Loss of Available Lysine in Protein in a Model Maillard Reaction System

20 Loss of Available Lysine in Protein in a Model Maillard Reaction System 1

BARBEE W. TUCKER, VICTOR RIDDLE, and JOHN LISTON

Downloaded by CORNELL UNIV on September 9, 2016 | http://pubs.acs.org Publication Date: April 29, 1983 | doi: 10.1021/bk-1983-0215.ch020

University of Washington, Institute for Food Science and Technology, College of Ocean and Fishery Sciences, Seattle, WA 98195

A model Maillard reaction system was developed to measure the loss of available lysine in a pure protein system after reaction with two keto-aldehydes normally present in food systems. Bovine plasma albumin and malonaldehyde or methylglyoxal reactions were measured in an aqueous solution. Conditions of reaction were varied over a range representative of conditions during handling and storage of food. pH was adjusted between 5 and 8. Temperatures in the range 20-60°C were tested. Reactant concentrations of 0.07 to 35.0 mM carbonyl compound and 1 to 10 g/L albumin were used. Loss of available lysine was measured by a trinitrobenzenesulfonic acid method for residual lysine. With malonaldehyde, loss of available lysine decreased as pH increased, but with methylglyoxal, an increased pH increased the lysine loss. Both carbonyl compounds caused significant increasing loss of lysine with increasing temperature and concentration. Results indicated that methylglyoxal reacts with protein to effect a loss of available lysine, but more slowly than does malonaldehyde. Temperature had the greatest effect on lysine loss, but the concentration of the carbonyl compound and the pH of the reaction mixture also influenced the rate.

Of the eight amino acids e s s e n t i a l f o r man and other animals, l y s i n e i s the most e a s i l y damaged by processing and/or storage of food. This damage or m o d i f i c a t i o n r e s u l t s i n a reduction i n n u t r i t i o n a l a v a i l a b i l i t y of l y s i n e . A v a i l a b l e l y s i n e can be 1

Deceased 0097-6156/83/0215-0395$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.


396

MAILLARD

REACTIONS

u t i l i z e d f o r metabolism and i s d i s t i n g u i s h e d from t o t a l l y s i n e , which i n c l u d e s damaged or bound l y s i n e . I t has been shown i n experiments with growing c h i c k s that no c o r r e l a t i o n e x i s t s between growth and t o t a l l y s i n e while there i s a good c o r r e l a t i o n between growth and unbound l y s i n e , suggesting that l y s i n e i s not used b i o l o g i c a l l y unless the ε-amino group i s f r e e (1). Plant p r o t e i n s are o f t e n low i n l y s i n e content and i t i s the l i m i t i n g amino a c i d i n most c e r e a l s . Processing damage compounds the problem. Although l y s i n e i s r e l a t i v e l y abundant i n animal p r o t e i n , heat treatment and/or storage may render i t n u t r i t i o n a l l y u n a v a i l a b l e by promoting i t s i r r e v e r s i b l e r e a c t i o n with carbonyl compounds (jÎ.e_. M a i l l a r d r e a c t i o n s ) to form i n d i g e s t i b l e c o l o r l e s s browning intermediates. Carbonyl compounds are present i n foods as reducing sugars and sugar breakdown products, l i p i d o x i d a t i o n products, or carbonyl groupé of p r o t e i n s . M a i l l a r d r e a c t i o n s may be d i v i d e d i n t o three stages as described by Mauron (2): e a r l y , advanced, and f i n a l . Early M a i l l a r d r e a c t i o n s i n v o l v e a condensation between a carbonyl compound and a f r e e amino group of a p r o t e i n — i n t h i s instance the ε-amino of of l y s i n e — w h i c h i s r a p i d l y converted v i a S c h i f f bases and the Amadori rearrangement to the b i o l o g i c a l l y u n a v a i l able deoxyketosyl compound (_3). Whatever the source of the carbonyl group, the b a s i c r e a c t i o n rendering the l y s i n e u n a v a i l able as a biochemical component i s the same. Although v i s i b l e browning may not occur, l y s i n e l o s s i s i r r e v e r s i b l e and t h i s early-forming product i s the major form of blocked l y s i n e i n foods. The second stage i n v o l v e s a number of r e a c t i o n s producing v o l a t i l e or s o l u b l e substances and the t h i r d gives i n s o l u b l e brown polymers. Although the e a r l y M a i l l a r d r e a c t i o n products are reported to have a n t i o x i d a n t p r o p e r t i e s and, i n f a c t , can be u t i l i z e d by processors to i n h i b i t l i p i d o x i d a t i o n i n animal p r o t e i n foods such as f i s h products, there i s an accompanying l y s i n e l o s s (4,·5). Malonaldehyde (MA) i s a major end product of o x i d i z i n g or r a n c i d l i p i d s and i t accumulates i n moist f o o d s t u f f s (6). Several MA-protein systems have been s t u d i e d . Chio and Tappel combined RNAase and MA to demonstrate f l u o r e s c e n c e a t t r i b u t e d to a conju gated imine formed by c r o s s l i n k i n g two ε-amino groups with the dialdehyde (7). Shin studied the same r e a c t i o n and found i t to be dependent on pH and reactant concentrations (8). Crawford reported the r e a c t i o n between MA and bovine plasma albumin (BPA) also to be pH dependent, and of f i r s t order k i n e t i c s with a maximum r a t e near pH 4.30. At room temperature 50-60% of the ε-amino groups were m o d i f i e d — 4 0 % i n the f i r s t eight hours, the remainder over a p e r i o d of days (9). Another carbonyl compound o f t e n found i n food products i s the keto aldehyde methylglyoxal (MGA). Having the same e m p i r i c a l formula and molecular weight as MA, MGA has been of i n t e r e s t p r i m a r i l y as a g l y c o l y t i c by-pass intermediate. In foods d e r i v e d from animal t i s s u e MGA i s probably formed from

