Characterization of Antioxidative Maillard Reaction Products from

16 Characterization of Antioxidative Maillard Reaction Products from Histidine and Glucose H. LINGNERT and C. E . ERIKSSON SIK-The Swedish Food Institute, Box 5401, S-402 29 Göteborg, Sweden

Downloaded by FUDAN UNIV on April 6, 2017 | http://pubs.acs.org Publication Date: April 29, 1983 | doi: 10.1021/bk-1983-0215.ch016

G. R. WALLER Oklahoma State University, Department of Biochemistry, Stillwater, OK 74078

Maillard reaction products (MRP) from histidine and glucose were fractionated by various methods to isolate the antioxidative products. Upon dialysis through membranes with a nominal molecular weight cut-off of 1000 daltons, antioxidative components were concentrated in the retentate. Further purification of the antioxidative material was obtained by isoelectric precipitation of the retentate at pH 5.0. The precipitate was considerably more antioxidative than the supernatant. The precipitate was fractionated by preparative electrophoresis. The most antioxidative electrophoresis fraction is now being analyzed by spectrometric methods. Its C, Η, Ν, O content was determined and EPR studies showed it to contain stable free radicals. EPR studies of several fractions of the histidine-glucose reaction mixture showed good agree ment between intensity of EPR signal and antioxidative effect. The free radicals are suggested to be of importance for the antioxidative mechanism of the MRP. U n s a t u r a t e d f a t t y acids i n foods a r e very s u s c e p t i b l e to o x i d a t i o n by oxygen in the a i r during processing and s t o r a g e . T h e o x i d a t i o n r e s u l t s i n i t i a l l y i n the f o r m a t i o n o f f a t t y a c i d hydroperoxides by a f r e e r a d i c a l chain mechanism. T h e h y d r o p e r o x i d e s a r e subject t o s e v e r a l f u r t h e r r e a c t i o n s f o r m i n g secondary products such as aldehydes, ketones, and o t h e r v o l a t i l e compounds, many o f w h i c h a r e odorous and cause r a n c i d f l a v o r i n the f o o d . This d e v e l o p m e n t o f r a n c i d f l a v o r l i m i t s t h e storage s t a b i l i t y o f a l a r g e number o f f o o d p r o d u c t s . The foods c a n be p r o t e c t e d against l i p i d o x i d a t i o n e i t h e r by t h e a d d i t i o n o f a n t i o x i d a n t s or by p a c k a g i n g i n v a c u u m or i n e r t gases t o e x c l u d e oxygen. T h e a n t i o x i d a n t s c a n be o f various types. They c a n work as " c h a i n - b r e a k e r s " t h a t i n t e r f e r e w i t h t h e f r e e r a d i c a l c h a i n r e a c t i o n , as " m e t a l i n a c t i v a t o r s " , t h a t bind o t h e r w i s e p r o - o x i d a t i v e m e t a l s , or as " p e r o x i d e d e s t r o y e r s " , w h i c h r e a c t w i t h h y d r o p e r o x i d e s t o give s t a b l e products by n o n r a d i c a l processes (1).

0097-6156/83/0215-0335$06.00/0 © 1983 American Chemical Society Waller and Feather; The Maillard Reaction in Foods and Nutrition ACS Symposium Series; American Chemical Society: Washington, DC, 1983.


