A New Mechanism of the Maillard Reaction Involving Sugar

2 A

New

Mechanism

of t h e

Maillard

Reaction

Involving Sugar Fragmentation and

Free

Radical

Formation

Downloaded by PURDUE UNIV on November 7, 2014 | http://pubs.acs.org Publication Date: April 29, 1983 | doi: 10.1021/bk-1983-0215.ch002

MITSUO NAMIKI and TATEKI HAYASHI Nagoya University, Department of Food Science and Technology, Nagoya, Japan 464 Analyses of the hyperfine structures of ESR spectra found at an early stage of the Maillard reaction led to the assignment that the radical products are Ν,Ν -disubstituted pyrazine cation radicals. Quantitative product determination at the reaction stage involving the free radicals indicates a sequential formation of glucosylamine, a 2-carbon and other sugar fragmentation products, then certain reducing products and the free radical, the Amadori product, and finally, glucosones. N,N' -dialkylpyrazines, or mixtures of glycolaldehyde with amino compounds, are shown to be highly active in free radical formation as well as in browning. Thus we propose the existence of a new pathway to browning in the Maillard reaction, involving sugar fragmentation and free radical formation prior to the Amadori rearrangement. '

The mechanism proposed by Hodge in 1953 (1) for the early stages of the Maillard reaction, involving the Amadori rearrange ment as a key step, has been accepted over a quarter of a century as a most apt description. Here we propose a new mechanism which involves cleavage of the sugar molecule with generation of a highly reactive two-carbon fragment at an early stage of the Maillard reaction, prior to the Amadori rearrangement. We were led to the idea of sugar fragmentation by our finding of the development of a novel free radical product at an early stage in the Maillard reaction, prior to browning Ç2). Based on the hyperfine structural analyses of ESR spectra of various sugaramino compound systems, the structures of the radical products were assigned as Ν,N -dialkylpyrazine cation radicals (3), and it was assumed that each alkyl-substituted nitrogen originated from the amino compound, and each two-carbon moiety of the pyrazine ring from the sugar fragment. By isolation and identification of glyoxal derivatives from the sugar-amine reaction system (4) we 3

1

0097-6156/83/0215-0021$07.50/0 © 1983 American Chemical Society In The Maillard Reaction in Foods and Nutrition; Waller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

22

MAILLARD

REACTIONS

have also e s t a b l i s h e d that the two-carbon fragmentation of the sugar molecule occurs p r i o r to Amadori rearrangement. The present paper describes our recent s t u d i e s on t h i s new mechanism of the M a i l l a r d r e a c t i o n (_5,6) .


