The Maillard Reaction in Foods and Nutrition - American Chemical

10

Characteristics Produced

of S o m e

by the

Maillard

New

Flavoring

Materials

Reaction

Downloaded by UNIV LAVAL on May 12, 2016 | http://pubs.acs.org Publication Date: April 29, 1983 | doi: 10.1021/bk-1983-0215.ch010

E. DWORSCHÁK and V. TARJÁN Institute of Nutrition, H-1097 Gyáliút3/a, Budapest, Hungary S. TUROS Kanold AB, Göteborg, Sweden Milk-crumbs and soya-crumbs are new aroma sources produced by the Maillard reaction and intended to be used in the food industry. During the production of the crumbs thermal reactions take place in the material (e.g. milk) and the added glucose and amino acid constituents. Fructose-lysine and lactulose-lysine, biologically active Amadori compounds, are formed in high levels in the milk-crumb, but much less in the soya-crumb, caused by lysine degradation. Extracts from the crumbs show only a low mutagenic activity measured by Ames test. Soya-crumb has a significant antioxidant effect; for this reason its possible role in the food industry is discussed. The KANOLD AB Company has developed a series of new materials, produced from mixtures of skimmed milk, glucose, and lysine (or other basic amino acids) by drying on a roller at a temperature of 125°C (1). Instead of skimmed milk, soya flour can be used as basic material. The new products, named milkand soya-crumbs respectively, have a very pleasant odor resembling that of caramel. The yellow to deep-brown coloration and the aroma characteristics are attributed to the Maillard reaction. According to experiments carried out in the confectionery industry, up to 50% of cocoa powder could be replaced by the crumbs without any significant changes in flavor, appearance, or consistency of food products, e.g. in chocolates. Our aim was to investigate some chemical properties as well as the possible mutagenic effects of the new materials. We determined the content of furosine, which is an amino acid derivative formed by acid hydrolysis of fructose-lysine or lactulose-lysine (2) (Fig. 1).

0097-6156/83/0215-0159$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.

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

Figure 1.

Browning

L—Fructose—lysine

Lysine + Glucose

Pyridosine

Formation of furosine and pyridosine by acid hydrolysis.

with 7.75 N HCI

Hydrolysis

Furosine


10.

DWORSCHAK E T AL.

Characteristics

of New Flavoring

Materials

161

Experimental


Material and S u p p l i e s M i l k - c r u m b and soya-crumb were p r e p a r e d by the KANOLD AB Co., G o t e b o r g , Sweden. N - f l ( - a c e t y l - L - l y s i n e . A Grade, Calbiochem. Tween 20 a n d 80 ( s o r b i t a n , p o l y o x y e t h y l a t e d , stearic ester), BDH C h e m i c a l s L t d . H e m o g l o b i n , h o r s e , p u r e . REANAL, H u n g a r y . S u n f l o w e r o i l , P h . Hg. V I p u r i t y . BHT ( 3 , 5 - d i t e r t - b u t y l - 4 - h y d r o x y t o l u e n e ) , A g r a d e REANAL

Preparation of furosine s o l u t i o n : N-a-acetyl-L-lysine ( 0 . 5 mmole) and g l u c o s e (1.0 mmole) are dissolved i n 1 ml 0.1 M K H P 0 ^ b u f f e r s o l u t i o n . The solution i s heated i n a c l o s e d v e s s e l f o r 5 h a t 100°C. Then t h e s o l u t i o n i s made 6 Ν w i t h HC1 and h e a t e d a t 105° for 24 h . The h y d r o l y s a t e i s e v a p o r a t e d i n v a c u o . A s o l u t i o n o f the r e s i d u e i n 20 ml w a t e r can be u s e d f o r t h e TLC production of furosine spots. For quantitative evaluation t h e same a b s o r b a n c e was assumed f o r the n i n h y d r i n r e a c t i o n p r o d u c t b o t h a r g i n i n e and f u r o s i n e . 2

