i d , these d i f f e r e n c e s may be a t t i b u t e d t o the amino group on the purine r i n g i n GMP. I n a study on the e f f e c t of heating on n u c l e o t i d e s i n beef, pork, and lamb, Maey

et a l (2?) r e p o r t e d the f o l l o w i n g c o n c l u s i o n s . I n the case o f b e e f , CMP was not g r e a t l y I n f l u e n c e d by h e a t i n g to 49°C and ?? G i n t e r n a l t e m p e r a t u r e . AMP content appreciably increase t u r e s , t h i s I n c r e a s e might be due to the h y d r o l y s i s o f adenosine diphosphate(ADP) and adenosine t r i p h o s p h a t e ( A T P ) . UNP and IMP c o n t e n t s d e c r e a s e d by h e a t i n g to 6

%^C and ?7°C.

The i n f l u e n c e o f r o a s t i n g t o ^9°C and

71 C i n t e r n a l t e m p e r a t u r e s , on p o r k n u c l e o t i d e s was s i m i l a r t o t h a t on b e e f . A l l n u c l e o t i d e s d e c r e a s e d d u r i n g r o a s t i n g except AMP which i n c r e a s e d i n q u a n t i t y 0 when h e a t e d t o 7 1 ° C . When lamb was heated t o 6O C i n t e r n a l t e m p e r a t u r e , GMP and UMP were d e s t r o y e d a f t e r 5 and 15 minutes r e s p e c t i v e l y . CMP d o u b l e d i t s o r i g i n a l c o n c e n t r a t i o n and t h e n degraded g r a d u a l l y . About 85% o f IMP content was l o s t a f t e r h e a t i n g f o r 30 m i n . ( F i g ure 7 ) . b - Amino Compounds and G u a n i d l n e s . Cooking caused s i g n i f i c a n t i n c r e a s e s i n c r e a t i n e and d e c r e a s e i n amino a c i d s , c r e a t i n e , non-amino n i t r o g e n and t o t a l c a r b o h y d r a t e s (28, 29) · I n the case o f b e e f , c r e a t i n e c o n t e n t l o s s was dependent on the h e a t i n g t e m p e r a t u r e . The l o s s amounted t o 10 and 20$ of the o r i g i n a l c o n c e n t r a t i o n , when h e a t e d up t o k^°t and 7 7 ° C , r e s p e c t i v e l y . D u r i n g c o o k i n g t o 77*C, a l l f r e e amino a c i d s o f b e e f r o a s t s i n c r e a s e d , exoept t h r e o n i n e , s e r i n e , g l u t a m i c , h i s t i d l n e , and a r g i n l n e , which d e c r e a s e d . The o v e r a l l e f f e c t o f c o o k i n g was an I n c r e a s e o f 38.3# I n the t o t a l amino a c i d s . T o t a l e x t r a c t a b l e amino n i t r o g e n i n c r e a s e d a p p r o x i m a t e l y 18# I n the same sample d u r i n g c o o k i n g . These i n c r e a s e s were p r o b a b l y due t o p r o t e i n h y d r o l y s i s and p o s s i b l y i n v o l v e d c a t h e p s i n s o r o t h e r p r o t e o l y t i c enzymes i n the t i s s u e , s i n c e i t has been shown t h a t most f r e e amino a c i d s d e c r e a s e d d u r i n g h e a t i n g when i s o l a t e d from the p r o t e i n s by d i a l y s i s ( T a b l e V I I I ) . T a u r i n e , a n s e r i n e , and c a r n o s i n e i n c r e a s e d to the g r e a t e s t e x t e n t d u r i n g c o o k i n g . These were the major

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

10. MABROUK

Nonvolatile Nitrogen and Sulfur Compounds in Red Meats 161

o

' 10

20

30

40

50

60

TIME , MIN

Figure 7. Effect of heating lamb to 60° C internal temperature on inosine 5''-monophosphate

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

PHENOLIC, SULFUR, AND NITROGEN COMPOUNDS I N FOOD FLAVORS

162

Table VIII.

Percent Change in Free Amino Compounds of Lyophilized Red Meats Diffusâtes

Amino Compounds Beef Phosphoserine Glycerophosphoethanolamine Phosphoethanolamine Taurine Urea Aspartic Threonine Serine+Asparagine Glutamic Acid Proline Glycine Alanine Cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Ornithine NH3 + Lysine Histidine Carnosine + Anserine 1-Methylhistidine aAmino-n-butyric acid Total

-19.4 -50.0 + 24.2 -

61.0 33.3 82.1 52.1

- 45.0 - 44.9 -100.0 - 50.8 - 62.7 - 57.4 - 38.6 - 56.8 - 28.7 + -

33.6 1.7 57.7 89.8

-55.77

Percent Change Lamb

- 26.7

- 100.0 +

65.2

- 10.5

- 56.7 - 52.3 - 53.1

- 72.8 - 47.6 _

-

28.9 18.9 59.1 35.4 33.0 45.6 15.7 39.5 31.6 46.8 26.2 36.3 47.8

- 37.3

Pork - 34.3 - 50.0 +320.0 - 67.2 - 6.3

-

82.0 39.0 62.5 42.5 33.2 57.8 - 33.3 + 44.9 - 19.4 - 40.5 - 33.9 - 23.5 -

4.9 22.0 - 14.1 -100.0

+

- 19.8

(-) decrease in value (+) increase in value From reference (9)

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

10.

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Nonvolatile Nitrogen and Sulfur Compounds in Red Meats 163

amino compounds present I n both, raw and cooked beef. The increase i n f r e e amino compounds content during cooking i s important f o r meat f l a v o r development due t o t h e i r p a r t i c i p a t i o n i n the browning r e a c t i o n . The r e c ­ ommended usage o f c a r n o s i n e i n soup p r e p a r a t i o n and c r e a t i n e i n meat e x t r a c t s a r e due t o t h e i r c o n t r i b u t i o n the m o u t h f e e l . anserine i s d e s i r e d as i t s f l a v o r l i n ­ gers i n the mouth and was therefore savored more s t r o n g l y than would be expected simply from a consider­ a t i o n of t a s t e i n t e n s i t y as measured by t h r e s h o l d d i l u t i o n technique (JO). A r g i n i n e , l y s i n e and h i s t I d i n e exert the same e f f e c t as the f l a v o r sensation clung t e n a c i o u s l y t o the mouth. The odor and t a s t e of cooked beef were improved by c o n t r a c t i n g raw beef w i t h b a s i c amino a c i d s . Smearin beef w i t h a r g i n i n befor broil­ ing or r o a s t i n g , r e s u l t e e v a l u a t i o n (31)· Seventeen amino a c i d s and ten peptides were sub­ jected t o p y r o l y s i s (Barber Coleman u n i t Model 4180) and the products were i d e n t i f i e d by mass spectrometry, Table I X (32). B e n z e n e , t o l u e n e , e t h y l benzene, and s t y r e n e were i d e n t i f i e d i n the p r o d u c t s o f the t h e r m a l d e g r a d a d a t l o n o f p h e n y l a l a n i n e ( 3 3 , 3 ^ ) . These r e p o r t s were r e s u l t s o f work done a t v e r y higK temperatures above 700° C . , which i s f a r h i g h e r than the temperature of the oven used i n c o o k i n g . U s u a l l y , the oven used f o r r o a s t i n g i s set between 1 7 6 ° and 1 9 0 ° C . D u r i n g b r o i l ­ ing, the temperature at the s u r f a c e o f the meat might r e a c h a degree as h i g h as 280~300°C. depending upon i t s d i s t a n c e from the s o u r c e o f h e a t . T h u s , my d i s c u s s i o n w i l l be l i m i t e d t o r e p o r t s i n the l i t e r a t u r e where the temperature d i d not exceed 300* C .

c - P y r o l y s i s of S u l f u r C o n t a i n i n g Amino A c i d s . P y r o l y s i s o f c y s t e i n e and c y s t i n e r e s u l t e d i n 7-8 v o l a ­ t i l e compounds i n c l u d i n g 2 - m e t h y l t h l a z o l o d l n e which i s c o n s i d e r e d to be a a p r o d u c t o f the r e a c t i o n of a c e t a l dehyde and m e r o a p t e t h y l a m i n e ( 3 5 ) · E l e v e n compounds were i d e n t i f i e d i n the p r o d u c t s o f m e t h i o n i n e p y r o l y s i s . B e s i d e these v o l a t i l e compounds, a l a n i n e , c y s t i n e and 1 s o l e u c ine ( n o n - v o l a t i l e s);and a l a n l n e , i s ο l e u c i n e , and m e t h i o n i n e were d e t e c t e d i n the p y r o l y z e d p r o d u c t s o f c y s t e i n e and c y s t i n e , r e s p e c t i v e l y , but no amino a c i d s were found among m e t h i o n i n e p r o d u c t s . The m i x t u r e o f the seven i d e n t i f i e d v o l a t i l e compounds produced from c y s t i n e d e v e l o p e d a p o p - c o r n l i k e aroma w i t h a r o a s t e d s e s a m e - l i k e o n e . M e t h y l mercaptan seemed to be the main c o n t r i b u t o r t o a p i c k l e d r a d i s h odor produced from the p y r o l y s i s of m e t h i o n i n e . In 1973, Kato et a l . ( 3 6 ) i d e n ­ t i f i e d f i f t e e n new v o l a t i l e compounds i n the p y r o l y s i s

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

PHENOLIC, SULFUR, AND NITROGEN COMPOUNDS IN FOOD FLAVORS

164

Table IX.