Development of Novel M a i l l a r d Reaction

Free R a d i c a l s i n the E a r l y

Stage

of

the

With the s i n g l e exception of the s t a b l e free radical observed i n melanoidin prepared from the g l y c i n e - g l u c o s e r e a c t i o n (7), there have been no r e p o r t s of f r e e r a d i c a l formation i n the e a r l y stages of the M a i l l a r d r e a c t i o n . We have observed the development of ESR spectra with c h a r a c t e r i s t i c hyperfine s t r u c t u r e s at an e a r l y stage i n the M a i l l a r d r e a c t i o n (_2). Figure 1 shows the changes with time i n i n t e n s i t i e s of the ESR signal with h y p e r f i n e s t r u c t u r e , the ESR s i g n a l w i t h broad s i n g l e t , and browning during the r e a c t i o n of D-glucose with a- or (3-alanine i n b o i l i n g water; the ESR s p e c t r a with hyperfine s t r u c t u r e are shown f o r each r e a c t i o n mixture at the maximum i n t e n s i t y . In the case of glucose-g-alanine the ESR s i g n a l with h y p e r f i n e s t r u c t u r e could be detected as soon as the r e a c t i o n mixture was heated; the relative intensity increased r a p i d l y during about ten min and then decreased rapidly, with simultaneous l o s s of h y p e r f i n e structure. The disappearance of h y p e r f i n e s t r u c t u r e i n the ESR spectrum was a l s o accompanied by a gradual i n c r e a s e i n browning and development of a melanoidin-type ESR s i g n a l with broad singlet. We are concerned here only with the ESR s p e c t r a with hyperfine s t r u c t u r e , due to the new f r e e r a d i c a l products. The ESR s p e c t r a of r e a c t i o n mixtures of glucose with aand β-alanine d i f f e r e d from each other i n the complexity of their hyperfine s t r u c t u r e , the former being s p l i t i n t o 19 l i n e s and the l a t t e r i n t o 25 l i n e s . T h i s d i f f e r e n c e was shown to depend on differences i n the s t r u c t u r e of amino a c i d , a- and 3-alanine, by comparison with s p e c t r a obtained from various sugar-amino acid systems as shown i n Table I. A l l the sugars and t h e i r r e l a t e d carbonyl compounds gave e s s e n t i a l l y the same type of ESR spectrum w i t h a given amino a c i d , with the exception of glyceraldehyde and dihydroxyacetone. The l a t t e r two showed s p e c t r a resembling each other and with more complicated h y p e r f i n e s t r u c t u r e s than the others. The carbonyl compounds most e f f e c t i v e i n formation of free radicals are a l s o most e f f e c t i v e i n browning, and the f a c t that glycolaldehyde has e s p e c i a l l y high a c t i v i t y i n both r e a c t i o n s i s p a r t i c u l a r l y s i g n i f i c a n t i n t h i s study. Such carbonyl compounds as furfural and crotonaldehyde showed high a c t i v i t y only i n browning, and examination of s t r u c t u r e s of v a r i o u s carbonyl compounds suggest that the presence of an e n e d i o l or a p o t e n t i a l e n e d i o l grouping i n the carbonyl compounds i s necessary f o r r a d i c a l formation. Investigations of v a r i o u s amino compounds i n d i c a t e d that

In The Maillard Reaction in Foods and Nutrition; Waller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.


2.

ΝΑΜίκι AND HAYASHI

New Mechanism of Maillard Reaction

23

Heating Time (min) Figure 1. Free radical formation and browning in the reaction of Ό-glucose with α-alanine or β-alanine (each 3 M), and ESR spectra of the reaction mixtures heated in a boiling water bath. Key: O, ESR signal with hyperfine structure; , ESR signal with broad line; and A, browning.


24

MAILLARD REACTIONS

Table I ESR S p e c t r a l Data on Free R a d i c a l s and Browning i n the Reaction of Sugars and Other Carbonyl Compounds w i t h a- or 3-Alanine*


ESR Spectra S p l i t t i n g Line No. Intensity

Browning

a-Alanine D-Glucose D-Fructose D-Arabinose D-Xylose D-Ribose Glycolaldehyde

19 19 19 19 19 19

+ + + + + +++

+ + 4+ ++ +++ -H-+

25 25 25 25 25 35 ~ 35 ~ 25 25

++ ++ ++ -H++ +++ +++ ++-H++

++ ++ -H-H-H-hf M M M M I M M I M MI M M ++

25

++

3-Alanine D-Glucose D-Fructose D-Arabinose D-Xylose D-Ribose Glyceraldehyde Dihydroxyacetone Glycolaldehyde 3-Deoxyglucosone 5-Hydroxymethylfurfural Furfural Glyoxal Crotonaldehyde Propionaldehyde

M M I ++HMil +

*Aqueous s o l u t i o n s (each 3 M) were heated i n b o i l i n g water bath.


2.

ΝΑΜίκι AND

HAYASHi


25

ability to develop an ESR spectrum with the characteristic hyperfine s t r u c t u r e was only observed i n compounds with a primary amino group, although c e r t a i n amino compounds, such as aniline, c y s t e i n e , and ethylenediamine?failed to show the spectrum (3).