Methods Determination of Furosine For the determination of furosine the f o o d crumbs were h y d r o l y z e d w i t h 6 N HC1 f o r 24 h at 105° C i n a c l o s e d g l a s s v e s s e l . The 0.5% h y d r o l y s a t e was chromatographed on a c a t i o n exchange r e s i n - c o a t e d t h i n l a y e r p l a t e ( s o d i u m c y c l e F I X I 0 N 50 X 8 p l a t e s , CHIN0IN P h a r m a c e u t i c a l Works, B u d a p e s t ) d e v e l o p e d by D e v e n y i , e t a l . (3). For t h e separation of furosine a 0.12 M c i t r a t e buffer (sodium concentration 400 meq/1, pH = 6.0) was u s e d . The chromatogram was developed a t 50° C. A f t e r the d r y i n g o f the p l a t e , t h e chromatogram was s p r a y e d w i t h 0.4%, n i n h y d r i n i n acetone. Furosine spots were evaluated with a VIS-UV-2 Chromatogram A n a l y z e r , FARRAND O p t i c a l Co., I n c . , N.Y. Test f o r Mutagenic A c t i v i t y M i l k - c r u m b s and soya-crumbs extracted i n two d i f f e r e n t ways were tested for possible m u t a g e n i c i t y by the Ames t e s t . A p o r t i o n o f the crumbs was e x t r a c t e d w i t h a 5 - f o l d amount of 5% Tween 80 aqueous s o l u t i o n . In another e x t r a c t i o n , a 5-g sample was k e p t i n 60 ml refluxing ethanol f o r one h . The f i l t e r e d ethanol was distilled from the dissolved fatty material a t a normal pressure. The d i s t i l l a t e was u s e d f o r the Ames t e s t . In t h e Ames t e s t the f o l l o w i n g Salmonella typhimurium s t r a i n s were u s e d : TA 1535 and TA 100, w h i c h a r e s e n s i t i v e t o


162

MAILLARD REACTIONS


mutagens c a u s i n g b a s e - p a i r s u b s t i t u t i o n s , and TA 1537, TA 1538, and TA 98, s e n s i t i v e t o mutagens c a u s i n g f r a m e - s h i f t m u t a t i o n s . Manipulation of the t e s t s t r a i n s , and p r e p a r a t i o n o f t h e m i c r o s o m i c f r a c t i o n o f the A r o c h l o r 1254 p r e t r e a t e d R-Amsterdam male rats, were c a r r i e d out as d e s c r i b e d by Ames, e t a l . ( 4 ) . The c o n t r o l mutagen was 2 - a m i n o f l u o r e n e i n a 0.1 m g / p l a t e d o s e . Determination of A n t i o x i d a n t E f f e c t The measurement was based on consumption of d i s s o l v e d o x y g e n by a s u n f l o w e r o i l emulsion in a closed system w i t h or w i t h o u t the presence of antioxidant material. The e m u l s i o n was f r e s h l y p r e p a r e d (f>) f r o m 10 ml s u n f l o w e r oil, 2 ml 30% aqueous s o l u t i o n o f Tween 20, and 88 ml 0.1 M phosphate buffer at various pH values. This mixture was h o m o g e n i z e d i n a BIOMIX-mixer (LABOR MIM, B u d a p e s t ) f o r 5 min. To 50 ml e m u l s i o n was added 0.1 g f i n e l y g r o u n d crumb sample, and the m i x t u r e homogenized w i t h a magnetic s t i r r e r . The oxygen c o n t e n t of the e m u l s i o n was evaluated with a RADELKIS (Budapest) p 0 -pC0 a n a l y z e r u s i n g an ΟΡ-9263-type oxygen membrane e l e c t r o d e . From the moment o f a d d i n g 5 ml 0.14% h e m o g l o b i n s o l u t i o n t o the s t i r r e d e m u l s i o n , the t i m e was measured w i t h a s t o p w a t c h . The t i m e r e q u i r e d f o r 50% r e d u c t i o n o f the d i s s o l v e d o x y g e n c o n c e n t r a t i o n was recorded. The antioxidant effect (Α.Ε.) i s c a l c u l a t e d f r o m the equation (5) A.E. = ( T - T ) T 2

2

a

c

c

where T i s t i m e e l a p s e d f o r 50% r e d u c t i o n o f t h e a v a i l a b l e gaseous oxygen i n the sample containing the a n t i o x i d a t i v e material, and T the corresponding time interval i n the c o n t r o l , w i t h o u t crumb s a m p l e . For comparison, a_j_strong a n t i o x i d a t i v e m a t e r i a l used i n the food i n d u s t r y , 10 mM BHT i n 0.1 ml s u n f l o w e r o i l , was added t o a p o r t i o n o f t h e e m u l s i o n . a

c

R e s u l t s and

Discussion

Table I shows the f u r o s i n e c o n t e n t o f t h e m i l k - c r u m b and soya-crumb samples i n comparison to o t h e r food p r o t e i n sources thermally processed. Milk-crumb has the highest furosine content, probably due t o the f r e e l y s i n e and g l u c o s e added i n the production; moreover, the milk p r o t e i n has also a c o n s i d e r a b l e l e v e l of p r o t e i n - b o u n d lysine. D a t a f r o m T a b l e I p o i n t out t h a t o t h e r m i l k - b a s e d products are likely to form Amadori products, in this case lactulose-lysine and f r u c t o s e - l y s i n e , even u n d e r moderate thermal treatments. The f u r o s i n e c o n t e n t , o r i g i n a t i n g f r o m the acid h y d r o l y s i s of Amadori p r o d u c t s , was h i g h i n a w h i p p i n g a g e n t made f r o m sugar and Na c a s e i n a t e . It is surprising, however, that c h e e s e s have also relatively high furosine levels.