Pyrolysis Products of Amino Acids and Dipeptides

Product

Compound Glycine Alanine B-alanine Valine Norvaline Leucine Isoleucine Serine Threonine Taurine Methionine Cystine Phenylalanine Tyrosine Tryptophan Proline Hydroxyproline Glycyl-glycine Glycyl-valine Glycyl-proline Glycyl-methionine Glycyl-serine Glycyl-tryptophan Glycyl-alanine Alanyl-glycine Glycyl-leucine Leucyl-glycine

Fran reference

Acetone Acetaldehyde Acetic Acid 2-Methyl propanal n-But anal 3-Methyl butanal Pyrazine 2-Ethylethyleneimine Thiophene Methyl propyl sulfide Methyl thiophene Benzene Toluene Ammonia, Carbon dioxide Pyrrole N-Methyl pyrrole Acetone Acetone, 2-Methyl propanal Acetone, Pyrrole Acetone, Methyl propyl sulfide Acetone, Pyrazine Acetone, Anmonia Acetone, 2-Methyl pyrrole Acetone, Acetaldehyde, anmonia Acetone, Cyclopentane Acetone, Acetic Acid

(32)

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

10.

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Nonvolatile Nitrogen and Sulfur Compounds in Red Meats 165

p r o d u c t s o f c y s t e i n e and f i v e more from c y s t i n e * The p o p c o r n - l i k e aroma most p r o b a b l y i s made up from t h e aroma o f many compounds such as hydrogen s u l f i d e , t h i a z o l e s , p y r i d i n e s and 2 - m e t h y l t h i a z o l i d i n e . T h i a z o l e s and r e l a t e d compounds g i v e a p y r i d i n e - o r p i c o l i n e - l i k e o d o r . û c - P i o o l i n e g i v e s a m i l d p y r i d i n e - l i k e aroma and resembles p o p c o r n - l i k e one produced from p y r o l y z e d s u l f u r c o n t a i n i n g amino a c i d s (35)· Thiophenes were d e t e c t e d i n the v o l a t i l e s o f p y r o l y z e d c y s t e i n e , b u t were not detected i n those of p y r o l y z e d c y s t i n e , t h e y g e n e r a l l y g i v e s l i g h t l y u n p l e a s a n t and c h a r a c t e r i s t i c odors* The d i f f e r e n c e s i n odor between c y s t e i n e and c y s t i n e may be p a r t l y a t t r i b u t e d t o t h e p r e s e n c e o f t h i o p h e n e s I n the v o l a t i l e s * T a b l e X l i s t s t h e compounds i d e n t i f i e d i n p y r o l y s i s p r o d u c t s o f s u l f u r - c o n t a i n i n g amino a c i d s . T h i a z o l e s and t h l a z o l l n e t i o n o f 2 - m e t h y l t h l a z o l l d l n e ; and c ^ - p l c o l i n e and 2 e t h y l - 5 - m e t h y l p y r i d i n e may produced by t h e r e a c t i o n o f a c e t a l d e h y d e and ammonia. 2 - M e t h y l t h l a z o l l d l n e I s thought t o be produced from cystearnine and a c e t a l d e h y d e (37)· I t i s q u i t e obvious t h a t s i m p l e compounds such as ammonia, a c e t a l d e h y d e , cysteamine appear t o be import a n t p r e c u r s o r s o f many v o l a t i l e compounds. d - P y r o l y s i s o f A r o m a t i c Amino A c i d s . Amine-like, p h e n o l - l i k e and i n d o l e - l i k e odors developed from p y r o l y z e d p h e n y l a l a n i n e , t y r o s i n e and t r y p t o p h a n , r e s p e c t i v e l y ( 3 8 ) . Twelve compounds, many o f which have a r o m a t i c r i n g s , were i d e n t i f i e d i n the v o l a t i l e s from t h e r m a l d e g r a d a t i o n o f p h e n y l a l a n l n e ( T a b l e X I ) . T y r o s i n e and t r y p t o p h a n produced some p h e n o l s and I n d o l e s , r e s p e c t i v e l y , a l o n g w i t h s e v e r a l o t h e r compounds. e- P y r o l y s i s o f Hydroxy Amino Compounds. Ten v o l a t i l e compounds i n c l u d i n g s e v e r a l p y r a z i n e s were i d e n t i f i e d i n the p y r o l y s i s products of L - s e r i n e ( 3 9 ) . P y r a z i n e s were a l s o i d e n t i f i e d i n t h e p y r o l y s i s p r o d u c t s o f L - t h r e o n i n e , b u t n o t from a l a n i n e and a r e c o n s i d e r e d t o be c h a r a c t e r i s t i c p y r o l y s i s p r o d u c t s o f ^ - h y d r o x y amino compounds. A l s o , d i k e t o p l p e r a z i n e s . amines and c a r b o n y l compounds were i d e n t 1 f i e d , T a b l e X I I . P y r a z i n e s a r e h e t e r o c y c l i c n i t r o g e n compounds which c o n t r i b u t e s i g n i f i c a n t l y t o t h e d e s i r a b l e unique f l a v o r and odor a s s o c i a t e d w i t h r o a s t i n g o r t o a s t i n g o f f o o d s . The odor o f p y r a z i n e s has been d e s c r i b e d as c h a r a c t e r i s t i c a l l y e a r t h y , n u t t y and r o a s t e d . Maga and Sizer(40) r e v i e w e d p y r a z i n e s i n f o o d s . P y r a z i n e s c o u l d n o t be f o u n d when g l y c i n e , a l a n i n e , p h e n y l a l a n i n e , β - a l a n i n e , l e u c i n e , i s o l e u c i n e , v a l i n e , methionine, c y s t i n e , hydro­ xy l y s i n e , t y r o s i n e , h i s t i d i n e , p r o l i n e , h y d r o x y p r o l i n e ,

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

166

PHENOLIC, SULFUR, A N D NITROGEN COMPOUNDS I N FOOD FLAVORS

Table Χ.

Pyrolysis Products of Sulfur-Containing Amino Acids

Compounds

Cystine

Cysteine

Ethylamine Propylamine Allylamine Crο ty lamine α-Alanine Isoleucine 2-Amino-4-methylthiobutyri 2- Methylthiazolidine Mercaptehylamine Hydrogen Sulfide Sulfur 3- Methylthiopropylamine Methional Acetaldehyde Propionaldehyde Isobutyladehyde Acetone Ammonia Ammonium carbonate 2-Methyl thiazoline a-Picoline 2 - Ethyl - 5-methylpyridine 2-Ethylthiazole Thiophene 2- Methyl thiophene 3- Methyltetrahydrothiophene 2,5-Dimethyl thiophene 2,3-Dimethyl thiophene 2 (or 3)-Ethylthiophene 2.3- Dihydro-4(or 5) ethylthiophene 2-Methyl-3(or 4)-ethylthiophene 2,3,5-Trimethylthiophene 3 - Methyl - η - propy 1 thiophene 2.4- Dimethyl-5-ethylthiophene 2-Methylthiazole 2-Methyl-5-ethyl thiazole

35 35 35

35 35 -

35,36 35 35 35 -

35,36 35 35

35 35 36 36 36 36 36

35 35 36 36 36 36 36 36 36 36 36 36 36 36 36 36 36

-

Methionine 35 35 35 35 35

35 35 35 35 35

From reference (35) and (36)

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

10. MABROUK

Nonvolatile Nitrogen and Sulfur Compounds in Red Meats 167

Table XI. Volatile Compounds Produced From The Pyrolysis of Aromatic Amino Acids

Volatile Compounds Benzylamine Bibenzyl p-Cresol m-Cresol Ethyl Benzene 3-Ethylindole Indole Phenol 3-Phenyl ethylamine 3-Phenylpropionitrile Skatole Stilbene Toluene Vinylbenzene Phenylacetaldehyde Acetaldehyde Benzaldehyde Ammonia Methylamine Aniline Tyramine Ethylamine

Phenylalanine

Tyrosine

Tryptophan

+ + + +

+ + +

+ +

+ +

+

+

+ +

+ + + + + +

+ +

+

+ +

From reference (38)

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

39,41 39,41 41

Pyrazine 2-Methylpyrazine 2,3-Dimethylpyrazine 2,5-Dimethylpyrazine Trimethylpyrazine 2-Ethylpyrazine 2-Ethyl-5-methylpyrazine 2 - Ethyl - 6 -methy lpyraz ine 2,6 - Die thy lpyraz ine 2.5- Dimethyl- 3-ethylpyrazine 2.6- Diethyl- 3-methylpyrazine Pyrazine, mol wt 136 Pyrazine, mol wt 136 pyrazine, mol wt 150 Pyrazine, mol wt 150 Pyrazine, mol wt 164 Pyrazine, mol wt 178 4-methyl-n-propylpyrazine 2,5-diketo-3,6-dimethylpiperazine Pyrrole 39 39

41 41 41

39,41 39,41 41 39

39,41

Serine

39

39

41

41 41 41 41 41 41 41 41

41

41

41 41 41

41 41 41

Glucosamine

41 41

Ethanolamine

39,41

39 39,41

41

Threonine

41

41

4-Amino-3hydroxybutyric

V o l a t i l e Compounds Produced From Hydroxy Amino Compounds

Compound

Table XII.

