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Influence of D-Glucose Configuration on the Kinetics of the Nonenzymatic; Browning Reaction
Influence of &Glucose Configuration on the Kinetics of the Nonenzymatic
Browning Reaction J6rg Haseler, Beate Beyerlein and Lothar W. Kroh TECHNISCHEUNIVERSITATBERLIN - INSTITUT FUR LEBENSMITTELCHEMIE GUSTAV-MEYER-ALLEE 25, D-13355 BERLIN, GERMANY Summary
The Linetics of the nonenzymatic browning d o n , especially caramelization. was influenced by the configuration of D-~~IJCOSC. Spearophotometric analysis suggests a higher reactivity of P-~glucose in comparison with a-D-glucosc during caramelization. More 5-hydroxymethyl-2-furaldehyde(HMF) and 2furaldehyde (FF) were obtained from b-~-glucose than from a-pglucose. In addition to the 3deoxyglucosonc pathway, an alternative mechanism via two C3-fragments was proposed. The formation of 1 , 6 - a n h y d r o - ~ - ~ - gand l ~ glucobioses was independent of the configuration of glucose because it paaes through a glycosyl cation. Due to steric preference, the 14-linked glucobioses isomaltose and gentiobiose an favoured. Under Maillard reaction canditions, the formation of HMF was also influenced by the configuration. At the start of the d o n the Amadori compound was formed slightly easier from a-r>-glucose.
Introduction The constitution and stereochemistry of carbohydrates influences their reactivity in thermolysidhydrothermolysisreactions fimdamentally, for example, the configuration of Dglucose af€ects the kinetics of nonenzymatic browning reactions.' Under given conditions the p-anomer shows higher reactivity. glucose is the main product of the thermolysis of a-glucans.2The individual steps in such reactions are important. Either glucose reacts via transglycosylation to branched maltodextrins or it is an intermediate in the formation of 1,6-anhydro-P-D-glucose(AHG). A glycosyl cation intermediate could exert a considerable influence on the reaction.' The preferred pathway is possibly influenced by stereochemicaleffects. It is known that enolization is a much slower reaction than mutarotation, nevertheless at temperatures above the melting point mutarotation takes place.3 While at 20 "C mutarotation is a matter of hours, at higher temperatures reaction will proceed magnitudes faster. If mutarotation dominates in the initial stage of the browning reaction no difference in the behaviour of the two anomers should be observable. Materials and Methods Reaction mixtures (100 mg) containing glucose or mixtures of glucose-DL-alanine (equimolar) were heated in a closed system at 160 "C or 180 "C for caramelization and in an open system for the Maillard reaction at 135 "C. After heating the samples were dissolved in 2 mL distilled water (filtered if necessary). Spectrophotometric measurements at 280 nm and 420 nm were used for the determination of browning intensity using a W O N 930 spectrophotometer(F'harmacia).
Kinetics and Analytical Aspects
173
Reaction products S-hydroxymethyI-2-fUraldehyde(HMF) and 2-furaldehyde (FF) were detected by HPLC: pump, Phannacia LKB 2248; column, 2 x RP-C 18, Spherisorb ODS2, 250 mm x 4.6 mm, 5 pm; eluent, 0.01 N hexanesulfonic acid-methanol(77:23), flow rate, 0.7 mL/min; detector, UVMS (VWM 2141), A=280 nm; oven temperature, 55 "C; injection volume, 20 pL. Carbohydratesand Amadori compoundswere analyzed by RI-HPLC: pump, Shimadzu LC9 4 column, IEC Aminex HPX 87C; eluent, 0.01 N Ca(NO& flow rate, 0.5 mL/min; detector, Shimadzu RID-64 oven temperature, 80 "C; injection volume, 20 pL. The non-volatile polar &action formed upon ~ g i u c o s ethermolysis was characterized by GC/EI-MS after reductive cleavage'", GC,HP 5890 Series 11 gas-liquid chromatograph; column, SE-54 (0.25 mm x 25 m); temperature, 100 "C for 3 min, 2 "C/min to 250 "C, hold for 10 min; MS,HP 59898; scan, 40-550 amu.