10.

DWORSCHAK E T AL.

Characteristics

of New

Flavoring

Materials

163

Table I F u r o s i n e c o n t e n t o f m i l k and s o y a - c r u m b , a n d o f o t h e r


f o o d s and b a s i c

materials

Furosine, mg/100 g p r o t e i n

Milk-crumb

4000

Soya-crumb

150

Milk treated at ultra-high temperature

180

Condensed sweet m i l k Infant formulae

(EGYT p r o d u c t s )

400 110-230

Whipping agent (from sodium caseinate) Whole m i l k powder

3000 70

Gruyère c h e e s e

210

Port

390

s a l u t cheese

Roasted

coffee

Nescafe UNIBIT T e x t u r e d Soya C o n c e n t r a t e S 202/24 (PURINA)

200 350

20



164

MAILLARD REACTIONS

Soya p r o d u c t s c o n t a i n much l e s s r e d u c i n g c a r b o h y d r a t e and also lower bound l y s i n e t h a n t h o s e from m i l k ; m o r e o v e r , o n l y small amounts o f f r e e lysine and g l u c o s e were added i n t h e production o f soya-crumb. The s m a l l e r f u r o s i n e v a l u e s f r o m soya products c a n be e x p l a i n e d by t h e s e f a c t s . The e f f e c t o f intensive heat treatment on t h e f o r m a t i o n of f u r o s i n e i s d e m o n s t r a t e d by r o a s t e d c o f f e e . Erbersdobler, e t a l . (6) found karyocytomegaly i n the kidneys o f r a t s f e d 16200 t o 23300 ppm f r u c t o s e - l y s i n e i n t h e diet. The d i e t d i d n o t c o n t a i n l y s i n o a l a n i n e ( F i g . 2) b u t t h e renal damage was s i m i l a r t o t h a t found a f t e r l y s i n o a l a n i n e administration by o t h e r a u t h o r s . The d e t a i l s o f t h i s q u e s t i o n a r e r e v i e w e d e l s e w h e r e (1). E r b e r s d o b l e r ' s r e p o r t has n o t been c o n f i r m e d w i t h new d a t a by o t h e r s . Among t h e v a l u e s g i v e n i n t h e T a b l e I o n l y m i l k crumb and t h e w h i p p i n g a g e n t have f u r o s i n e l e v e l s t h a t c a n be compared t o the fructose-lysine (or lactulose-lysine) content used i n Erbersdobler's experiment. The test f o r m u t a g e n i c a c t i v i t y o f t h e d e t e r g e n t (Tween) e x t r a c t d i d n o t i n d u c e any r e v e r t a n t s ; a l l p l a t e c o u n t s were i n the range o f s p o n t a n e o u s m u t a t i o n r a t e , w i t h and w i t h o u t S-9 mix. From t h e e t h a n o l d i s t i l l a t e we g o t a p o s i t i v e r e s p o n s e demonstrated i n Table I I . I n the case of milk-crumb the numbers o f i n d u c e d r e v e r t a n t s were about a h u n d r e d f o l d t h o s e o f the spontaneously r e v e r t e d c o l o n i e s w i t h t h e most s e n s i t i v e s t r a i n s (TA 98, 100) o n l y w i t h o u t m e t a b o l i c a c t i v a t i o n . In the same e x p e r i m e n t the t e s t strain TA 100 r e s p o n d e d t o the soya-crumb s a m p l e , a l s o o n l y w i t h o u t m e t a b o l i c a c t i v a t i o n . Thus, a c c o r d i n g to our r e s u l t s , t h e crumbs have o n l y s l i g h t m u t a g e n i c a c t i v i t y as compared t o o t h e r h e a t - t r e a t e d and M a i l l a r d products described i n the l i t e r a t u r e ( 8 ) . Our e x p e r i m e n t s on t h e a n t i o x i d a t i v e e f f e c t o f soya-crumbs are summarized i n T a b l e I I I . S u r p r i s i n g l y m i l k - c r u m b had no e f f e c t a t any pH v a l u e , a l t h o u g h t h e o p p o s i t e c o u l d be e x p e c t e d from i t s h i g h l e v e l of Amadori p r o d u c t s . Soya-crumb has a s i g n i f i c a n t a n t i o x i d a t i v e e f f e c t i n t h e sunflower o i l emulsion s y s t e m , a l t h o u g h l e s s t h a n BHT i n t h e concentration g e n e r a l l y used i n the food i n d u s t r y (Table I I I ) . The antioxidative effect i s higher i n the a c i d region w i t h a maximum o b s e r v e d between pH v a l u e s o f 4 and 5. Our results confirm the a n t i o x i d a t i v e effect o f some Maillard products. The l o w t o x i c i t y o f t h e s e p r o d u c t s may permit using them i n c e r t a i n f o o d p r o d u c t s ( e . g . i n s a u s a g e s ) as m i l d a n t i o x i d a n t s o f n a t u r a l o r i g i n (_5) . Conclusions Intensive search has been s t a r t e d r e c e n t l y t o f i n d b a s i c materials o f m o d e r a t e p r i c e t o be u s e d i n t h e f o o d i n d u s t r y .