41 41 41

41 41

41

41 41

Alanylserine

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

(41).

39 39 39 39 39 39 39 39 39

2-Methylpyrrole Dime thylpyrrole 3-Methyl- 4- ethylpyrrole Ethylamine Ethanolamine Ammonia Acetaldehyde Propionaldehyde Paraldehyde

From references (39} and

Serine

39 39

39

39

Threonine

Ethanolamine

Glucosamine

4-Amino-3hydroxybutyric Alanylserine

V o l a t i l e Compounds Produced from Hydroxy Amino Compounds (cont'd)

Compound

Table XII.

170

P H E N O L I C ,

S U L F U R ,

A N D

N I T R O G E N

C O M P O U N D S

I N

F O O D

F L A V O R S

tryptophan, l y s i n e , a s p a r t i c , a s p a r a g i n e , glutamic, adenine, and adenosine were i n d i v i d u a l l y to p y r o l y s i s * On the other hand, pyrazines were obtained by heating i n d i v i d u a l amino-hydroxy compounds, notably those having amino and hydroxy groups i n adjacent c a r b o n p o s i t i o n s without the p a r t i c i p a t i o n of s u g a r s . I t i s obvious that the amino-hydroxy compounds themselves serve as a s o u r c e of b o t h carbon and n i t r o g e n i n the pyrazine molecule. Thus,these compounds glutamlne, subjected

are

important p r e c u r s o r s i n f o o d s , e . g . ,

ethanolamlne,

glucosamine, s e r i n e , threonine, 4 - a m i n o - 3 - h y d r o x y b u t y r i e a c i d , and a l a n y l s e r l n e . The composition of pyraz i n e mixtures and the r e l a t i v e amounts of each pyrazine p r o d u c e d v a r i e d from one amino-hydroxy compound t o a n o t h e r * F o r example, p y r a z i n e 2-methyl p y r a z i n e , 2 , 5 - d i -

methyl p y r a z i n e , 2 , 3 - d l m e t h y p y r a z l n e w i t h ( 2 - m e t h y l p y r a z i n e f o r the l a r g e s t peak) r e s u l t e d from g l u c o s a m i n e ; w h i l e t r i m e t h y l - , 2 , 5 - d i m e t h y l - , and 2 , 5 - d i m e t h y l - 3 - e t h y l p y r a z i n e ( w l t h 2 , 5 - d i m e t h y l p y r a z i n e f o r the l a r g e s t peak were o b t a i n e d from 4 - a m i n o - 3 - h y d r o x y - b u t y r l c a c i d (hi). Dawes and Edwards (42) s p e c u l a t e d on the mechanism o f f o r m a t i o n o f s u b s t i t u t e d p y r a z i n e s i n s u g a r - a m i n e systems.

f - Nonenzymlo Browning R e a c t i o n . Reactions i n duced by h e a t i n g amino a c i d s and sugars a r e known as nonenzymic browning o r M a i l l a r d r e a c t i o n s . Meat f l a v o r s a r e a l s o g e n e r a t e d i n r e a c t i o n s o f t h i s t y p e . T h i s had been the s u b j e c t o f s e v e r a l r e v i e w s (43, 44, 4 5 , 46). The M a i l l a r d r e a c t i o n i s the r e a c t ! o n ^ b e t w e e n an amino compound (amine, amino a c i d , p e p t i d e , or a p r o t e i n ) and a g l u c o s i d i c hydroxy group i n a sugar · Hodge (44) p r o posed a s e v e n - s t e p mechanism: 1 - Sugar-amino a c i d c o n d e n s a t i o n ( f o r m a t i o n o f N - s u b s t i tuted glycosylamine ). 2- Amadori rearrangement ( rearrangement t o produce a substituted l-amino-l-deoxy-2-ketose)· 3 - Sugar d e h y d r a t i o n . 4- Sugar f r a g m e n t a t i o n . 5 - Amino a c i d d e g r a d a t i o n . 6- A l d o l c o n d e n s a t i o n . ?- A l d e h y d e - a m i n e p o l y m e r i z a t i o n . P r o d u c t s of the M a i l l a r d r e a c t i o n i n c l u d e a l i p h a t i c a l d e h y d e s , f u r f u r a l , f u r f u r a l d e r i v a t i v e s , k e t o n e , and 1 , 2 - d l c a r b o n y l compounds. The a l i p h a t i c a l d e h y d e s a r e p r o d u c e d by the o x i d a t i v e d e g r a d a t i o n o f the amino acide (known as S t r e c k e r d e g r a d a t i o n ) . The r e s u l t i n g a l d e h d e s c o n t a i n one c a r b o n atom l e s s t h a n t h e i n i t i a l amino a c i d . The S t r e o k e r d e g r a d a t i o n can o c c u r by r e a c t i o n o f amino a c i d s e i t h e r w i t h Amadori rearrangement p r o d u c t s

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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Nonvolatile Nitrogen and Sulfur Compounds in Red Meats 171

o r w i t h d l c a r b o n y l compounds p r e s e n t i n the r e a c t i o n m i x t u r e * F u r f u r a l and i t s d e r i v a t i v e s a r e produced by

dehydration of the amadori rearrangement products, and they can p a r t i c i p a t e i n the S t r e o k e r degradation. There i s no d e f i n i t e r e l a t i o s h i p between the odor produced and the temperature t o which v a r i o u s i n d i v i d u a l amino a c i d s were h e a t e d w i t h g l u c o s e ( 4 7 ) · For example , w i t h l e u c i n e and threonine the odor was"pleasant at 100? but u n p l e a s a n t a t 1 8 0 ° C . With h l s t i d l n e an odor a p p e a r ­ ed a t 1 8 0 ° C . I r r e s p e c t i v e of the temperature, g l y c i n e , c i - a l a n l n e , / ^ - a l a n i n e and glutamic a c i d produced only the odor of b u r n t sugar when h e a t e d w i t h g l u c o s e ; w h i l e o

tryptophan, tyrosine, a r g l n i n i n e , and«-amlnobutyric a c i d produced t h e b u r n t sugar odor o n l y a t 1 8 0 ° C T a b l e X I I I l i s t s the v o l a t i l e compounds produced by the r e a c t i o n o f c y s t e i n and p y r u v a l d e h y d e (48) , t h e r e a r e not too many d i f f e r e n c e s between the aroma produced by t h e r e a c t i o n o f p y r u v a l d e h y d e and g l u c o s e • P y r a z i n e s had been i d e n t i ­ f i e d as the v o l a t i l e s c o n t r i b u t i n g to the f l a v o r s p r o d ­ uced b y the sugar-amino a c i d r e a c t i o n . 2 , 5 - D l m e t h y l - , and 2 , 5 - d i m e t h y l - 3 - e t h y l p y r a z l n e were d e t e c t e d i n the v o l a t i l e s r e s u l t i n g from h e a t i n g c y s t e i n e and c y s t i n e w i t h p y r u v a l d e h y d e . Trime t h y I p y r a ζ i n e was found o n l y i n the p r e s e n c e o f c y s t e i n e . W h i l e m e t h y l - , 2 , 5 - d i m e t h y l - , 2- m e t h y l - 3 - e t h y l - , 2 - m e t h y l - 6 - e t h y l - , and 2 , 5 - d l m e t h y l 3- e t h y l p y r a ζ ine were p r o d u c t s o f h e a t i n g g l u c o s e w i t h c y s t e i n e ; m e t h y l p y r a z i n e was the o n l y p y r a z i n e p r e s e n t in the v o l a t i l e s r e s u l t i n g from a m i x t u r e o f g l u c o s e and c y s t i n e . S e v e r a l t h l o p h e n e s were d e t e c t e d i n t h e v o l a t i l e s p r o d u c e d b y the r e a o t i o n o f c y s t e i n e w i t h p y r u v a l d e h y d e except 2 - t h l o p h e n o l c a d d w h i c h e x i s t e d o n l y i n the p r e s e n c e o f c y s t i n e . Thiophene and h y d r o x y t h i o p h e n e were found i n the v o l a t i l e s p r o d u c e d from heat­ ed g l u c o s e - o y s t e i n e . T h l o p h e n e s g e n e r a l l y g i v e a l i t t l e u n p l e a s a n t and c h a r a c t e r i s t i c o d o r . T h i a z o l e s were p r o d ­ uced from s u l f u r - c o n t a l n i n g amino a c i d s i n the p r e s e n c e of e i t h e r g l u c o s e o r p y r u v a l d e h y d e , b u t t h e i r s p e c i e s were d i f f e r e n t . #

g- Peptides. I n f l a v o r c h e m i s t r y , p e p t i d e s have been s t u d i e d as components w h i c h c o n t r i b u t e a b i t t e r , s o u r and sweet t a s t e . R e p o r t s on p e p t i d e s as o o n t r t r i b u t o r s t o d i s c o l o r a t i o n and aroma o f f o o d s was seldom r e p o r t e d . R e l a t i n g t o the p r o d u c t i o n o f f o o d f l a v o r and nonenzymic b r o w n i n g , s t u d i e s were c o n c e n t r a t e d on amino a c i d s , amines and p r o t e i n s . The o r d e r o f the browning r a t e o f amino compounds i s : t e t r a g l y c i n e > t r i g l y c i n e > diglycine^> DL-alany 1-DL-alanine]> g l y c i n e > D L - a l a n i n e , and the r e a c t i v i t y o f p e p t i d e i s much h i g h e r t h a n t h a t

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

172

PHENOLIC, SULFUR, AND NITROGEN COMPOUNDS I N FOOD FLAVORS

Table XIII.