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


20.

TUCKER E T A L .

Loss of Lysine

397

in Protein

hydrated glyoxalate (dihydroxyacetate, DHA), the l a t t e r accumul a t i n g as a r e s u l t of impaired g l y c o l y s i s f o l l o w i n g death and subsequent f r e e z i n g , thawing, and/or storage (10). Riddle and Lorenz c l e a r l y demonstrated that MGA can be produced nonenzym a t i c a l l y as the r e s u l t of a polyvalent-anion-catalyzed r e a c t i o n (11). I t a l s o occurs as a product o f autoclaved glucose, unsaturated f a t t y a c i d o x i d a t i o n , and food i r r a d i a t i o n (12,13,14). Hodge demonstrated i t s p a r t i c i p a t i o n i n browning r e a c t i o n s and i t or i t s precursor DHA i s c u r r e n t l y a component of quick- or i n s t a n t - t a n n i n g l o t i o n s (15). At the pH of most foods and i n moist systems MA e x i s t s as the enol tautomer and an enamine d e r i v a t i v e would be the i n i t i a l product while MGA would form, more slowly, the more h i g h l y colored imine d e r i v a t i v e . This study was undertaken to f u r t h e r i n v e s t i g a t e the r e l a t i o n s h i p s between food system conditions and damage to l y s i n e . Experimental A model system demonstrating the n u t r i t i o n a l d e s t r u c t i o n of l y s i n e i n bovine plasma albumin (BPA) by r e a c t i o n with e i t h e r a dialdehyde (MA) or a keto-aldehyde (MGA) was studied i n r e l a t i o n to r e a c t i o n rates as a f f e c t e d by pH, temperature, r e a c t i o n time and carbonyl concentration. The BPA was F r a c t i o n V obtained from Schwartz/Mann and had a molecular weight of 69 x 1 0 with s i x t y l y s i n e residules/mole, an assayed content of 11.4%. I t was d i s s o l v e d i n 0.0200 M phosphate-citrate b u f f e r adjusted to the d e s i r e d pH. Malonaldehyde was prepared by a c i d h y d r o l y s i s of i t s b i s - ( d i m e t h y l a c e t a l ) . An aqueous s o l u t i o n of pyruvic aldehyde was d i l u t e d with d i s t i l l e d water and phosphate-citrate b u f f e r to give an MGA s o l u t i o n of the d e s i r e d pH (16). A v a i l a b l e l y s i n e i n the model system was determined by an adaptation of the method of Kadade and Leiner (17)» A f t e r prel i m i n a r y rate s t u d i e s the parameters chosen f o r measurement were: pH—5,6,7,8; temperature—20, 40 and 60°C i n a water bath; t i m e — u s u a l l y 24 hours with samplings a t 10 min and 1,2,4, and 8 or 10 hours ( o c c a s i o n a l l y a f t e r 2 o r 4 days @ 20°C); BPA concent r a t i o n — 1 g/L, 10 g/L; carbonyl compound c o n c e n t r a t i o n — 0 . 0 7 to 35.0 mM or c a r b o n y l : l y s i n e — 1 : 1 0 to 50:1. Experimental procedures employed g e n e r a l l y were as f o l l o w s : The BPA s o l u t i o n was measured i n t o a c u l t u r e tube, the carbonyl compound at the desired concentration added and the two gently mixed. Zero time samples were withdrawn and the capped tubes were placed i n a Beckman thermocirculator at the experimental temperature. Blank (no carbonyl) and c o n t r o l (no BPA) tubes were included with each run. Samples were withdrawn a t s p e c i f i e d i n t e r v a l s and assayed f o r a v a i l a b l e l y s i n e . In a d d i t i o n t o determining p r o t e i n damage as i n d i c a t e d by per cent l o s s o f a v a i l a b l e l y s i n e , the amount of carbonyl compound' 3


