Hi-Res PDF - American Chemical Society

Chapter 16

Downloaded by KTH ROYAL INST OF TECHNOLOGY on December 3, 2015 | http://pubs.acs.org Publication Date: October 3, 1989 | doi: 10.1021/bk-1989-0409.ch016

Heat-Induced Flavor Formation from Peptides George P. Rizzi Miami Valley Laboratories, The Procter & Gamble Company, Cincinnati, OH 45239-8707

Peptides can degrade during food processing to form novel taste compounds like diketopiperazines (DKPs) or react with reducing sugars to produce volatile Maillard products. Eleven DKPs were detected in commercial cocoas and model studies substantiated a DKP formation mechanism involving intramolecular cyclization of linear peptide precursors. Model Maillard reactions of peptides and fructose generated Strecker degradation products from amino acids with blocked amine and carboxyl functionalities.

P e p t i d e s have l o n g been r e c o g n i z e d a s i m p o r t a n t f l a v o r compounds i n p r o c e s s e d f o o d s . The t a s t e o f p e p t i d e s p e r s e i s v e i l known i n cheeses ( 1 ) , meat ( 2 ) , 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 i n c l u d i n g s o y p r o d u c t s ( 3 ) , cocoa ( 4 , 5 ) and t o a l e s s e r e x t e n t i n r o a s t e d malt ( 6 ) , c o r n s t e e p l i q u o r ( 7 ) and aged sake ( 8 ) . S t r u c t u r a l l y , food p e p t i d e s c a n o c c u r a s l i n e a r p r o t e i n fragments ( 1 - 3 , 5 ) , c y c l i c d i m e r s ( d i k e t o p i p e r a z i n e s , DKPs)[4, 6-8] and c y c l i c t r i m e r s ( 7 ) . P e p t i d e s formed e n z y m i c a l l y o r by m i n e r a l a c i d h y d r o l y s i s o r t h e r m a l d e g r a d a t i o n o f h i g h e r m o l e c u l a r weight p r o t e i n c a n a l s o s e r v e a s f l a v o r p r e c u r s o r s i n t h e r m a l l y i n d u c e d r e a c t i o n s . The r e a c t i v i t y o f p e p t i d e s i s e v i d e n c e d by t h e i r b e h a v i o r d u r i n g p y r o l y s i s / G C ( 9 ) , h e a t i n g i n a i r ( 1 0 ) , r e a c t i o n s w i t h mono- ( 1 1 ) , and d i c a r b o n y l (12, 13) compounds and r e a c t i o n s i n hot a c e t i c a c i d ( 1 4 ) . The types o f r e a c t i o n s observed f o r p e p t i d e s i n c l u d e : s i d e - c h a i n t h e r m o l y s i s , f r a g m e n t a t i o n i n t o amino a c i d s , DKP f o r m a t i o n and M a i l l a r d r e a c t i o n w i t h ambient c a r b o h y d r a t e s . Besides t h e i r general f l a v o r forming p o t e n t i a l peptides a r e a l s o r e p o r t e d t o be u n i q u e p r e c u r s o r s o f c o m p o s i t e f o o d aromas. P e p t i d e s formed i n the f e r m e n t a t i v e s t a g e o f cacao p r o c e s s i n g have been l i n k e d t o r o a s t g e n e r a t e d c h o c o l a t e aroma ( 5 ) . A l s o , a m e t h i o n i n e r i c h p o l y p e p t i d e has been a s s o c i a t e d w i t h r o a s t e d peanut v o l a t i l e s (15). 0097-6156/89/0409-0172$06.00/0 ο 1989 American Chemical Society

In Thermal Generation of Aromas; Parliment, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

16.

RIZZI

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173

The purpose o f t h i s work was t o i n v e s t i g a t e the o c c u r r e n c e and h e a t - i n d u c e d o r i g i n o f c y c l i c d i p e p t i d e s (DKPs) i n cocoa; and s e p a r a t e l y , t o s t u d y t h e mechanism o f DKP f o r m a t i o n i n s i m p l i f i e d r e a c t i o n systems. A l s o , the f o r m a t i o n o f v o l a t i l e s was measured i n a s e r i e s o f model M a i l l a r d r e a c t i o n s o f p e p t i d e s , amino a c i d m i x t u r e s and f r u c t o s e .