Volatile Compounds Produced by the Reaction of Sulfur-Containing Amino Acids with Glucose or Pyruvaldehyde.

Volatile Compounds

Glucose and Cysteine Cystine

Thiophene Hydroxy thiophene 2- Methylthiophene 3- Methyl Thiophene 2,5-Dimethyl thiophene 2.3- Dihydro-4(or 5)-ethylthiophene 2 (or 3-)Ehtylthiophene 2- Methyl-3(or 4-)ethyl Thiophene 2,3,5-Trimethy1thiophene 3- Methyl-n-propylthiophene 2.4- Dimethyl-5-ethylthiophene 2-Thiophenoic acid 2-Methyltetrahydrothiopehen-3-one Thiazole 2-Ethylthiazole 2-Methylthiazole 5 (or 4)-Methyl thiazole 5 (or 4)-Ethylthiazole Trimethyl thiazole 2-Methylthiazoline 2-Acetyl-4-methylthiazole Pyridine α-Picoline β-Picoline 2-Methyl-5-ethylpyridine Methylpyrazine 2.5- Dimethylpyrazine 2-Methyl-6-ethylpyrazine 2-Methyl-3-ethylpyrazine 2,5-Dimethyl- 3-ethylpyrazine Trimethylpyrazine Furfural 2-Methyl-5-ethylfuran 2-Acetylfuran 2-Acetyl- 5-methylfuran Furfural alcohol 5-Methylfurfural 2,5-Dimethyl-3-ethylfuran 2-Furoic acid Phenol p-Nfethylbenzoic Acid

X X -

Pyruvaldehyde § Cysteine Cystine

-

X

-

-

X X

-

-

-

-

X X

X X X X X -

X X -

_ X X Χ X X X χ X X -

X

X

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X

X

-

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-

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X -

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X X X X

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

10. MABROUK Table XIII.

Nonvolatile Nitrogen and Sulfur Compounds in Red Meats 173 V o l a t i l e Compounds Produced by the Reaction of Sulfur-Containing Amino Acids with Glucose or Pyruvaldehyde. (cont'd)

V o l a t i l e Compounds Ethyl Alcohol 2,4 (or 5) Dimethyl thiazole 2-Methyl-4(or 5) ethylthiazole 2-Ethylthiazoline Benzoic Acid p-Methylbenzoic Acid Acetic Acid 2-Methylthiazoline Furfuryl alcohol Benzene

Glucose and Cysteine Cystine X Χ -

X X -

Pyruvaldehyde ξ Cysteine Cystine

X

-

-

X

X Χ Χ -

X X X

X -

X

-

From reference (48)

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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ducts r e s u l t i n g from the r e a c t i o n o f amino a c i d s w i t h o a r b o n y l compounds and by the p y r o l y s i s o f some amino a o i d s were considered t o be s t a b l e , e s p e c i a l l y when i t s s u b s t i t u e n t s are a l k y l groups* Pyrazines w i t h s u b s t i ­ t u e n t s o t h e r t h a n a l k y l group such as i n the c a s e of d i h y d r o p y r a ζ i n e was shown t o be r e a c t i v e * The a d d i t i o n

of pyrazinone to d i p e p t i d e - g l y o x o l s o l u t i o n Increased the browning w h i l e amino a c i d - p y r a z i n o n e o r g l y o x a l p y r a z i n o n e s o l u t i o n caused no browning ( 4 9 ) » One c a n assume t h a t p y r a z i n o n a c t i o n , and i t probably r e a c t s w i t h n e i t h e r the amino a c i d n o r w i t h g l y o x a l , b u t w i t h the p r o d u c t produced i n the r e a c t i o n . The e x p e r i m e n t a l r e s u l t s i n d i c a t e d t h a t 2 - ( 3 - m e t h y l - 2 - o x o - p y r a z l n - l - y l ) p r o p l o n i c a c i d was more a c t i v e i n browning t h a n 2-(2 -oxopyrazln-1 -yl) i s o c a p r o l c a c i d , t h e s u b s t i t u e n t may be c o n s i d e r e d as a causative factor. I n a study on l i p i d browning,Dugan and Rao (50) r e p o r t e d t h a t the r e a c t i o n s p r o c e e d r e a d i l y at low mois­ t u r e l e v e l s ( 2 . 5 $ ) and ambient o r e l e v a t e d t e m p e r a t u r e s . The r e a c t i o n between p h o s p h a t i d y l e tha n o l a m i n e ( P E ) and n o n a n a l ( T a b l e XV) on p r o t e i n m a t r i x ( l l p l d - f r e e b e e f muscle f i b e r s ) i n d i c a t e d t h a t the c a r b o n y l r e a c t i o n s prooeeded w i t h b o t h amino groups from PE and from the p r o t e i n m a t r i x . C e r t a i n amino a c i d s , such a s , l y s i n e , a l a n i n e , p h e n y l a l a n i n e , and t y r o s i n e r e a c t e d w i t h a l ­ dehydes (nonanals o r o x i d a t i o n p r o d u c t s o f u n s a t u r a t e d Ρ Ε ) , s u f f i c i e n t l y t o reduce t h e i r q u a n t i t y i n the t o t a l amino a c i d c o n t e n t i n the s y s t e m . The r e a c t i o n s were c o m p e t i t i v e t h a t the p r e s e n c e o f PE had a s p a r i n g e f ­ f e c t on amino a c i d s i n the p r o t e i n m a t r i x . T h i s o b s e r ­ v a t i o n may p r o v i d e a r a t i o n a l e f o r the changes I n t e x ­ t u r e , c o l o r , f l a v o r , and n u t r i t i v e v a l u e o f d r i e d f o o d s c o n t a i n i n g p r o t e i n s and p h o s p h o l i p i d s . ,

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In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

MABROUK

Nonvolatile Nitrogen and Sulfur Compounds in Red Meats 175

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10.

MABROUK

Nonvolatile Nitrogen and Sulfur Compounds in Red Meats 177

ana 3-meroaptothiophenes i n v a r i o u s degrees of s a t u r â t i o n ( 5 2 ) . T h e t h i o p h e n e analogs of the h y d r o x y f u r e n o n e s s t a r t i n g m a t e r i a l s were a l s o formed i n t h e s e r e a c t i o n s . These a n a l o g s i n t u r n a f f o r d e d s t i l l more m e r o a p t o t h i o phenes and m e r c a p t o t h i o p h e n o n e s . T h e r e a c t i o n systems and many o f p u r i f i e d i s o l a t e d compounds had m e a t - l i k e o d o r s . As hydroxyfuranones and hydrogen s u l f i d e had been r e p o r t e d i n b o i l e d b e e f ( 5 1 ) , i t i s expected t h a t t h e i r r e a c t i o n p r o d u c t s a r e a l s o p r e s e n t , though i n conc e n t r a t i o n s u n d e t e c t e d by the a v a i l a b l e i n s t r u m e n t a l techniques. Taste

of F l a v o r

Precursors

The o v e r a l l flavor" s e n s a t i o n may c o n v e n i e n t l y be d i vided into sensatio case o f cooked meats, the c o n t r i b u t i o n o f a r o m a t i c compounds would be l a r g e i n comparison w i t h t h a t due t o the n o n - v o l a t i l e t a s t e - b e a r i n g components. A l l D-amino a c i d s have sweet t a s t e . W i t h the e x c e p t i o n o f L - p h e n y l a l a n l n e which i s b i t t e r , a n d 1 - a l a n i n e w h i c h i s s l i g h t l y sweet, a l l L-amino a c i d s a r e t a s t e l e s s * F r e e L - g l u t a m l c a c i d has a w e l l known b r o t h y t a s t e . Some o f the L - g l u t amyl d i p e p t i d e s formed from c o u p l i n g a n amino a c i d t o the ( X - o a r b o x y l group o f L - g l u t a m i c a c i d were found t o t a s t e b r o t h y : glutamyla spart1ο , glutamylthreonine , g l u t a m y l s e r i n e , and g l u t a m y l g l u t a m i c (53)· G l u t a m y 1 g l y c y l s e r i n e i s r e s p o n s i b l e f o r the b r o ï B y t a s t e o f a n e n z y m a t l o a l l y m o d i f i e d soybean p r o t e i n . About t h i r t y a c i d i c o l i g o p e p t i d e s were i s o l a t e d and I d e n t i f i e d i n a f l a v o r p o t e n t i a t i n g f r a c t i o n from f i s h p r o t e i n h y d r o l y s a t e (5*0 · F o u r d i p e p t i d e s ( g l u t a m y l a spar11c, g l u t a my I g l u t a m l o , g l u t a m y l s e r i n e , and t h r e o n y l g l u t a m i c ) , and five tripeptldes (aspartylglutamylserine,glutamylaspart y l g l u t a m i c , glutamylglutanrineglutamic, g l u t a m y l g l y c y l s e r i n e , and s e r y l g l u t a m y l g l u t a m i c ) had a f l a v o r q u a n t i t a t i v e l y r e s e m b l i n g t h a t o f monosodium glutamate(MSG). The t h r e s h o l d l e v e l o f g l u t a m y I g l y c y l s e r i n e , f o r examp l e , was e s t i m a t e d t o be a p p r o x i m a t e l y 0.2$ i n water a t pH 5.0 , whereas t h a t o f MSG was almost one t e n t h o f t h i s l e v e l under the same c o n d i t i o n s . Some i s o l a t e d p e p t i d e s were r e p o r t e d t o e x h i b i t b i t t e r taste(glutamylaspartylvaline, aspartylleucine. isoleucylglutamylg l u t a m i c , and i s o l e u o y l g l u t a m i c ) w h i l e o t h e r s had a f l a t t a s t e . A strong s y n e r g i s t i c r e l a t i o n s h i p exists between n u c l e o t i d e s and MSG w i t h the r e s u l t t h a t b l e n d s of MSG and n u c l e o t i d e s exceed the p o t e n c y and v e r s a t i l i t y o f e i t h e r m a t e r i a l a l o n e . T h e optimum b l e n d w h i c h o f f e r s the g r e a t e s t e f f e c t i v e n e s s f o r t h e l e a s t c o s t i s a b l e n d o f 95% MSG and 5% n u c l e o t i d e s . D e s i r a b l e f l a tt