398

MAILLARD REACTIONS

remaining a f t e r r e a c t i o n with BPA was measured. MA was assayed by Buttkus's m o d i f i c a t i o n of the method of Sinnhuber and Yu (18,19). MGA was assayed by the method of Vogt as modified by Riddle (20,11).


Results and d i s c u s s i o n Among the v a r i a b l e s t e s t e d (pH, temperature, and reactant concentration) temperature was the most i n f l u e n t i a l parameter with each carbonyl compound at a l l l e v e l s tested and reactant concentration was s i g n i f i c a n t when c a r b o n y l - l y s i n e r a t i o s were above 1. In c o n t r a s t , a l t e r a t i o n of pH w i t h i n the pH 5-8 range t e s t e d (that commonly found i n p r o t e i n food systems) had a n e g l i g i b l e e f f e c t . Although p r e l i m i n a r y s t u d i e s i n d i c a t e d optimal pH values f o r these r e a c t i o n s to be c l o s e to 5 f o r MA and near 8 f o r MGA, r a t e changes when the experiments were conducted at pH 6 were s l i g h t — e s p e c i a l l y as compared with r a t e changes o c c u r r i n g with increases i n temperature and reactant c o n c e n t r a t i o n . Theref o r e , the graphs presented are based upon data u s i n g a pH of 6. F i g . 1 shows the l o s s of a v a i l a b l e l y s i n e a f t e r 12 hours of r e a c t i o n of BPA with e i t h e r MA or MGA as a f u n c t i o n of temperature and carbonyl concentration. As the r e a c t i o n temperature was increased the l y s i n e l o s s was s i g n i f i c a n t l y increased with each carbonyl compound although the l o s s with MA was c o n s i d e r a b l y greater than with MGA. At 60°C, not an u n l i k e l y temperature f o r a v a r i e t y of p r o c e s s i n g procedures, as much as 80% of the l y s i n e was l o s t w i t h MGA and 90% with MA—much of t h i s l o s s o c c u r r i n g during the f i r s t 2-4 hours as shown i n F i g . 2. These are s i g n i f i c a n t n u t r i t i o n a l l o s s e s and i l l u s t r a t e the damage p o s s i b l e to foods e i t h e r through prolonged heat p r o c e s s i n g o r , perhaps more important, by storage i n hot c l i m a t e s . Even a f t e r 10 min the l o s s was more than 20% while i t exceeded 50% i n 4 hours. Studies of the MA-lysine r e a c t i o n by other authors, as d i s c u s s e d p r e v i o u s l y , were conducted at 37°C or lower. There are a few reports of l y s i n e l o s s e s measured as a f u n c t i o n of i n c r e a s i n g temperature to more than 100°C; some of these reports c o r r e l a t e these l o s s e s with browning and others with moisture content (21-26). However, most s t u d i e s of high temperature e f f e c t s have been on browning r a t h e r than n u t r i e n t l o s s . The e f f e c t s of carbonyl c o n c e n t r a t i o n are i l l u s t r a t e d i n F i g s . 3 and 4. MA e x h i b i t e d a more obvious r e l a t i o n s h i p between carbonyl c o n c e n t r a t i o n increases and l y s i n e l o s s than d i d MGA. I t i s evident that l o s s occurs to a greater extent with the d i rather than the keto-aldehyde. T h i s i s c o n s i s t e n t with the p r e v i o u s l y mentioned theory that the enamine d e r i v a t i v e forms more r e a d i l y than the imine. The c a r b o n y l : l y s i n e r a t i o used i n f l u e n c e d the l y s i n e l o s s . MA at a r a t i o of 50:1 caused a s i g n i f i c a n t l y greater l y s i n e l o s s than equimolar amounts of MGA, but each demonstrated a d e f i n i t e , though not l i n e a r , increase i n r e a c t i v i t y with i n c r e a s i n g


20.