Experimental

Part

Amino a c i d s and p e p t i d e s were a n a l y t i c a l grade c o m m e r c i a l samples o b t a i n e d from Sigma C h e m i c a l Company, S t . L o u i s , MO. S o l v e n t s and c h e m i c a l r e a g e n t s were a l l c o m m e r c i a l , a n a l y t i c a l grade m a t e r i a l s . Commercial cocoa powders were d e f a t t e d w i t h hexane a t room t e m p e r a t u r e and a i r d r i e d b e f o r e a n a l y s i s . D r i e d , u n r o a s t e d cocoa beans were e x t r a c t e d w i t h m e t h a n o l - v a t e r a c c o r d i n g t o Rohan and S t e w a r t (16) and a l i q u o t s o f f r e e z e - d r i e d bean e x t r a c t s were s e a l e d i n t o g l a s s v i a l s and r o a s t e d i n a t h e r m o s t a t e d o i l b a t h . P r i o r to DKP a n a l y s i s cacao p r o d u c t s were c l e a n e d up t o remove theobromine, p o l y p h e n o l s and f i b e r by t h e method o f P i c k e n h a g e n e t a l . ( 4 ) , but o m i t t i n g the s i l i c a - g e l chromatography s t e p . DKPs were a n a l y z e d by h i g h r e s o l u t i o n gas chromatography (HRGC) on a R e s t e k (RTX-5) 60 M X 0.32 mm i . d . f u s e d s i l i c a column ( l u DB-5 f i l m ) i n t e r f a c e d t o a F i n n e g a n model 4500 mass s p e c t r o m e t e r w i t h an INCOS d a t a p r o c e s s i n g system. Mass s p e c t r a were o b t a i n e d a t 1 s e c . s c a n speed w i t h n o m i n a l mass r e s o l u t i o n o f 1.0 amu. I n l e t and t r a n s f e r l i n e t e m p e r a t u r e s were 250 and 3 0 0 C r e s p e c t i v e l y . A t y p i c a l s e p a r a t i o n o f DKP homologs and c i s / t r a n s isomers i s shown i n F i g u r e 1. DKPs were q u a n t i t a t e d u s i n g peak a r e a i n t e g r a l s and r e f e r e n c e t o a complete s e t o f e x t e r n a l s t a n d a r d s . S t a n d a r d DKPs w i t h two r i n g s u b s t i t u e n t s were m i x t u r e s o f c i s / t r a n s i s o m e r s t h e r e b y a l l o w i n g q u a n t i t a t i o n o f i s o m e r i c DKPs i n c o c o a . Model r e a c t i o n s i n v o l v i n g p e p t i d e s and amino a c i d s i n a c e t i c a c i d were worked up by d i l u t i n g the p r o d u c t s w i t h w a t e r and removing unchanged s t a r t i n g m a t e r i a l s w i t h B i o - r a d AG 50V-X8 c a t i o n exchange r e s i n (50-100 mesh, H - f o r m ) . A l i q u o t s were a n a l y z e d f o r DKPs a f t e r a d d i n g c ( A l a - G l y ) as an i n t e r n a l s t a n d a r d . DKP s t a n d a r d s were s y n t h e s i z e d by r e f l u x i n g 1.3 M s o l u t i o n s o f d i p e p t i d e s i n a c e t i c a c i d f o r two h o u r s . Treatment w i t h a c e t i c a c i d l e d t o p a r t i a l r a c e m i z a t i o n o f e n a n t i o m e r i c a l l y pure p e p t i d e s . A f t e r removal o f unchanged s t a r t i n g m a t e r i a l s w i t h c a t i o n exchange r e s i n , p r o d u c t s were i s o l a t e d by vacuum e v a p o r a t i o n and p u r i f i e d by r e c r y s t a l l i z a t i o n . R e c r y s t a l l i z a t i o n p r o v i d e d a n a l y t i c a l l y pure p r o d u c t s , but DKPs w i t h two r i n g s u b s t i t u e n t s s t i l l c o n t a i n e d m i x t u r e s o f c i s / t r a n s isomers. S t r u c t u r e s o f p u r i f i e d p r o d u c t s were v e r i f i e d by e l e m e n t a l a n a l y s i s and by r e f e r e n c e t o p u b l i s h e d MS d a t a ( 1 7 ) . F o r M a i l l a r d r e a c t i o n s , aqueous s o l u t i o n s o f amino a c i d s , p e p t i d e s and f r u c t o s e were f r e e z e - d r i e d i n 50 mL serum b o t t l e s and septum-sealed b e f o r e i n c u b a t i n g i n a t h e r m o s t a t e d o i l b a t h . C o o l e d m i x t u r e s were t r e a t e d w i t h w a t e r t o make 10X w/v m i x t u r e s and e q u i l i b r i u m headspaces were sampled a t 50°C f o r HRGC a n a l y s i s a c c o r d i n g t o p r o c e d u r e s d e v e l o p e d by Haynes and S t e i m l e ( 1 8 ) . e