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

178

PHENOLIC, SULFUR, AND NITROGEN COMPOUNDS IN FOOD FLAVORS

vors were enhanced by a d d i t i o n of sodium lnosinate r e gardless of the type of the meat or cooking method (5§)« Disodium lnosinate oonsistently produced an Impression of greater v i s c o s i t y and Increased f l a v o r . Amino acids play an Important part In the p a l a t a b l e taste of foods* When glycine was added to a 1# NaCl s o l u t i o n containing L-glutamlo or L-aspartlo and IMP plus GMP at 0.1# , the palatable taste of the medium was s i g n i f i c a n t l y Improved. The ternary synergism among L-glutamic or L-aspartic,5'-nucleotides and glycine Is d i f f e r e n t from the binary synergism already known between L-glutamlo a d d and 5 ' - n u c l e o t i d e s (56). The t e r nary synergism i s s i g n i f i c a n t at the concentration of the stimulus threshold of these components. The ternary synergism wa t du t th t tast f glyolne. No binary synergis served between glyolne and L-glutamic or L-aspartlo, or g l y c i n e and 5'-nucleotides.Extending the study to i n clude other o(-amlno a d d s , Indicated the absemoe of any binary synergism of palatable taste between IMP plus GMP or MSG and glyolne, L-alanine, L-serine, L - h l s t l dine-HCl, L-methlonine, or DL-tryptophan In 1$ NaCl sol u t i o n i s ? ) •However, L - a l a n l n e ( 0 . 0 5 ) , L - e y s t i n e ( 0 . 0 5 ) , Çlyclne(O.lO), L-histldlne-HCl(0.004), ^methionine 1 0 . 0 3 ) . L - p r o l l n e ( 0 . 2 0 ) , L-serIne(0.10), DL-tryptophan (0*015), or L-valine(0.15g./dl.) gave a ternary synergism with IMP plus GMP and MSG(0.01#, respectively) In 1# NaCl s o l u t i o n . When two or three of t h i s group of amino aoids were added to 1# NaCl s o l u t i o n containing MSG and IMP plus GMP at 0.01JÉ, the ternary synergism caused by these amino acids was estimated as the algeb r a i c sum of the a c t i v i t y of the Individual amino acids ( 5 8 ) ·

PfcaymaoolgKlogl E f f e c t ? Bed meat f l a v o r precursors have w e l l known functions In human n u t r i t i o n . The evidence of t h e i r pharmacological properties has been known f o r many decades before t h e i r contributions to f l a v o r were established. Suoh propert i e s of amino a c i d s , peptides, sugars,nucleotides..etc have been reviewed In human physiology ,human biochemi s t r y and human n u t r i t i o n books and reviews (££,60). Meat extract, which Is a concentrate of aqueous beef extract, Is olalmed to be a stimulant. Products containing meat e x t r a c t , have been regarded as general t o n i c s and stimulants, a s s i s t i n g recovery from exhaust i o n and f a t i g u e . Preoise information about these e f f e c t s are lacking. I t Is, of course possible that the well-known promotion of g a s t r i c and I n t e s t i n a l seore-

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

10. MABROUK

Nonvolatile Nitrogen and Sulfur Compounds in Red Meats 179

tion and motility might cause secondary,more generalized effects and result In a real or apparent stimulation. Injecting creatine i n frogs before setting them to work on a treadmill, proved to be responsible for Improving muscular performance and hastened recovery after work(61). In his classical fistulas experiments with dogs,. Pavlov (62) had Indicated that meat extract was a powerful stimulant of gastric secretion. He evaluated some of meat extract components and concluded that creatIne,hypoxanthlne, xanthine,leucine and a mixture of inoslne and hypoxanthlne were Ineffective* Later, Krimberg and Komarov (63,64,6j>) and Korohow (66) demonstrated that carnosine at a concentration of 0*02 g./kg was the compound responsible for the gastric stimulating properties* Using a dose of 0*005 g./kg Schwarz and Goldschmld retion following th dogs with gastric f l s t u l a s ( 6 £ ) · Evaluating pure samples of carnosine, carnitine and methyl guanidlne for their effect as stimulant of gastric secretion, proved that carnosine was the most powerful one. Intravenous injection of carnosine was most effective than subcutaneous and oral administration. The principal funotlon of anserine and carnosine Involves the coupling of phosphorylation with glycolysis and the synthesis of adenosine triphosphate. Carnitine which had been reported in meat extracts is Identical with vitamin % ( 6 8 ) . Methionine has been reported to be beneficial In the prevention and treatment of l i v e r Injury due to poisoning by arsenic, chloroform, carbon tetraohoride, or t r i n i t r o toluene.lt has been recommended in the treatment of eclampsia, shook, infectious hepatitis and cirhosls of the l i v e r . It has been prescribed In the management of obese, and patients with severe burns(69)· "The whole f i e l d of flavor precursor chemistry is In i t s Infancy, and It is reasonable to say that no study — be i t academic or commercial — of a natural flavor can be considered oomplete unless i t includes the precursors of that flavor. An approach to any f l a vor problem which recognizes the importance of the two complementary approaches is more likely to give a complete picture of the flavor and the mechanism of Its formation than attempts to Interpret i t s chemical constitution , Rohan s statement(70) expresses my views. 11

1

ABSTRACT

Red meats e x h i b i t d i s t i n c t t a s t e and f l a v o r which are c h a r a c t e r i s t i c of t h e i r animal speies. Age of the animal, type of feed, animal c o n d i t i o n , type of meat

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

180

PHENOLIC, SULFUR, AND NITROGEN COMPOUNDS IN FOOD FLAVORS

cuts, meat processing, etc., strongly affect the taste and flavor of individual meats. Non-volatile nitrogen and sulfur compounds play an important role in the development of the characteristic flavors of red meats. Their pharmacological effect, relative importance, and their role i n the formation of flavor components were discussed. Speculations on the reasons for differences in red meat flavors that intrigue flavor chemists were reported. Literature Cited 1. Wong, Ε., Nixon, L . N . , Johnson, C . B . , J. Agr. Food Chem. (1975), 23, 495. 2. Patterson, R.L.S., J. Sci. Food Agr. (1968), 19, 31. 3. Crocker, E.C., Food Res. (1948), 13, 179. 4. Bender, A.E., Wood, T., Palgrave,J.A., J. S c i . Food Agr. (1958), 9, 812. 5. Wood, T., J. Sci. Food Agr. (1961), 12, 61. 6. Batzer,O.F., Santoro, A . T . , Landmann, W.A., J. Agr. Food Chem. (1962), 10, 94. 7. Batzer, O . F . , Santoro, A.T., Tan, M.C., Landmann, W.A., Schweigert, B . S . , J. Agr. Food Chem. (1960), 8, 498. 8. Hornstein, J., "Chemistry and Physiology of F l a ­ vors" ,Schults, H.W., Day, E . A . , Libbey, L . M . , Eds. pp. 228-250, Avi Publishing Co., Westport, Conn. 1967. 9. Macy, R . L . , Jr., Naumann, H.D., Bailey, M.E., J. Food Sci. (1964), 29, 136. 10. Macy, R . L . , Jr., Naumann, H.D., Bailey, M.E., J. Food Sci. (1964), 22, l42. 11. Wasserman, A.E., Gray, N . , J. Food Sci. (1965), 30, 801. 12. Zaika, L.L., Wasserman, A.E., Monk, C . B . , Jr., Salay, J., J. Food Sci. (1968), 33, 5 3 · 13. Mabrouk, A.F., Jarboe, J.K., O'Connor, E.M., J. Agr. Food Chem. (1969), 17, 5. 14. Mabrouk, A.F., J. Agr. Food Chem. (1973), 21, 942. 15. Mabrouk, A.F., Kramer, R . , Jarboe, J.K., Alabran, D.H., Unpublished data. 16. Paulson, J.C., Deatherage, F.E., Almy, E . P . , J. Am. Chem. Soc. (1953), 75, 2039. 17. Jarboe, J.K., Mabrouk, A.F., J. Agr. Food Chem. (1974), 22, 787. 18. Dvorak, Z., Vognarova, I., J. Sci. Food Agr. (1969), 20, l46. 19. Johnson, A.R., Viokery, J . R . , J. Sci. Food Agr. (1964), 15, 695.

