Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors

to commercial tea products such as green tea (not fermented), oolong tea ...... course, has the added advantage of contributing a touch of lemon flavo...
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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

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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, George, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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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 noteworthy that the c a f f e i n e c o n c e n t r a t i o n i n the b l a c k t e a beverage s t u d i e d i n t h i s i n v e s t i g a t i o n (Table 1) was 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, George, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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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-gallate (II) (-)-Epigallocatechin ( I I I ) (-)-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 0.3 1.5

15 68 04 41

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, George, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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Table 1, (continued)

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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

1.5

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 4.56 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, George, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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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 , ( - ) - E p i g a l l o c a t e c h i n ; R]=OH; R =H ( - ) - E p i g a l l o c a t e c h i n - 3 - g a l l a t e ; Ri=OH; R =VIIb 2

X

2

2

I V >

2

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, George, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

Polyphenolic Compounds in the Taste of Tea

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SANDERSON E T A L .

XI, XII, XIII, XIV,

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, George, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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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 c m a t e r i a l , and other substances, but c o n s i d e r a b l e s o l i d matter remains e x t r a c t a b l e w i t h b o i l i n g water. 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, George, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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Polyphenolic Compounds in the Taste of Tea

IH

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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, George, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

2

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, George, 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)

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In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, George, 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)

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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.

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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 s t h a t the a s t r i n g e n c y was best described as having two components; namely, a tangy component that was sharp and puckery w i t h l i t t l e a f t e r t a s t e e f f e c t ( t h i s i s a d i f 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, George, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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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 are removed from the e x t r a c t ( F r a c t i o n 6 ) . Removal of the c a f f e i n e from a polyphenol-free b l a c k tea e x t r a c t (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, George, 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 (%)

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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, George, 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

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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, George, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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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 n 6) had a weak, s l i g h t l y b i t t e r , greenish t a s t e that was not recognized as a b l a c k tea t a s t e . 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, George, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

SANDERSON ET AL.

Polyphenols Compounds in the Taste of Tea

29

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2.

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, George, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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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

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

C

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

15

837

206

9.4

219

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, George, 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

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Astringency b Tangy Non-Tangy

Black Tea Aroma

Black Tea Taste

0 0

1 4

0 0

None None

0

4

0

None

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 Very green, h a r s h , bitter 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

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, George, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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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 e amount of t h e a r u b i g i n s i s formed i n the macerated tea l e a f p r i o r to the s t a r t of the formal tea fermentat i o n p e r i o d : This must be due to the tea fermentation 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, George, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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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) are a s t r i n g e n t i n a d d i t i o n to having a b i t t e r t a s t e . Of p a r t i c u l a r importance was the f i n d i n g t h a t i n no case was the a s t r i n g e n c y shown by the p u r i f i e d t e a polyphenols 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, George, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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Table 4:

SULFUR,

AND

NITROGEN

COMPOUNDS

Polyphenol (-)-Epicatechin ( I ) (-)-Epicatechin gallate (II) ( - ) - E p i g a l l o c a t e c h i n Not (III) (-)-Epigallocatechin g a l l a t e (IV) Not (+)-Catechin (V) Crude T h e a f l a v i n s , a n a t u r a l mixture (XI-XIV) T h e a f l a v i n (XI) 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 ) Theaflavin d i g a l l a t e (XIV) G a l l i c acid (Vila) Thearubigins (XVII- Not Not XX, others) Tannic a c i d b

c

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

a

IN

astringent 60 astringent 60

80 36

12.5

Tea

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

35

Trace

30

16-18

60 70

Trace 5-11

0.6 1.8

75- 100 30- 50

1.2 3.7

Not determined 2.4 -

4.8

c 3- 5 bitter a s t r i n g e n t ^ Not determined^ 95- 120 determined" Not 20 80 u

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, George, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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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 the i n f u s i o n s obtained were analyzed f o r the amount of s o l i d s e x t r a c t e d and t h e i r o r g a n o l e p t i c q u a l i t i e s . 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, George, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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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.

B.

E f f e c t of length of Tea l e a f i n f u s i o n -

infusion period: 1 minute 3 minutes 5 minutes

490 590 670

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, George, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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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

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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

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

Black Tea Taste

Mild Strong Very s t r o n g

2 3 4

41 48 54

48 48 0 48

Aroma

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, George, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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NITROGEN COMPOUNDS IN FOOD FLAVORS

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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 y i n the i n f u s i o n from the tangy type, which 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 , to a non-tangy type. Furt h e r , the r e s u l t s (Table 2, p a r t D) show that d e g a l l a t i o n of the 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, George, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

2.

SANDERSON ET A L .

Polyphenolic Compounds in the Taste of Tea

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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 experiments was complete w i t h the 1-min. and 3-min. i n f u s i o n s and almost complete w i t h the 5-min. i n f u s i o n . 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-

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

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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 astringency i s a f f e c t e d most suggests t h a t i t i s the c a f f e i n e / p o l y p h e n o l complex that i s most a l t e r e d by the pH 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, George, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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2. SANDERSON ET AL.

Polyphenolic Compounds in the Taste of Tea

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 tea aroma was found to be an e s s e n t i a l comple­ ment to the b l a c k t e a s o l i d s f o r the expression of f u l l b l a c k tea 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

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

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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 , by the consumers i n these other c o u n t r i e s . 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

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

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Polyphenolic Compounds in the Taste of Tea

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 r some d i s t i l l e d water was added t o t h a t f r a c t i o n . Added water was removed from the polyphenol f r a c t i o n by f r e e z e - d r y i n g . The c a f f e i n e and polyphenol f r e e aqueous 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 fermented tea flush was frozen by mixing with crushed dry ice and freeze dried, and one sample was fired 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.

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