+



174

THERMAL GENERATION OF AROMAS

ι

456 I I I

1

2

Peak No.

60 m χ 0.32 mm RTX-5 2 0 0 ° C @ 4 ° / m i n t o 280°C

3

11 1 0

in

1 2 3 4 5 6 7 8 9 10 11

DKP Ala-Gly Ala-Val Ala-Leu Ala-Pro Gly-Leu Gly-Pro Leu-Pro Ala-Phe Gly-Phe Phe-Val Pro-Phe

1 1

U 10

15

20

25

30

35

Retention Time (minutes) F i g u r e 1. Separation

HRGC Chromatogram o f S y n t h e t i c DKPs S h o v i n g o f C i s / T r a n s Isomers


40

16.

RIZZI


175


Discussion of Results O c c u r r e n c e and F o r m a t i o n o f DKPs i n Cacao P r o d u c t s . Thermal d e g r a d a t i o n o f p e p t i d e s ( o r p r o t e i n s ) d u r i n g c o c o a bean r o a s t i n g i s e v i d e n c e d by the p r e s e n c e o f DKPs i n c o c o a powder ( 4 , 1 7 ) . Cocoa DKPs a r e i m p o r t a n t f l a v o r components t h a t have been shown t o produce a c h a r a c t e r i s t i c b i t t e r t a s t e i n c o n j u n c t i o n w i t h n a t u r a l l y o c c u r r i n g theobromine ( 4 ) . I n t h i s s t u d y we u t i l i z e d HRGC a n a l y s i s c o u p l e d w i t h a s e n s i t i v e n i t r o g e n s e l e c t i v e d e t e c t o r and a mass s p e c t r o m e t e r t o compare t y p e s and amounts o f DKPs i n v a r i o u s l y processed cocoas. S i m i l a r a n a l y t i c a l t e c h n i q u e was used t o f o l l o w the f o r m a t i o n o f DKPs d u r i n g i n v i t r o r o a s t i n g o f c h o c o l a t e f l a v o r p r e c u r s o r s i s o l a t e d from u n r o a s t e d beans. Q u a n t i t a t i v e d a t a ranges f o r DKPs found i n f o u r cocoas a r e e x p r e s s e d i n mg o f DKP/Kg o f f a t f r e e - c o c o a as f o l l o w s : c ( G l y - L e u ) , 0-25; c ( G l y - P r o ) , 2-7; c ( G l y - P h e ) , 0-11; c ( A l a - G l y ) , 0-12; c ( A l a - V a l ) , 56-143, c ( A l a - L e u ) , 22-69; c ( A l a - P r o ) , 19-44; c ( A l a - P h e ) , 2-19; c ( L e u - P r o ) , 39-115 and c ( P h e - P r o ) , 0-26. Toward the end o f the s t u d y an e l e v e n t h DKP was q u a l i t a t i v e l y i d e n t i f i e d i n c o c o a and showed to be c ( V a l - P h e ) . Our r e s u l t s c o n f i r m e d the DKPs found by p r e v i o u s a u t h o r s (4, 17) e x c e p t f o r c ( A s p - P r o ) and c ( A s p - P h e ) which were a p p a r e n t l y not amenable t o d i r e c t GC analysis. Two new DKPs , c ( G l y - L e u ) and c ( A l a - L e u ) were o b s e r v e d f o r the f i r s t time i n c o c o a . Cocoas c o n t a i n e d s i m i l a r k i n d s o f DKPs, however q u a n t i t a t i v e d i f f e r e n c e s s u g g e s t e d t h a t DKP p r o f i l e s o b t a i n e d by HRGC might s e r v e as a s e n s i t i v e f i n g e r p r i n t t e c h n i q u e . A l