TUCKER E T A L .

lOOr

Loss of Lysine

399

in Protein

96.5 CARBONYL-BPA: I2hrs., pH 6 corb. lys :


S

ι • ι

MA

MGA

MA

20°C

Figure 1.

MGA 60°C

Variation of lysine loss with temperature, nature of carbonyl reagent, and ratio of reagents.

hours Figure 2. Rate of lysine loss at fixed reactant ratio with change of temperature and nature of carbonyl reagent. Key: —, malonaldehyde (MA ); and , methyl glyoxal (MGA).


400

MAILLARD REACTIONS

lOOr-

CARBONYL-BPA: 24hrs. pH 6, 4 0 ° C t

80h

78.6

MA

I

2 60h ο σ 40h

I

MGA

45.7


g

31.0

26.0

^ 20\11.0

I

: 10

I : I

10 : I

50: ι

c a r b o n y l : lysine

Figure 3.

Variation of lysine loss with ratio of reactants.

lOOrC A R B O N Y L - B P A pH 6, 4 0 ° C :

corb

:

lys

50 : I

Figure 4.

Rate of lysine loss at various ratios of malonaldehyde and methylglyoxal to bovine plasma albumin.



20.

TUCKER E T A L .

Loss of Lysine in Protein

401

concentrations. At molar r a t i o s of c a r b o n y l : l y s i n e near 1 very l i t t l e e f f e c t was noted with an i n c r e a s e i n temperature ( F i g . 1 ) — an i n d i c a t i o n that foods with a low content o f carbonyl compounds might maintain l y s i n e s t a b i l i t y even under p r o c e s s i n g c o n d i t i o n s . Loss of the carbonyl component was measured as an i n d i c a t i o n of i t s r e a c t i o n with BPA. Results are i l l u s t r a t e d as f u n c t i o n s of the v a r i o u s parameters i n Tables I and I I . At each concen t r a t i o n l e v e l of carbonyl compound, i n c r e a s i n g temperatures were accompanied by increases i n l o s s of the carbonyl component. Increasing the lysine:MGA to 100:1, i n e f f e c t c r e a t i n g c o n d i t i o n s f o r a f i r s t order r e a c t i o n , decreased the i n f l u e n c e of tempera ture, yet even a t 20°C more than 75% of the MGA reacted. C o n t r o l samples of MGA i n which no BPA was present showed a l o s s of 42.7% of the MGA a t 60°C, but no s i g n i f i c a n t l o s s at 20 or 40°C. These l a r g e l o s s e s of MGA at the higher temperature are a t t r i b u t e d i n part to p o l y m e r i z a t i o n and i n t r a m o l e c u l a r Cannizzaro-type r e a c t i o n s . Corrected r e s u l t s , taking t h i s pheno menon i n t o account, are shown i n Table I I . Even a f t e r c o r r e c t i n g f o r s e l f - r e a c t i o n s , both carbonyl compounds r e f l e c t e d l o s s e s i n excess of the l y s i n e l o s s (mole f o r mole), which might be due to r e a c t i o n s o c c u r r i n g between the carbonyl compounds and other a c t i v e s i t e s of the p r o t e i n such as the α-amino group of the N-terminal aspartate and/or t h i o l residues (27). Therefore, i t i s d i f f i c u l t to c o r r e l a t e l o s s e s of MGA with decreased l y s i n e availability. Because t h i s model system employed d i l u t e aqueous s o l u t i o n s , the e f f e c t of water a c t i v i t y (A ) on the l o s s of a v a i l a b l e l y s i n e was not a parameter. During storage o f d r i e d foods A^ as w e l l as temperature i s a c o n s i d e r a t i o n . Labuza reported a loss of a v a i l a b l e l y s i n e i n dry milk a t A^ = 0.44 to 0.68 (28). Conclusions In general both d i - and keto-aldehydes r e a c t with l y s i n e residues of BPA a t temperatures which may occur during storage under t r o p i c a l c o n d i t i o n s or r e l a t i v e l y mild heat processing to e f f e c t i v e l y decrease the a v a i l a b l e l y s i n e . The di-aldehyde, through formation of an enamine d e r i v a t i v e , r e a c t s more r a p i d l y and to a greater extent than the keto-aldehyde a t pH values l i k e l y to be encountered i n foods. The conjugated imine d e r i v a t i v e of the keto-aldehyde, however, contained a more h i g h l y c o l o r e d chromophore. Therefore, damage to the l y s i n e by MGA may be accompanied by more "browning" than the greater l y s i n e damage with MA. Temperature as w e l l as reactant concentration s i g n i f i c a n t l y increases these l o s s e s . Rapid c o o l i n g and low storage temperatures a f t e r heat processing of p r o t e i n f o o d s t u f f s appear to be r e q u i r e d i n minimizing n u t r i t i o n a l l y s i n e l o s s .