336

MAILLARD

REACTIONS

The most c o m m o n a n t i o x i d a n t s are phenols, such as b u t y l a t e d h y d r o x y a n i s o l e ( B H A ) or b u t y l a t e d h y d r o x y t o l u e n e (BHT). Increasing i n t e r e s t has, however, been d i r e c t e d t o w a r d s the u t i l i z a t i o n of n o r m a l food c o n s t i t u e n t s w i t h a n t i o x i d a t i v e p r o p e r t i e s (2). A m o n g those, the M a i l l a r d r e a c t i o n products ( M R P ) m i g h t be of s p e c i a l i m p o r t a n c e , since they are so widespread in foods. The f i r s t r e p o r t on a n t i o x i d a t i v e e f f e c t of M R P was made by F r a n z k e and Iwainsky (3). S h o r t l y a f t e r w a r d G r i f f i t h and Johnson (4) r e p o r t e d that the a d d i t i o n of glucose to c o o k i e dough r e s u l t e d in a b e t t e r s t a b i l i t y against o x i d a t i v e r a n c i d i t y during storage of the c o o k i e s . R e s e a r c h on a n t i o x i d a t i v e M R P was then m a i n l y p e r f o r m e d by groups in J a p a n . A s y m p o s i u m on M a i l l a r d R e a c t i o n s in F o o d held in U d d e v a l l a , Sweden, 1979 i n c l u d e d also the aspect of a n t i o x i d a t i v e p r o p e r t i e s . The c o n t r i butions on t h i s subject c o n t a i n e d also b r i e f r e v i e w s (5, 6, 7). M o s t of the work has been done on m o d e l systems. Some a p p l i c a t i o n s in food systems have, h o w e v e r , also been r e p o r t e d (8 - 11). I d e n t i f i c a t i o n of a n t i o x i d a t i v e M R P K n o w l e d g e about the c h e m i c a l s t r u c t u r e of the a n t i o x i d a t i v e M R P is very l i m i t e d . O n l y a f e w a t t e m p t s have been made to c h a r a c t e r i z e t h e m . Evans, et a l . (12) d e m o n s t r a t e d t h a t pure r e d u c t o n e s produced by the r e a c t i o n b e t w e e n hexoses and secondary amines were e f f e c t i v e in i n h i b i t i n g o x i d a t i o n of v e g e t a b l e oils. The i m p o r t a n c e of r e d u c t o n e s f o r m e d f r o m a m i n o acids and r e d u c i n g sugars is, h o w e v e r , s t i l l obscure. E i c h n e r (6) suggested t h a t r e d u c t o n e - l i k e compounds, 1,2-enaminols, f o r m e d f r o m A m a d o r i r e a r r a n g e m e n t p r o d u c t s c o u l d be responsible f o r the a n t i o x i d a t i v e e f f e c t of M R P . The m e c h a n i s m was c l a i m e d to i n v o l v e i n a c t i v a t i o n of l i p i d h y d r o p e r o x i d e s . On the other hand, Y a m a g u c h i , et a l . (5) found most of the a n t i o x i d a t i v e e f f e c t in the m e l a n o i d i n f r a c t i o n . By various c h r o m a t o g r a p h i c methods they p u r i f i e d an a n t i o x i d a t i v e p r o d u c t f r o m g l y c i n e and xylose having a m o l e c u l a r weight of a p p r o x i m a t e l y 4500. P o s s i b l y s e v e r a l d i f f e r e n t compounds f o r m e d by the M a i l l a r d r e a c t i o n can e x h i b i t a n t i o x i d a t i v e p r o p e r t i e s . T h e i r f o r m a t i o n m i g h t be d e p e n dent on what r e a c t a n t s are used and on the r e a c t i o n c o n d i t i o n s ( t e m p e r a t u r e , t i m e , w a t e r c o n t e n t etc.) The a i m of the present work was to c h a r a c t e r i z e the a n t i o x i d a n t s f o r m e d in the r e a c t i o n b e t w e e n h i s t i d i n e and glucose in order to e l u c i d a t e the m e c h a n i s m of t h e i r a n t i o x i d a t i v e a c t i o n . The c o m b i n a t i o n h i s t i d i n e - g l u c o s e was chosen since it p r e v i o u s l y was found to be one of the most e f f e c t i v e c o m b i n a t i o n s in m o d e l s y s t e m s (13). Synthesis of M R P M a i l l a r d r e a c t i o n products w e r e o b t a i n e d by r e f l u x i n g 100 m l of d i s t i l l e d w a t e r c o n t a i n i n g 0.1 m o l L - h i s t i d i n e m o n o h y d r o c h l o r i d e m o n o h y d r a t e and 0.05 m o l D - g l u c o s e f o r 20 h. The p H of the r e a c t i o n m i x t u r e was adjusted to 7.0 w i t h potassium h y d r o x i d e b e f o r e s t a r t i n g the reaction.