s o , dutched cocoas t y p i c a l l y had lower l e v e l s o f DKPs compared to o r d i n a r y processed cocoas. HRGC p r o v i d e d a second a n a l y t i c a l d i m e n s i o n by r e s o l v i n g c i s / t r a n s DKP i s o m e r s i n compounds c o n t a i n i n g two r i n g substituents. F o r s i x o f the DKPs found i n c o c o a p a i r s o f compounds c o r r e s p o n d i n g t o g e o m e t r i c i s o m e r s were o b s e r v e d and they were c o n f i r m e d t o be i s o m e r s by t h e i r i d e n t i c a l mass s p e c t r a . I t was i n t e r e s t i n g t h a t c ( A l a - V a l ) , c ( A l a - L e u ) , c ( A l a - P r o ) and c ( P h e - P r o ) o c c u r r e d as s i n g l e geometic i s o m e r s i n some c o c o a s , a r e s u l t that i s i n d i c a t i v e of s t e r e o r e t e n t i v e p r o t e i n degradation during roasting. I n d u t c h p r o c e s s c o c o a the l a t t e r DKPs appeared as m i x t u r e s o f i s o m e r s a p p a r e n t l y due t o p a r t i a l r a c e m i z a t i o n caused by more a l k a l i n e p r o c e s s c o n d i t i o n s . Heat Induced DKP F o r m a t i o n i n Cocoa Bean E x t r a c t s . E x t r a c t s of d r i e d , u n r o a s t e d cocoa beans were p r e p a r e d by the method o f Rohan and Stewart ( 1 6 ) , r o a s t e d a t 120°C f o r 30 minutes and a n a l y z e d by HRGC to p r o v i d e the d a t a i n T a b l e I . DKPs were found f o r the f i r s t time i n u n r o a s t e d beans, p o s s i b l y as a r e s u l t o f h e a t o r enzyme a c t i o n d u r i n g f e r m e n t a t i o n o r d r y i n g . I n g e n e r a l , DKP l e v e l s i n c r e a s e d a f t e r r o a s t i n g and more DKPs were produced i n e x t r a c t s o f the more h i g h l y fermented Ghana and B a h i a v a r i e t i e s . I t was c u r i o u s t h a t c ( A l a - P h e ) and c ( P h e - P r o ) d i s a p p e a r e d d u r i n g Ghana e x t r a c t r o a s t i n g but e v i d e n t l y s u r v i v e d a s i m i l a r t r e a t m e n t i n the Bahia extract.


176

THERMAL GENERATION OF AROMAS T a b l e I . Heat Induced DKP F o r m a t i o n


DKP Observed

Arriba U R

Bean Sanchez U R

i n Cocoa Bean E x t r a c t s Variety Ghana R U

U

Bahia R

480 1560 940 c(Ala-Val) 450 0 0 0 470 c(Ala-Leu) 880 560 0 0 270 0 0 0 1160 730 480 42 c(Ala-Pro) 125 450 116 430 120 0 69 c(Gly-Leu) 0 0 0 0 0 1720 1110 1000 c(Leu-Pro) 146 800 63 190 470 280 c(Ala-Phe) 104 T r a c e T r a c e 0 0 0 0 480 0 240 T r a c e T r a c e c(Phe-Pro) 0 0 0 6200 1960 105 3410 Totals 271 2040 306 1640 U = U n r o a s t e d , R = Roasted a t 120°C f o r 30 min.; d a t a i n mg/kg o f unheated e x t r a c t rounded t o t h r e e s i g n i f i c a n t f i g u r e s