402

MAILLARD REACTIONS

TABLE 1 V a r i a t i o n of l o s s of carbonyl compound (by r e a c t i o n with BPA at 1:10 r a t i o ) with temperature, source of carbonyl group, and r a t i o of reagents ι


LOSS CARBONYL (%)

20°C

40°C

60°C

MA - pH 5

2.7

8.6

16.0

MGA - pH 7

6.0

47.0

92.5

TABLE 2 V a r i a t i o n of l o s s of MGA (by r e a c t i o n with BPA i n 24 hr at pH 7) with r a t i o of reactants and temperature

% LOSS MGA MGA:lysine 20°C 1:10

6

40°C

60°C

47

93 53

1:100

76

84

#

100 57

Corrected f o r s e l f - r e a c t i o n .


#

20.

TUCKER E T A L .

Loss of Lysine

in Protein

403

Acknowledgments We wish to acknowledge support f o r some p u b l i c a t i o n costs from the Egtvedt Food Research Fund, U n i v e r s i t y of Washington.

Literature 1.


2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.

cited

Roach, A.G.; Sanderson, P.; Williams, D.R. J. Sci. Food Agr. 1967, 18, 274. Eriksson, C., Ed.; "Maillard Reactions in Food"; Pergamon Press: Oxford, 1981. Ibid; p. 159-176. Ibid; p. 421-428. Ibid; p. 441-442. Meyer, L.H. "Food Chemistry"; Reinhold Book Corp.: NY, 1960. Chio, K.S.; Tappel, A.L. Biochemistry 1969, 8, 2827. Shin, B.K.; Huggins, J.W.; Carrasay, K.L. Lipids 1972, 7, 229. Crawford, D.L.; Yu, T.C.; Sinnhuber, R.O. J. Food Sci. 1967, 32, 332. Fujimaki, S. Agr. Biol. Chem. 1968, 32, 46. Riddle, V.; Lorenz, F.W. J. Biol. Chem. 1968, 243, 2718. Englis, D.T.; Hanahan, D.J. J. Am. Chem. Soc. 1945, 76, 51. Cobb, W.Y.; Day, E.A. J. Am. Oil Chem. Soc. 1965, 42, 110. Scherz, H. Radiat. Res. 1970, 43, 12. Hodge, J.E. J. Agr. Food Chem. 1953, 1, 928. Tucker, B.W. M.S. Thesis, Univ. of Washington, 1974. Kakade, M.L.; Leiner, I.E. Analyt. Biochem. 1969, 27, 273. Buttkus, H. J. Food Sci. 1967, 32, 432. Sinnhuber, R.O.; Yu, T.C. Food Res. 1958, 23, 626. Vogt, M. Biochim. Z. 1929, 211, 17. Taira, H.; Sukurai, Y. Jap. J. Nutr. Food 1966, 18, 359. Miller, E.L.; Carpenter, K.T.; Morgan, C.B. Brit. J. Nutr. 1965, 19, 249. Miller, E.L.; Carpenter, K.J.; Milner, C.K. Brit. J. Nutr. 1965, 19, 547. Carpenter, K.J.; Morgan, C.B.; Lea, C.G.; Parr, L.J. Brit. J. Nutr. 1962, 16, 451. deGroot, A.P. Food Tech. 1963, 17, 339. Mauron, J.; Egli, R.H. Arch. Biochem. Biophys. 1955, 59, 433. Buttkus, H. J. Am. Oil Chem. Soc. 1972, 49, 613. Labuza, T.P. CRC Crit. Rev. Fd. Tech. 1972, 3, 217-240.

RECEIVED November 2, 1982


Loss of Available Lysine in Protein in a Model Maillard Reaction System

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