E f f e c t of Reaction Free Radicals Ç2)

Conditions on Development and

Stability

of

pH seemed the most important f a c t o r i n the M a i l l a r d r e a c t i o n for determining the rates of browning as w e l l as r e a c t i o n processes. As shown i n F i g . 2a, f r e e r a d i c a l development was observed even at n e u t r a l pH, and, l i k e browning, was enhanced markedly by increase i n pH, although the ESR s i g n a l disappeared r a p i d l y at pH above 11. The f r e e r a d i c a l formed i s known to be fairly s t a b l e i n weakly a c i d i c r e a c t i o n mixture, although i t i s unstable i n moderately a l k a l i n e s o l u t i o n ( F i g . 2b). It i s known that oxygen plays an important r o l e i n f r e e r a d i c a l r e a c t i o n s , and r a d i c a l s u s u a l l y disappear r a p i d l y i n the presence of oxygen. I n t e r e s t i n g l y , the f r e e r a d i c a l formed i n the early stages of the M a i l l a r d r e a c t i o n i s f a i r l y s t a b l e i n the r e a c t i o n mixture, as the ESR s i g n a l could be observed i n the mixture on heating i n an open t e s t tube without e x c l u s i o n of a i r , although i t was abolished r a p i d l y by bubbling a i r . Attempts to i s o l a t e the r a d i c a l product are as yet unsuccessful. A n a l y s i s of Hyperfine

Structure i n ESR

Spectra

(3)

To e l u c i d a t e the s t r u c t u r e of the f r e e r a d i c a l products, analyses were made of the hyperfine s t r u c t u r e s of ESR spectra of various r e a c t i o n systems of sugar w i t h amino compounds. Figure 3 shows analyses of r e p r e s e n t a t i v e ESR spectra of the reaction mixtures of D-glucose with α-alanine or β-alanine. The hyperfine s t r u c t u r e s of (A) could be resolved i n t o 8.41 G q u i n t e t , 2.86 G quintet and 2.93 G t r i p l e t due to two equivalent nitrogens, four equivalent protons and two equivalent protons, r e s p e c t i v e l y . On the other hand, (B) was resolved i n t o the 8.17 G q u i n t e t , 2.84 G quintet and 5.36 G q u i n t e t , due to two equivalent nitrogens, four equivalent protons and four equivalent protons, r e s p e c t i v e l y , as i n d i c a t e d by the s t i c k diagrams and s p l i t t i n g constants i n each figure. The r e s u l t s of analyses of the ESR spectra of various r e a c t i o n systems are summarised i n Table I I . I t was shown that all spectra have i n common the s p l i t t i n g s arising from two equivalent nitrogens (about 8.2 G) and four equivalent protons (about 3.0 G), and a d d i t i o n a l l y from an even number of equivalent protons with d i f f e r e n t s p l i t t i n g constants. These assignments l e t to the reasonable assumption that the r a d i c a l products are N,N disubstituted pyrazine c a t i o n r a d i c a l d e r i v a t i v e s , as shown i n F i g . 4. This assumption was s t r o n g l y supported by the f a c t that the hyperfine s t r u c t u r e as w e l l as the g-value of the ESR !


MAILLARD REACTIONS


26

Figure 3. ESR spectra of the reaction mixtures of (Λ) glucose-a-alanine and (B) glucose-β-alanine. (Reproduced from Ref. 3. Copyright 1977, American Chemical Society.)


2.

NAMIKI AND HAYASHI


27

Table I I A n a l y s i s of Hyperfine Structures of ESR Spectra

Amino acide or Amine

Splitting

constants (G)


(a-H) Glycine a-Alanine β-Alanine Valine Phenylalanine Other amino a c i d s * t-Butylamine Methylamine Me t hy l-d_ 3-amine Ethylamine N,N -Diethylpyrazinium s a l t f

4.69 2.93 5. 36 1.43 1.31 1.5±0.1

(4H) (2H) (4H) (2H) (2H) (2H)

7.98 1.25 5. 37 5. 33

(6H) (6D) (4H) (4H)

(2-N)

(4-H)

8.15 8.41 8.15 8.40 7.99 8.310.15 8.74 8.35 8.38 8.35 8.37

3.04 2.86 2.84 2.88 3.03 2.9±0.1 2.74 2.83 2.86 2.85 2.82

Sugar; D-glucose, D-arabinose, D-xylose or glycolaldehyde. *; s e r i n e , methionine, l e u c i n e , i s o l e u c i n e , t y r o s i n e , arginine.

H

A New Mechanism of the Maillard Reaction Involving Sugar

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