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

10.

Mabrouk

Nonvolatile Nitrogen and Sulfur Compounds in Red Meats 181

20. Dahl, O., J. Agr. Food Chem. (1963), 11, 350. 21. Bendall, J.R., J. Soc. Chem. Ind. (1946), T226. 22. Nakajima, N., Ichikawa, K., Kamada, M., Fujita, Ε., J. Agr. Chem. Soc. Japan (1961), 35, 797· 23. Mabrouk, A.F., Abstracts of Papers. 86, Division of Agricultural And Food Chemistry, 160th ACS National Meeting, Chicago, I l l i n o i s , September 1970. 24. Mabrouk, A.P., Abstracts of Papers. 39, Division of Agricultural And Food Chemistry, 162nd ACS National. Meeting, Washington, D . C . , September 1971. 25 Terasaka, Μ., Takeda Scientific Information Bulle­ t i n No. 26. 26. Fujlmakl, M . , Chuyen, Ν. V . , Matsumoto. T., Kurata, T., J. Agr. Chem. Soo. Japan (1970), 44, 275. 27. Maoy, R . L . , Jr., Food S c i . (1970) 28. Maoy, E.L., Jr., Neumann, H.D., Bailey, M . E . , J. Food Sci.(1970), 35, 81. 29. Maoy, R . L . , Jr., Neumann, H.D., Bailey, M . E . , J. Food S c i . (1970), 35, 83. 30. Craske, J.D., Beuter, P . H . , J. Sci Food Agr. (1965), 16, 243. 31. Miyake, Μ., Tanaka, Α . , J.Food S c i . (1971), 36, 674. 32. Merrltt, C . , Jr., Bobertson, D . H . , J.Gas Chromatog. (1967), 4, 96. 33. Giacabbo, Η . , Simon, W., Pharm. Acta Helv. (1964), 32, 162. 34. Vollmin, J., Kriemler, P . , Omura, J., Selble, J., Simon, W., Microchem. J. (1966), 11, 73. 35. Fujimaki, M . , Kato, S., Kurata, T., Agr. Biol. Chem. (1969), 22, 1144. 36. Kato, S., Kurata, Τ . , Fujimaki, Μ., Agr. B i o l . Chem. (1973), 37, 1759. 37. Tondeur, Β . , Slon, Β . , Deray, Ε . , Bull. Soc. Chim. France (1964), 2493. 38. Kato, S., Kurata, T., Fujimaki, M . , Agr. B i o l . Chem. (1971), 21, 2106. 39. Kato, S., Kurata, T., Ishitsuka, B . , Fujimaki. Μ., Agr. B i o l . Chem. (1970), 34, 1826. 40. Maga, J.A., Sizer. C . E . , CBC C r i t i c a l Rev. Food Technol., (1973), 4, 39. 41. Wang, P . , Odell, G . V . , J. Agr. Food Chem.(1973), 21, 868. 42. Dawes, I.W., Edwards, R . A . , Chem. Ind. (London), (1966), 2203. 43. Hodge, J.E., J. Agr. Food Chem. (1953), 1, 928. 44. Hodge, J.E., "The Chemistry and Physiology of F l a ­ vors" , Sohultz, H.W., Day, Ε . Α . , Libbey, L.N., Eds., pp 465-491, Avl Publishing C o . , Westport, Conn. , 1967. In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

182

45.

PHENOLIC, SULFUR, AND NITROGEN COMPOUNDS IN FOOD FLAVORS

Reynolds, T.M., Advan. Food Res. (1965), 14, 167. 46. Reynolds, T.M., Advan Food Res. (1963), 12, 1. 47. Herz, W.J., Shalleriberger, R.S., Food Res. (1960), 25, 491. 48. K a t o , S., Kurata, T., Fujimaki, M., Agr. Biol. Chem. (1973), 37,539. 49. Chuyen,N.V., Kurata, T., Fujimaki, M., Agr. Biol. Chem. (1973), 37, 327. 50. Dugan, R.L., Jr., Rao, G.V. "Evaluation of The F l a ­ vor Contribution of Products of the Maillard Reac­ tion ". Technical Report 72-27F, ρ 110,US Army Natick Development Center, Natick, MA ,1972. 51. Tonsbeek, C . H . T . , Blancken, A.J., van de Weerdhof, T., J. Agr. Food Chem. (1968), 16,10l6. 52. van den Ouweland Brothers Co.),U.S 53. Arai, S., Yamashlta, M., Noguohi, M., Fujimaki, M., Agr. Biol.Chem. (1973), 37, 151. 54. Noguchi, Μ., A r a i , S., Yamashita, M., Kato, H., Fujimaki, M.. J.Agr. Food Chem. (1975), 23, 49. 55. Kuninaka, A.,"The Chemistry and Physiology of F l a ­ vors ", Sohultz, H.W., Day, E . A . , Llbbey, L.N., Eds., pp 515-535, Avi Publishing Co., Westport, Conn., 1967. 56. Yokotsuka, T., Saito, N . , Okuhara. Α . , Tanaka, T., Nippon Nogei Kagaku Kaishi (1969), 43, 165. 57. Tanaka, T.,Saito, Ν.,Okuhara,A.,Yokotsuka, T., Nip­ pon Nogei Kagaku Kaishi (1969), 43,171. 58. Tanaka, T., Salto, N . , Okuhara, Α . . Yokotsuka, T., Nippon, Nogei Kagaku Kasshi (1969), 43, 263. 59. Brobeck, J.R., Ed. " Best & Taylor's Physiological Basis of Medical Practice", The Williams & Wilkins Company, Baltimore,1973 60. Babkin, Β . P . "Secretory Mechanism of the Digestive Glands" 2nd Ed. ,p 1027, Paul B.Hoeber, Inc.,New York, 1950. 61. Kobart, E . H . , Arch. Exp.Path. Pharmak. (1882-3),15, 56. 62. Pavlov, I.P."The Work of the Digestive Glands",2nd English Ed. by W.H.Thompson,C.Griffin & Co.,1910. 63. Krimberg, R., Komarov, S . A . , Biochem. Z.(1926),171, 169. Thru CA,20,3313 (1926). 64. Krimberg, R., Komarov, S.A..Biochem.Z. (1927),184. 442. Thru CA,2l,2309 (1927). 65. Krimberg, R., Komarov, S . A . . Biochem. Z.(1928),194, 410. Thru CA,22,2779 (1928). 66. Korchow, A. Blochem.Z.(1927),190, 188. Thru CA,22, 1625 (1928).

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

10.

Mabrouk

Nonvolatile Nitrogen and Sulfur Compounds in Red Meats 183

67. Schwarz, C., Goldschmidt, Ε., Arch. ges. Physiol. (Pfluger's) (1924), 202, 435. Thru CA,18, 2027, (1924). 68. Carter, H.E., Bhattacharyya, P . K . , Weidman, K . R . , Fraenkel,G., Arch. Biochem. Biophys. (1952) 405. 69. Osol, A.E., Pratt, R . , Altsohule, M . D . , " The United States Dispensatory And Phyelolans' Phar­ macology",pp 715-716, J.P.Lippincott Company, Philadelphia & toronto,1972. 70. Rohan, T . A . , The Flavour Industry (1971), 2, 147.

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

11 F u r a n s S u b s t i t u t e d at t h e T h r e e P o s i t i o n w i t h S u l f u r WILLIAM J. EVERS, HOWARD H. HEINSOHN, JR., BERNARD J. MAYERS, and ANNE SANDERSON International Flavors and Fragrances, Inc., 1515 Highway 36, Union Beach, N. J. 07735

D u r i n g r e c e n t y e a r s the a n a l y s i s of cooked meats and of model systems has been r e p o r t e d by numerous investigators; leading references include Wilson et al ( 1 ) , Mussinan and K a t z ( 2 ) , Persson and von Sydow ( 3 ) , S c h u t t e ( 4 ) , and van den Ouweland and Peer ( 5 ) . We now r e p o r t the isolation, identification and s y n t h e s i s o f two important meat f l a v o r and aroma chemicals, 2 - m e t h y l - 3 - f u r a n t h i o l (1) and b i s (2-methyl-3furyl) disulfide (2).