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

16.

LiNGNERT E T A L .

Characterization of Histidine-Glucose Products

337

Measurement of antioxidative e f f e c t The a n t i o x i d a t i v e e f f e c t o f the various f r a c t i o n s of M R P was e v a l u a t e d by a p r e v i o u s l y described p o l a r o g r a p h i c m e t h o d (14).


Separation of antioxidative M R P In the course o f t r y i n g to i s o l a t e the a n t i o x i d a t i v e M R P , we found that part o f t h e a n t i o x i d a t i v e e f f e c t was lost during t h e i s o l a t i o n processes. This could be e x p l a i n e d in t w o ways. E i t h e r the a n t i o x i d a n t s were unstable or t h e r e were d i f f e r e n t a n t i o x i d a n t s i n t h e r e a c t i o n m i x t u r e a c t i n g s y n e r g i s t i c a l l y , r e s u l t i n g in a l o w e r e f f e c t when such a n t i o x i d a t i v e components had been s e p a r a t e d . Since r e c o m b i n a t i o n of t h e various f r a c t i o n s f a i l e d to r e s t o r e the o r i g i n a l a n t i o x i d a t i v e e f f e c t , w e studied the s t a b i l i t y of the a n t i o x i d a n t s in more d e t a i l . The a n t i o x i d a n t s w e r e then found to be s e n s i t i v e to oxygen (15). W i t h t h e knowledge o f the i n s t a b i l i t y of the a n t i o x i d a n t s , s p e c i a l c a r e was used t o a v o i d c o n t a c t w i t h oxygen i n e v e r y single s e p a r a t i o n step. A l l solvents were degassed and bubbled w i t h n i t r o g e n p r i o r t o use and t h e various f r a c t i o n s w e r e f r o z e n as soon as possible. Dialysis P r e v i o u s l y we r e p o r t e d that the a n t i o x i d a n t s f r o m h i s t i d i n e and glucose c o u l d be c o n c e n t r a t e d by u l t r a f i l t r a t i o n (7). T h e r e s u l t s i n d i c a t e d that the a n t i o x i d a t i v e compounds had a m o l e c u l a r weight of more than 1000. T h e r e f o r e , we f u r t h e r studied t h e p o s s i b i l i t y o f s e p a r a t ing the a n t i o x i d a n t s w i t h r e s p e c t to t h e i r m o l e c u l a r s i z e by dialysis. D i a l y s i s tubing w i t h a m o l e c u l a r w e i g h t c u t - o f f o f 1000 daltons was used (Spectrapor 6, S p e c t r u m M e d i c a l Industries Inc., L o s A n g e l e s , C A ) . A 1 5 - m l p o r t i o n of t h e M a i l l a r d r e a c t i o n m i x t u r e was t r a n s f e r r e d to e a c h of f i v e dialysis tubes. T h e contents of t h e f i v e tubes were d i a l y z e d t o g e t h e r against 6.5 1 o f degassed, n i t r o g e n - b u b b l e d , d i s t i l l e d w a t e r a t 5 C . A f t e r 12 h one o f the tubes was w i t h d r a w n and the other four w e r e t r a n s f e r r e d t o a new d i a l y s i s tank c o n t a i n i n g 6.5 1 of w a t e r and d i a l y s i s c o n t i n u e d f o r another 1 2 - h p e r i o d . A g a i n one of t h e tubes was w i t h d r a w n w h i l e d i a l y s i s was r e n e w e d f o r t h e other t h r e e f o r another 12-h period and so on. F i g u r e 1 shows t h e a n t i o x i d a t i v e e f f e c t and t h e t o t a l amount of m a t e r i a l in each o f t h e f i v e r e t e n t a t e s and i n e a c h o f the f i v e d i a l y s a t e s . The t o t a l amounts o f m a t e r i a l (open bars) a r e given as p e r c e n t a g e o f t h e o r i g i n a l m a t e r i a l in one d i a l y s i s tube. The a n t i o x i d a t i v e e f f e c t ( f i l l e d bars) is c o m p a r e d on an e q u a l - w e i g h t basis. A f t e r the t h i r d d i a l y s i s no f u r t h e r i m p r o v e m e n t of the a n t i o x i d a t i v e e f f e c t o f t h e r e t e n t a t e was o b t a i n e d , even though s m a l l a m o u n t s of less a n t i o x i d a t i v e m a t e r i a l s t i l l passed t h e m e m b r a n e and w e r e found in t h e d i a l y s a t e . A f t e r d i a l y s i s f o r 3 χ 12 h only 3.5 % o f t h e o r i g i n a l m a t e r i a l r e m a i n e d in the r e t e n t a t e . Precipitation A f t e r t h e f o u r t h dialysis some p r e c i p i t a t e could be observed i n t h e r e t e n t a t e in t h e dialysis tube. T h e p r e c i p i t a t i o n was found to be p H dependent; the p r e c i p i t a t e was r e v e r s i b l y dissolved when p H was e i t h e r i n c r e a s e d or d e c r e a s e d . T h e i n f l u e n c e o f p H on t h e