DWORSCHAK ET AL.

165 Characteristics of New Flavoring Materials


10.


MAILLARD REACTIONS

166

Table I I Number o f r e v e r t a n t c o l o n i e s p r o d u c e d by e t h a n o l

extracts


of m i l k crumb and s o y a crumb

Materials mg/plate

S-9

Salmonella

t y p h i m u r i u m TA

98

100

20 13

29 22

1100 66

160 13

Soya-crumb 62.5 mg

18 0

5 2700

Milk-crumb 62.5 mg

7 1200

3 2200

1535

1537

1538

(spontaneous reversion)

2-Aminofluorene 0.1 mg

14 4

130 6

The column S-9 shows w h e t h e r t h e e x p e r i m e n t was c a r r i e d o u t w i t h (+) o r w i t h o u t (-) m e t a b o l i c a c t i v a t i o n .


DWORSCHAK ET AL.

Characteristics

of New Flavoring

Materials


Table I I I Antioxidative effect

(A.E.) o f soya-crumb a t

different

pH v a l u e s

Soya-crumb, 0.1 g

A.E.

pH = 7.47

0.84

5.92

1.30

5.03

1.83

4.15

1.90

3.20

1.45

2.25

1.23

5

BHT 1 0 " mmole pH = 7.47

2.18



168

MAILLARD REACTIONS

Milk-crumbs and soya-crumbs belong to this group. Their favorable organoleptic characters and good adaptability to further technological processes have been established, but more scientific investigations are s t i l l needed. The analysis of the aroma constituents of the crumbs is a task for the future. The potential toxicity of the Amadori products (e.g. fructose-lysine) is s t i l l uncertain. The high levels of furosine in the milk-crumb add to the need to settle this question. Our experiments on the mutagenicity of these materials revealed that a water extract of the crumbs did not give a positive Ames test. On the other hand the ethanol extract after distillation produced reverted colonies in the range of the positive control (2-AF) with the most sensitive strains, but only without metabolic activation. The Maillard products responsible for mutations seemed to be metabolized in the living organism. In view of our experiments and the present international evaluation on heat-processed foods, the crumbs present no more detrimental risk to human health than other heat-processed foods, when consumed. The model experiments on the antioxidative effect of crumbs revealed the potential ability of the soya-crumb to prevent lipid oxidation in certain foods. The mild acid medium of meat products seems to be favorable for fulfilling this hope. However, further investigations in food products are necessary to obtain a decisive answer. As the case of milk crumb shows, a Maillard product is not necessarily an antioxidant. Literature Cited 1. 2. 3. 4. 5. 6. 7. 8.

Kanold AB Co., Swedish Patent Application 10, 811/72. Finot, P. Α.; Bricout, J . ; Viani, R.; Mauron, J . Experientia 1968, 24, 1097. Devenyi, T.; Hazai, I.; Ferenczi, S.; Bati, J . Acta Biochem. Biophys. Acad. Sci. Hung. 1971, 6, 385. Ames, Β. Ν.; McCann, J.; Yamasaki, E. Mutat. Res. 1976, 31, 347. Lingnert, H. "Antioxidative effect of Maillard reaction" products". ISBN 91-7290-079-2 (SIK-Rapport), Göteborg, 1979. Ebersdobler, H. F.; Brandt, Α.; Scharrer, E . ; von Wangenheim, B. Prog. Food Nutr. 1981, 5, 257. Dworschak, E. CRC Crit. Rev. Food Sci. Nutr. 1980, 13, 1. Sugimura, T. CRC Crit. Rev. Toxicol. 1979, 6, 189.

RECEIVED November

22, 1982


The Maillard Reaction in Foods and Nutrition - American Chemical

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