2

1

While p r e p a r i n g s m a l l b a t c h e s (1000 g) of a p r o c e s s e d meat f l a v o r which c o n s i s t e d of a m i x t u r e of L c y s t e i n e h y d r o c h l o r i d e , thiamine c h l o r i d e h y d r o c h l o r i d e , h y d r o l y z e d v e g e t a b l e p r o t e i n and water heated at reflux for four hours (6), it was noted t h a t the c o n densate possessed an i n t e n s e r o a s t e d aroma. The material r e s p o n s i b l e f o r the aroma proved t o be e x t r a c t a b l e w i t h methylene c h l o r i d e but when the methylene c h l o r i d e was removed, o n l y a minute amount of m a t e r i a l remained. In o r d e r t o o b t a i n workable q u a n t i t i e s of aroma m a t e r i a l f o r a n a l y s i s a large scale preparation was u n d e r t a k e n . T h i s r e a c t i o n t o t a l e d 4,000 l b s . and gave 40 g a l l o n s condensate from which 16 liters of methylene c h l o r i d e e x t r a c t was o b t a i n e d . Careful s o l vent removal o f the e x t r a c t under m i l d vacuum and f i n a l c o n c e n t r â t i o n w i t h a stream of n i t r o g e n gave 184

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

11.

EVERS E T A L .

Furans Substituted

with

185

Sulfur

50 ml of c o n c e n t r a t e p o s s e s s i n g a p o w e r f u l r o a s t aroma. T h i n - l a y e r chromatography of the c o n c e n t r a t e i n ­ d i c a t e d the presence of a r e a d i l y i s o l a b l e compound near the s o l v e n t f r o n t . Repeated p r e p a r a t i v e t h i n l a y e r chromatography gave, from 2.4 g of c o n c e n t r a t e , a 66 mg sample of the d i s u l f i d e 2. The s t r u c t u r e i s a s s i g n e d on the b a s i s of mass s p e c t r a l , p r o t o n magnetic resonance and i n f r a r e d d a t a . When the c o n c e n t r a t e was s u b j e c t e d t o e x a m i n a t i o n by GLC, i t gave a s u c c e s s i o n of peaks b l e n d i n g i n t o a complex chromotogram. One p a r t i c u l a r s e c t i o n of the GLC e f f l u e n t when o r g a n o l e p t i c a l l y e v a l u a t e d had what was d e s c r i b e d as a pot r o a s t " type aroma. I s o l a t i o n of the compound or com­ pounds r e s p o n s i b l e f o r t h i s odor was u n d e r t a k e n . As a r e s u l t of r e p e a t e d t r a p p i n g and rechromatography i t became apparent t h a p r e s e n t i n v e r y minute amounts. With much t r i a l and e r r o r , a scheme f o r the i s o l a t i o n of t h i s compound was worked o u t . T h u s , from 4 ml of c o n c e n t r a t e we o b t a i n e d 2 - 3 λ of the f u r a n t h i o l 1. As i n the c a s e of the d i ­ s u l f i d e , the s t r u c t u r e proposed was based on mass s p e c t r a l , p r o t o n magnetic resonance and i n f r a r e d d a t a . In o r d e r t o c o n f i r m the proposed s t r u c t u r e s of 1 and 2, t h e i r s y n t h e s i s was u n d e r t a k e n and i s o u t l i n e d i n Table I. S u l f o n a t i o n of 5 - m e t h y l - 2 - f u r o i c a c i d (3) w i t h fuming s u l f u r i c a c i d gave the known 2 - m e t h y l 3 - s u l f o - 5 - f u r o i c a c i d (4) (7) which was d e c a r b o x y l a t e d with mercuric c h l o r i d e to y i e l d 2 - m e t h y l - 3 - f u r a n s u l f o n i c a c i d (5). Treatment of 5 w i t h t h i o n y l c h l o r i d e gave the s u l f o n y l c h l o r i d e 6, which was reduced w i t h l i t h i u m aluminum h y d r i d e , g i v i n g 2 - m e t h y 1 - 3 - f u r a n t h i o l (1). T h i s m a t e r i a l has an i d e n t i c a l mass s p e c t r u m , p r o t o n magnetic resonance and i n f r a r e d spectrum as the m a t e r i a l s i s o l a t e d from the r e a c t i o n meat f l a v o r . Oxi­ d a t i o n of 1 w i t h i o d i n e gave b i s ( 2 - m e t h y l - 3 - f u r y l ) d i s u l f i d e (2) which a l s o has i d e n t i c a l s p e c t r a l p r o ­ p e r t i e s as the i s o l a t e d m a t e r i a l . These s y n t h e s e s thus c o n f i r m the s t r u c t u r e s proposed from the a n a l y t i c a l d a t a f o r the i s o l a t e d m a t e r i a l s (8). S i m i l a r compounds were s y n t h e s i z e d i n o r d e r t o compare the f l a v o r p r o p e r t i e s of t h e s e compounds. The c o r r e s p o n d i n g 2 , 5 - d i m e t h y l - 3 - f u r a n t h i o l (8) was p r e ­ pared by h y d r o l y s i s of 2 , 5 - d i m e t h y l - 3 - t h i o a c e t y l f u r a n (7)(10). Treatment of 2 , 5 - d i m e t h y l f u r a n w i t h e i t h e r s u l ­ f u r m o n o c h l o r i d e or s u l f u r d i c h l o r i d e gave, i n low y i e l d , a m i x t u r e c o n t a i n i n g both b i s ( 2 , 5 - d i m e t h y l - 3 f u r y l ) s u l f i d e (10c) and b i s ( 2 , 5 - d i m e t h y l - 3 - f u r y l ) d i ­ sulfide (lOd). The c o r r e s p o n d i n g t r i - and t e t r a s u l f i d e s of 2 were a l s o p r e p a r e d . Thus the r e a c t i o n of 1 11

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

PHENOLIC, SULFUR, A N D NITROGEN COMPOUNDS I N FOOD FLAVORS

TABLE I

3

4

5

1

7

1 , R=H

2, R » H , n - 2 10a,R=-H,n=-3 10b,R-H,n~4 10c,R=»Me,n=0. 10d,R-Me,n=»2

6

9

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

11.

Furans Substituted with Sulfur

EVERS E T A L .

187

w i t h s u l f u r d i c h l o r i d e gave b i s (2-methyl-3-furyl) t r i s u l f i d e (10a) and the r e a c t i o n of 1 w i t h s u l f u r m o n o c h l o r i d e gave b i s ( 2 - m e t h y 1 - 3 - f u r y 1 ) t e t r a s u l f i d e (10b). For comparison, 5 - m e t h y l - 2 - f u r a n t h i o l (12) and b i s ( 5 - m e t h y l - 2 - f u r y l ) d i s u l f i d e (13) were prepared. Treatment of the l i t h i o d e r i v a t i v e of s y l v a n (U) w i t h s u l f u r gave the t h i o l 12 which was o x i d i z e d w i t h i o d i n e t o g i v e 13.

11

1

A summary o f the o r g a n o l e p t i c e v a l u a t i o n s of the v a r i o u s compounds i s c o n t a i n e d i n T a b l e I I . It i s apparent from a b r i e f s t u d y of the t a b l e t h a t a l l of the f u r a n s where the s u l f u r atom i s bound t o the β c a r b o n have meaty aroma and t a s t e c h a r a c t e r i s t i c s . However, when the s u l f u r atom i s i n the a p o s i t i o n o n l y hydrogen s u l f i d e l i k e , burnt and c h e m i c a l n o t e s are o b s e r v e d . It i s somewhat s u r p r i s i n g t h a t a one c a r b o n s h i f t i n s u b s t i t u t i o n would produce t h i s s u b ­ s t a n t i a l change i n f l a v o r c h a r a c t e r . Experimental I s o l a t i o n of 1 and 2. In a s u i t a b l e v e s s e l a m i x ­ t u r e o f 35 l b s . of t h i a m i n e c h l o r i d e h y d r o c h l o r i d e , 35 l b s . of L - c y s t e i n e h y d r o c h l o r i d e , 1,238 l b s . of v e g e ­ t a b l e p r o t e i n h y d r o l y s a t e ( c a r b o h y d r a t e f r e e ) and 2,692 l b s . of water was heated a t r e f l u x f o r f o u r h o u r s . A f t e r 45 m i n u t e s , 40 g a l l o n s of condensate was removed d u r i n g 3^ h r s . Each g a l l o n was e x t r a c t e d w i t h 400 ml of CHgClg e x t r a c t s were combined and d r i e d w i t h sodium s u l f a t e . S o l v e n t removal i n m i l d (100 mm) vacuum and f i n a l c o n c e n t r a t i o n w i t h a stream of dry n i t r o g e n gave 50 ml o f c o n c e n t r a t e . 2 - M e t h y l - 3 - f u r a n t h i o l (1) : P r e p a r a t i v e GLC of the c o n c e n t r a t e ( 2 x 2 ml) on an 8' χ 3/4", 25% SE 30 on A/W DMCS column, programmed from 7 0 ° t o 2 2 5 ° C at l ° / r a i n y i e l d s about 300 λ c o n t a i n i n g a s t r o n g pot r o a s t aroma. Rechromatography of the 300 λ on a 10 χ 3/8", 20% CBW 20M A/W DMCS column programmed from 8 0 ° t o 2 2 5 ° C at l ° / m i n y i e l d s about 100 λ of m a t e r i a l c o n t a i n ­ i n g a v e r y i n t e n s e pot r o a s t aroma. F i n a l chromato­ graphy of t h i s m a t e r i a l on a 10 χ 1/4" g l a s s , 20% Apiezon L column gave 2-3 A of 2 - m e t h y l - 3 - f u r a n t h i o l a

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In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

188

PHENOLIC, SULFUR, AND NITROGEN COMPOUNDS IN FOOD FLAVORS

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11. EVERS ET AL.