338

MAILLARD REACTIONS


Original material

Retentate

Dialysate TIME OF DIALYSIS

100

50 AMOUNT

0

OF MATERIAL

4 ANTIOXIDATIVE

2

0

0

EFFECT

I

OF MATERIAL

2 ANTIOXIDATIVE

I

100

50 AMOUNT

(%)

=

AMOUNT

=

ANTIOXIDATIVE

(%)

4 EFFECT

OF MATERIAL EFFECT

Figure 1. Antioxidative effect and amount of material in retentates and dialysates after dialysis of histidine-glucose reaction mixture up to five times 12 h through dialysis tubing with a molecular weight cut-off of 1000 daltons.



16.

LINGNERT

ET AL.


339

p r e c i p i t a t i o n is shown in F i g u r e 2, where the absorption a t 450 n m o f t h e s o l u t i o n is given as a f u n c t i o n o f p H . Since t h e p r e c i p i t a t e w a s b r o w n c o l o r e d , t h e c o l o r i n t e n s i t y o f t h e supernatant could i n this w a y be used as a measure o f t h e e x t e n t o f p r e c i p i t a t i o n . It c a n be seen t h a t m a x i m u m p r e c i p i t a t i o n was o b t a i n e d a t p H 5.0. A t p H l o w e r than 3 or higher t h a n 7 no p r e c i p i t a t i o n c o u l d be observed. This p r e c i p i t a t i o n o c c u r r e d only w i t h t h e d i a l y z e d m a t e r i a l . S m a l l amounts o f p r e c i p i t a t e w e r e o b t a i n e d a l r e a d y w i t h t h e f i r s t r e n t e n t a t e , when adjusting t h e p H to 5.0. The crude r e a c t i o n m i x t u r e was, h o w e v e r , f u l l y soluble a l l over this p H range. Obviously salts o r o t h e r m a t e r i a l o f l o w m o l e c u l a r w e i g h t prevent t h e p r e c i p i t a t i o n . A d d i t i o n o f salts t o t h e d i a l y z e d m a t e r i a l was also shown to decrease t h e e x t e n t o f t h e i s o electric precipitation. Since t h e p r e c i p i t a t e was by f a r more a n t i o x i d a t i v e than the s u p e r natant or t h e o r i g i n a l r e t e n t a t e , when c o m p a r e d on a w e i g h t basis, t h e i s o e l e c t r i c p r e c i p i t a t i o n c o u l d be used as one f u r t h e r step i n t h e p u r i f i a c t i o n sequence. Electrophoresis A s o l u t i o n o f the p r e c i p i t a t e was f r a c t i o n a t e d by p r e p a r a t i v e paper e l e c t r o p h o r e s i s at p H 1.9 ( a c e t i c a c i d - f o r m i c a c i d b u f f e r ) . A 3 0 - m g p o r t i o n of t h e p r e c i p i t a t e was dissolved i n 0.45 m l of the b u f f e r and a p p l i e d ^ s a 2 0 - c m band on t h e paper ( W h a t m a n C h r o m a t o g r a p h y P a p e r 3 -j^j). T h e sample was c h r o m a t o g r a p h e d a t 3000 V f o r 30 m i n .