DKP F o r m a t i o n i n Model P e p t i d e Systems. The f a c t t h a t DKPs a r e formed a t r e l a t i v e l y low temperatures suggested t h a t c a t a l y z e d p e p t i d e d e c o m p o s i t i o n o c c u r s i n the weakly a c i d i c m i l i e u produced by cacao f e r m e n t a t i o n . R e a c t i o n s o f s i m p l e p e p t i d e s were f o l l o w e d a t 0.02 M ( i n i t i a l c o n c e n t r a t i o n ) i n r e f l u x i n g a c e t i c a c i d (120°C) t o s i m u l a t e t h e i n t e r i o r o f a bean d u r i n g r o a s t i n g ( T a b l e II)· Under these c o n d i t i o n s V a l - A l a was almost q u a n t i t a t i v e l y c o n v e r t e d i n t o c ( V a l - A l a ) a c c o r d i n g t o t h e p o s t u l a t e d r e a c t i o n mechanism ( 4 ) , F i g u r e 2 whereas an e q u i m o l a r m i x t u r e o f v a l i n e and a l a n i n e f a i l e d t o r e a c t . The i n t r a m o l e c u l a r n a t u r e o f DKP f o r m a t i o n was f u r t h e r e s t a b l i s h e d by r e a c t i o n s o f t r i p e p t i d e s . Each t r i p e p t i d e s t u d i e d produced a s i n g l e DKP t h a t had t o r e s u l t from a t t a c k o f t h e N - t e r m i n a l amino group a c c o r d i n g t o F i g u r e 2. DKP f o r m a t i o n by

Table I I .

DKP F o r m a t i o n

i n Model Systems

Initial Reactant(s) Conc.(M) Time, h r s Phe-Val 1.9 2.0 Phe, V a l 1.9 2.0 Val-Ala 0.020 0.50 Val, Ala 0.020 0.50 Gly-Leu-Ala 0.020 0.50 Ala-Leu-Gly 0.020 0.50 Leu-Gly-Phe 0.020 0.50 Pro-Gly-Gly 0.50 0.020 S o l v e n t = A c e t i c A c i d , Temperature * normal

Product c(Phe-Val) c(Phe-Val) c(Val-Ala) No R e a c t i o n c(Gly-Leu) c(Ala-Leu) c(Leu-Gly) c(Pro-Gly) reflux

X Yield 74 0.076 94 0 7.1 20 23 15



16. RIZZI

Heat-Induced Flavor FormationfromPeptides

F i g u r e 2.