Furans Substituted with Sulfur

189

h a v i n g : i r ( n e a t , ΚBr p l a t e s ) 3120, 2960, 2925, 2540 ( - S H ) , 1595, 1514, 1222, 1136, 1092, 938, 888 and 730 cm ; nmr ( C C 1 ) 8 2.32 ( s , 3 , a r i n g CHo), 2 . 4 7 ( s , l β S H ) , 6.19 ( d , l , J = 1 . 5 H z , β r i n g Η ) , 7715 ppm ( d , l , J=1.5Hz, a r i n g Η ) ; mass spectrum (70 ev) m/e ( r e l . i n t e n s i t y ) 116 ( 5 ) , 115 ( 8 ) , 114 ( 1 0 0 ) , 113 ( 2 9 ) , 86 ( 1 0 ) , 85 ( 2 3 ) , 71 ( 1 4 ) . bis (2-Methyl-3-furyl) d i s u l f i d e (2): Repeated p r e p a r a t i v e t h i n - l a y e r chromotography on 8" χ 8" p l a t e s c o a t e d w i t h 1.25 mm o f s i l i c a - g e l G (200 X p o r t i o n s per p l a t e , developed w i t h 1:10 e t h e r / h e x a n e ) gave from 2 . 4 g o f c o n c e n t r a t e 0.066 g o f b i s ( 2 - m e t h y 1 - 3 - f u r y l ) d i ­ s u l f i d e h a v i n g : i r ( n e a t , ΚBr p l a t e s ) 3120, 2955, 2920, 1580, 1512, 1222, 1120, 1085, 936, 885, 730 c m " ; nmr ( C C 1 ) * 2 . 0 8 ( s , 3 , a r i n g CHo), 6.27 ( d , 1 , J = 1 . 5 H z , β r i n g H ) , and 7.16 spectrum (70 ev) m/e ( r e l i n t e n s i t y ) 228 ( 6 ) , 227 ( 9 ) , 226 ( 6 0 ) , 115 ( 5 ) , 114 ( 1 7 ) , 113 ( 1 0 0 ) , 85 ( 1 3 ) . 2-Methyl-3-sulfo-5-furoic acid (4): The barium s a l t of 4a was prepared i n 93% y i e l d from 3 a c c o r d i n g t o S c u l l y and Brown ( 7 ) . The f r e e a c i d was i s o l a t e d f o r nmr by t r e a t i n g the barium s a l t w i t h HgSO^ f o l l o w e d by f i l t r a t i o n and e v a p o r a t i o n under vacuum. Nmr ( D 0 ) $ 2 . 6 2 ( s , 3 , a r i n g CHg) and 7.47 ppm ( s , l , β r i n g H) . 2-Methyl-3-furansulfonic acid (5): A s o l u t i o n of 33 g o f the barium s a l t o f 4a i n 450 ml o f HoO was t r e a t e d w i t h 20% H S 0 u n t i l no f u r t h e r p r e c i p i t a t i o n was o b s e r v e d . A f t e r f i l t r a t i o n , the f i l t r a t e was a d ­ j u s t e d t o pH 7 w i t h s o l i d NaHCOg. A f t e r a d d i t i o n of 26.3 g o f m e r c u r i c c h l o r i d e the r e s u l t i n g m i x t u r e was heated at r e f l u x and m o n i t o r e d f o r C 0 e v o l u t i o n . When C 0 e v o l u t i o n had s t o p p e d , the m i x t u r e was c o o l e d t o room temperature and t r e a t e d w i t h hydrogen s u l f i d e . A f t e r standing overnight p r e c i p i t a t e d mercuric s u l f i d e was removed by d é c a n t a t i o n and f i l t r a t i o n . The s o l u t i o n was e v a p o r a t e d t o d r y n e s s iri vacuo and the r e s i due r e c r y s t a l l i z e d from a minimum of b o i l i n g water t o g i v e 4 . 2 5 g o f 2 - m e t h y 1 - 3 - f u r a n s u l f o n i c a c i d as the sodium s a l t . Ir ( n u j o l m u l l ) 3140, 1240, 1229, 1190, 1110, 1075, 1018, 898 cm"" ; nmr ( D „ 0 ) 6 2 . 4 8 ( s , 3 , a r i n g C H ) , 6.62 ( d , l , J = 2 H z , β r i n g Η) and 7.43 ppm (d,l,J-2Hz, a ring H). 2-Methyl-3-furansulfonyl chloride (6): A mix­ t u r e o f 80 g o f 5 (sodium s a l t ) , 880 g t h i o n y l c h l o ­ r i d e and 15 drops o f d i m e t h y l formamide was heated a t r e f l u x f o r 100 m i n , c o o l e d t o room temperature and filtered. A f t e r washing the f i l t e r cake w i t h benzene (20 ml) and combining w i t h the f i l t r a t e , concentration i n vacuo gave 58 g o f a brown o i l . D i s t i l l a t i o n of the o i l gave 4 5 . 4 g o f 2 - m e t h y 1 - 3 - f u r a n s u f o n y l c h l o ­ r i d e as a l i g h t y e l l o w l i q u i d b o i l i n g a t 5 5 ° C a t 0.55 4

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In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

190

PHENOLIC, SULFUR, AND NITROGEN COMPOUNDS I N FOOD FLAVORS

t o r r . and h a v i n g : i r ( n e a t , ΚBr p l a t e s ) 3115, 1570, 1520, 1380, 1360, 1220, 1158, 1129, 1092, 1024, 948, 880, 740 c m " : nmr ( C C 1 ) 6 2.71 ( s , 3 , a r i n g CHo), 6.86 ( d , l , J - 2 H Z , β r i n g Η) and 7.52 ppm ( d , l , J = » 2 H z , a r i n g Η ) ; mass spectrum (70 ev) m/e ( d e c r e a s i n g i n ­ t e n s i t y , m o l , ion) 145, 97, 53, 43, 180, and i n t e n s i t y of P+2 (182) i s 43% of P. 2 - M e t h y l - 3 - f u r a n t h i o l (1): A s o l u t i o n of 45.4 g of 2 - m e t h y l - 3 - f u r a n s u l f o n y l c h l o r i d e (6) i n 540 ml o f anhydrous e t h e r was added dropwise t o a s o l u t i o n of l i t h i u m aluminium h y d r i d e i n 1200 ml e t h e r . After h e a t i n g at r e f l u x one h o u r , e x c e s s h y d r i d e was d e s ­ t r o y e d by the a d d i t i o n of e t h y l a c e t a t e . The r e s u l t ­ i n g m i x t u r e was poured i n t o 1,500 ml water and the ether layer separated. The aqueous phase was e x t r a c t e d w i t h e t h e r (4 χ 250 combined, washed w i t h 5% NaHCOg s o l u t i o n (150 m l ) , water (2 χ 200 ml) and d r i e d over sodium s u l f a t e . Sol­ vent removal i n vacuo gave 18 g crude 1 . Distillation gave 11.6 g of 2 - m e t h y 1 - 3 - f u r a n t h i o l b o i l i n g a t 575 8 . 5 ° at 36 t o r r and h a v i n g : i r , ( n e a t , KBr p l a t e s ) 3110, 2955, 2925, 2540 Î S H ) , 1594, 1512, 1223, 1134, 1092, 937, 889, 732 c m " ; nmr ( C C I . ) 6 2.34 (s,3, a r i n g CHo), 2.47 ( s , l , β r i n g S H ) , 6.19 ( d , l , J = 1 . 5 Hz, β r i n g H) and 7.15 ppm ( d , l , J - 1 . 5 H z , a r i n g H ) ; mass spectrum (70 eu) m/e ( r e l . i n t e n s i t y ) 116 ( 5 ) , 115 (8), 114 ( 1 0 0 ) , 113 (29), 86 (10), 85 (23), 71 (13). b i s (2-Methy1-3-fury1) d i s u l f i d e (2): A solution of 7.4 g of sodium h y d r o x i d e , 10 g of sodium c a r b o n a t e and 21 g o f 2 - m e t h y 1 - 3 - f u r a n t h i o l (1) i n 344 ml of water was p r e p a r e d . To t h i s s o l u t i o n was added a s o l u ­ t i o n of 77.3 g of potassium i o d i d e and 23.2 g o f i o ­ d i n e i n 780 ml o f water u n t i l the i o d i n e c o l o r p e r ­ sisted f o r 1 m i n . A f t e r r e n d e r i n g the s o l u t i o n c o l o r ­ l e s s w i t h 0.1 sodium t h i o s u l f a t e s o l u t i o n , the m i x t u r e wasextracted w i t h pentane (3 χ 150 m l ) . The pentane e x t r a c t s were combined, washed w i t h water (2 χ 100 ml) and d r i e d o v e r sodium s u l f a t e . S o l v e n t removal i n vacuo gave 10 g of an amber o i l . D i s t i l l a t i o n of the o i l gave 15.4 g of b i s ( 2 - m e t h y 1 - 3 - f u r y l ) d i s u l f i d e (2) b o i l i n g at 7 7 - 7 8 ° at 0.3 t o r r and h a v i n g : i r ( n e a t , KBr p l a t e s ) 3120, 2955^,2920, 1580, 1512, 1222, 1122, 1082, 938, 885, 730 cm ; nmr ( C C I . )