T a b l e I.

A n t i o x i d a t i v e e f f e c t and amount o f m a t e r i a l i n e a c h f r a c t i o n a f t e r f r a c t i o n a t i o n of p r e c i p i t a t e by paper e l e c t r o p h o r e s i s .

Fraction No.

Amount of material (%)

Original precipitate

100

Antioxidative effect

2.0

1

8

0.6

2

5

0.5

3

31

1.1

4

43

1.7

5

22

0.7



340

MAILLARD REACTIONS

0.6

o

ο Η δ 2

>

3 >

16.

LiNGNERT E T A L .


345

of l o w m o l e c u l a r w e i g h t , possibly f r e e r a d i c a l s , adsorbed on the m a i n c o m p o n e n t o f higher m o l e c u l a r w e i g h t . C a u t i o n must t h e r e f o r e be used when drawing c o n c l u s i o n s about the a n t i o x i d a t i v e m e c h a n i s m based on i n f o r m a t i o n about t h e m a i n c h e m i c a l s t r u c t u r e s in the f r a c t i o n .

Literature cited 1. 2.


3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

Frankel E.N. Prog. Lipid Res. 1980, 19, 1-22. Dugan, L.R. "Autoxidation in Food and Biological Systems"; Plenum Press: New York, 1980; p. 261. Franzke, C ; Iwansky, H. Deut. Lebensm.-Rundschau 1954, 50, 251-254. Griffith, T.; Johnson, J.A. Cereal Chem. 1957, 34, 159-169. Yamaguchi, N.; Koyama, Y.; Fujimaki, M. Prog. Food Nutr. Sci. 1981, 5, 429-439. Eichner, K. Prog. Food Nutr. Sci. 1981, 5, 441-451 Lingnert, H.; Eriksson, C . E . Prog. Food Nutr. Sci. 1981, 5, 453-466. Tomita, Y. Kaqoshima Daigaku Noqakubu Gukujutusu Hokoku 1972, 22, 115-121; Chem. Abstr. 1973, 78, 96215h. Lingnert, H. J. Food Process. Preserv. 1980, 4, 219-233. Lingnert, H.; Lundgren, B. J . Food Process. Preserv. 1980, 4, 235-246. Vandewalle, L.; Huyghebaert, A. Med. Fac. Landbouww. Rijksuniv. Gent 1980, 45, 1277-1286. Evans, C.D.; Moser, H.A.; Cooney, P.M.; Hodge, J.E. J . Am. Oil Chem. Soc. 1958, 35, 85-88. Lingnert, H.; Eriksson, C.E. J . Food Process. Preserv. 1980, 4, 161-172. Lingnert, H.; Vallentin, K.; Eriksson, C.E. J . Food Process. Preserv. 1979, 3, 87-103. Lingnert, H.; Waller, G.R. J . Agric. Food Chem. In press. Namiki, M.; Hayashi, T. J . Agric. Food Chem. 1975, 23, 487-491. Lessig, U.; Baltes, W. Z. Lebensm. Unters. Forsch. 1981, 173, 435-444. Kajimoto, G.; Yoshida, H. Yukaqaku 1975, 25, 297-300; Food Sci. Techn. Abstr. 1976, 8, 6N222. Gomyo, T.; Horikoshi, M. Agric. Food Chem. 1976, 40, 33-40.

RECEIVED November 4, 1982


Characterization of Antioxidative Maillard Reaction Products from

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