DKP F o r m a t i o n Mechanism


177

178



i n t e r m o l e c u l a r r e a c t i o n o f amino a c i d s was d e t e c t e d f o r a m i x t u r e o f p h e n y l a l a n i n e and v a l i n e , but o n l y under e x a g g e r a t e d r e a c t i o n c o n d i t i o n s o f 2 h r s . r e f l u x and i n i t i a l c o n c e n t r a t i o n s o f 1.9 M. S t e r e o c h e m i s t r y o f DKP F o r m a t i o n . C y c l i z a t i o n o f peptides that c o n t a i n c h i r a l c a r b o n atoms c a n y i e l d c i s / t r a n s DKP i s o m e r s which a r e s e p a r a b l e by HRGC. E n a n t i o m e r i c a l l y pure p e p t i d e s w i l l t h e o r e t i c a l l y form s i n g l e g e o m e t r i c i s o m e r s , i . e . D ( L ) - D ( L ) d i p e p t i d e s ==> c i s DKPs and D ( L ) - L ( D ) d i p e p t i d e s « > t r a n s DKPs. A c c o r d i n g t o o u r o b s e r v a t i o n s some c o c o a DKPs were p r e s e n t a s n e a r l y pure, s i n g l e geometric isomers. Our r e s u l t s s u g g e s t e d a s t e r e o r e t e n t i v e c y c l i z a t i o n o f p e p t i d e s c o n t a i n i n g o p t i c a l l y pure ( p r e s u m a b l y L ) amino a c i d s d u r i n g bean r o a s t i n g . An attempt t o d u p l i c a t e s t e r e o r e t e n t i v e DKP f o r m a t i o n i n a model system was o n l y p a r t i a l l y s u c c e s s f u l . C y c l i z a t i o n s t u d i e s on L - P h e - L - A l a i n a c e t i c a c i d s t r o n g l y s u g g e s t e d t h a t t h e L - A l a m o i e t y had undergone p a r t i a l racemization. The t o t a l DKP p r o d u c t ( 6 1 % y i e l d ) was a 91/9 i s o m e r i c mixture c o n t a i n i n g mostly c i s c(L-Phe-L-Ala) as evidenced by i t s o p t i c a l r o t a t i o n and m e l t i n g p o i n t ( 1 9 ) . The h i g h o p t i c a l p u r i t y o f the e n r i c h e d c i s product precluded the presence o f a s i g n i f i c a n t amount o f c(D-Phe-D-Ala) w h i c h would have r e s u l t e d by p a r t i a l r a c e m i z a t i o n o f b o t h L-Phe and L - A l a . The t r a n s isomer o b s e r v e d by HRGC was t h e r e f o r e c ( L - P h e - D - A l a ) ( 1 9 ) r e s u l t i n g from p a r t i a l racemization of alanine prior to or during c y c l i z a t i o n . In a s e p a r a t e e x p e r i m e n t c ( L - P h e - L - A l a ) d i d n o t i s o m e r i z e under c y c l i z a t i o n c o n d i t i o n s . The h i g h d e g r e e o f i s o m e r i c p u r i t y f o r some c o c o a DKPs i s n o t y e t e x p l a i n e d b u t i t may be a r e s u l t o f p r e c u r s o r s t h a t c o n t a i n s e v e r a l c o n t i g u o u s o p t i c a l l y pure amino acids. Formation o f V o l a t i l e s i n Peptide/Fructose Reactions. Typical M a i l l a r d r e a c t i o n v o l a t i l e s were o b s e r v e d when v a r i o u s f o o d p r o t e i n s were r o a s t e d a t 250°C (20) o r a u t o c l a v e d i n water a t 121-140°C (21, 2 2 ) . S i n c e p r o t e i n i s known t o b r e a k down i n t o amino a c i d s and p e p t i d e s under s i m i l a r c o n d i t i o n s ( 2 3 ) , i t seemed r e a s o n a b l e t h a t p e p t i d e s c o u l d be r e a c t i v e i n t e r m e d i a t e s i n t h e M a i l l a r d process. I n view o f t h e l a t t e r p o s s i b i l i t y e x p e r i m e n t s were d e s i g n e d t o t e s t t h e e x t e n t o f S t r e c k e r d e g r a d a t i o n f o r s p e c i f i c amino a c i d s c o n t a i n e d w i t h i n s m a l l p e p t i d e s . Mixtures c o n t a i n i n g f r u c t o s e and e q u i m o l a r amounts o f p e p t i d e s o r m i x t u r e s o f c o r r e s p o n d i n g f r e e amino a c i d s were r o a s t e d a t 120°C f o r 30 m i n u t e s and headspace v o l a t i l e s were a n a l y z e d by HRGC ( T a b l e I I I ) . I n g e n e r a l , d i - and t r i p e p t i d e s r e a c t e d d i f f e r e n t l y than m i x t u r e s o f amino a c i d s a s e v i d e n c e d by t o t a l FID a r e a s and v o l a t i l e profiles. D i p e p t i d e s c o n t a i n i n g v a l i n e o r l e u c i n e produced s i g n i f i c a n t amounts o f S t r e c k e r a l d e h y d e s , 2 - m e t h y l p r o p a n a l and 3 - m e t h y l b u t a n a l i n s p i t e o f b l o c k e d amino o r c a r b o x y l g r o u p s . A l s o , 3 - m e t h y l b u t a n a l was a major p r o d u c t i n r e a c t i o n s o f t r i p e p t i d e s c o n t a i n i n g l e u c i n e as t h e c e n t r a l amino a c i d . P y r a z i n e s were minor r e a c t i o n p r o d u c t s t h a t were more e a s i l y


16. RIZZI


179


T a b l e I I I . Headspace A n a l y t i c a l Data f o r R e a c t i o n s o f F r u c t o s e P l u s P e p t i d e s o r Amino A c i d s

T o t a l FID Peptide or Amino A c i d s Area/1000 41.0 Val,Gly 14.9 Val-Gly 83.0 Gly-Val Leu,Gly 890 343 Leu-Gly 134 Gly-Leu Ala,Leu,Gly 710 Ala-Leu-Gly 600 12.0 Gly-Leu-Ala 2-MP = 2 - m e t h y l p r o p a n a l , 3-MB ionization detector

X o f FID A r e a Major P y r a z i n e s S t r e c k e r RCH0 2,5 + 2,6-DiMe 3-MB 2-MP 0.1 1.2 81 Trace 2.4 16 Trace Trace 10 0.001 Trace 93 Trace 97 Trace Trace 90 Trace 0.005 88 Trace 0.01 1.2 4.3 0.2 69 Trace = 3-me t h y l b u t a n a l , FID = flame

measured by a n i t r o g e n s e l e c t i v e d e t e c t o r ( T a b l e I V ) . P e p t i d e r e a c t i o n s l e d t o d i f f e r e n t p y r a z i n e d i s t r i b u t i o n s f o r each s u b s t r a t e suggesting that p a r t i c u l a r peptides could, at l e a s t i n t h e o r y s e r v e a s u n i q u e p r e c u r s o r s o f c o m p o s i t e f o o d aromas. C h e m i c a l l y , i t v a s i n t e r e s t i n g t h a t the p e p t i d e s G l y - V a l , G l y - L e u and A l a - L e u - G l y produced r e l a t i v e l y more p y r a z i n e s than r e s p e c t i v e amino a c i d m i x t u r e s . Based on t h e e x t e n s i v e S t r e c k e r a c t i v i t y i t v o u l d appear t h a t f r u c t o s e r e a c t i o n s f a c i l i t a t e p e p t i d e h y d r o l y s i s , a n o r m a l l y s l o v p r o c e s s a t 120°C. Paper c h r o m a t o g r a p h i c a n a l y s i s o f p e p t i d e / f r u c t o s e r e a c t i o n m i x t u r e s shoved o n l y t r a c e s o f u n r e a c t e d s t a r t i n g m a t e r i a l and no e v i d e n c e f o r f r e e amino a c i d s . A l s o , c o n t r o l r e a c t i o n s i n v h i c h s o r b i t o l vas s u b s t i t u t e d f o r f r u c t o s e l e d t o no e v i d e n c e f o r S t r e c k e r d e g r a d a t i o n .

T a b l e IV. Headspace A n a l y t i c a l D a t a f o r R e a c t i o n s o f F r u c t o s e P l u s P e p t i d e s o r Amino A c i d s Peptide or T o t a l NPD Amino A c i d s Area/1000 4.8 Val,Gly 2.4 Val-Gly 4.2 Gly-Val 5.7 Leu,Gly 2.0 Leu-Gly 4.0 Gly-Leu 8.5 Ala,Leu,Gly 10.5 Ala-Leu-Gly 2.8 Gly-Leu-Ala NPD = N i t r o g e n - p h o s p h o r u s

A l k y l p y r a z i nés (X o f NPD) TetraMe TriMe Me DiMe(3) E t h y l 0.8 1.1 12 0.3 5.1 0.4 0 0 2.7 0 10 3.1 1.3 25 3.8 2.5 0.2 7.6 4.1 11 0.5 1.3 0 5 0 0.9 9.4 27 1.3 3.7 0 9.6 0 15 4.6 0 13 57 0 3.5 0 14 27 0 0 detector


Total 19 3 47 31 9 46 38 84 44

180


Acknowledgment t s I thank L. V. Haynes and A. R. S t e i m l e f o r HRGC method development and a n a l y s e s , P. R. K e l l e r and G. J . Harvey f o r o b t a i n i n g t h e mass s p e c t r a and P. R. Bunke f o r t e c h n i c a l a s s i s t a n c e .

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21. Qvist, I. H.; Von Sydov, E. C. F. J. Agric. Food Chem. 1974, 22, 1077-1084. 22. Nakanishi, T.; Itoh, T. Agric. Biol. Chem. 1967, 31, 1066-1069. 23. Fujimaki, M.; Kato,H.;Hayase, F. Agric. Biol. Chem. 1972, 36, 416-425.


Received January 6, 1989


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