Results and Discussion The chosen compounds allow different reaction pathways and mechanisms during the nonenzymatic browning reaction to be studied. At 280 nm intermediates such as enediols, dicarbonyls and aldehydes were measured while at 420 nm the higher molecular & d o n 8s well as coloured compounds and caramel were detected. The typical products of carbohydrate degradation during caramelition and Maillard reaction are represented by the firan derivatives 5-hydroxymethyl-2-furaldehyde(HMF) and 2-firaldehyde (FF).
time [min]
time [min]
Rflre 1. Browning intens@ of@ and pDgrvcose (I60 "C)at 280 nm (A; diluted 1:200) and 420 nm (B; diluted 130). eD-glucose, *; ~o-glvcose,Q
The potential influence of different configurationsof carbohydrate in the early phase of the Maillard reaction may be highest in the case of glycosylamine and Amadori compounds as typical intermediates. Reactions of the Amadori compound are influenced more by the
The Maillard Reaction in Foods and Medicine
I74
degradation of the carbohydrate than by carbohydrate configuration, therefore differences due to the configuration are rather small in Maillard reaction. Finally, the kinetics of the formation of AHG and the glucobioses were studied in caramelization, since the formation of these products is an important step toward hrther reactions. Similar amounts of AHG were formed, therefore it might be concluded that the mechanism yielding AHG is not influenced by the position of the anomeric hydroxyl group. Caramelization At 160 "C, the j3-D-configuration produces higher browning intensity (Figure 1). This can be explained by the initiating reactions of nonenzymatic browning. Ring opening, enolization, hrther reactions like abstraction of water, oxidative processes and retroaldolreactions were faster in P-&glucose. During the observed time (4 % h) the p-Dglucose shows always a higher absorbance at 420 nm (Figure 1B). It also results in higher formation of hran derivatives (Figure 2). The amount of HMF formed from a-&glucose was approximately half as much as formed from P-&glucose. Furthermore it was interesting that the amount of HMF increases markedly from the start of the reaction, while for the formation of FF an induction phase was observed. These results were consistent with the spectrophotometric data. The higher reactivity of the P-configuration could be explained by the following considerations. In the early phase, configuration and conformation may be decisive. The velocity of ring opening might differ between the a-and p-anomers or, if the velocity of ring opening is equal, the difference might be explained by favoured fragmentation of the ring structure of the two anomers; for example, in the C3-C4 position. 30 I
lBO 140
25
120
J
2 100 0
c
80
3
0
c.
L '0
fu E"
8 ca a
ir" F
20
15
0
60
0
cR 10 a
40
20 -
0
0
50
100
150 200 250 300
I0
time [min] time [min] Figure 2. Formation o$HMF (A) and FF (B) during heoting o$a- and PO-grucose at 160 "C. a-&glucose, 0; @-glucose, o
I75
Kinetics and Analylical Aspects
The formation of AHG appears not to be influenced by the configuration of the starting material. Both graphs show that neither configuration was preferred (Figure 3B). This suggests that the glucosyl cation might be here an imponant intermediate. In contrast, the amount of degraded glucose depended on the configuration of the starting material; the loss of P-glucose was greater than that of a-glucose (Figure 3A). Figure 4 shows the chromatograms of carbohydrate derivatives after reductive cleavage. The proportion of single acetylated derivatives is presented. Therefore the formation of dfierent l i e d glucobioses upon transglycosilation,was not influenced by the configuration of the starting material. 100
-E P)
8o
-0
%
r
0
E
m
60
0
m
E
0 0
\ F
40
m E
20
u
' 0
50
100
150
time [min]
200
250
Figure 3. A , Degradation o$ a-/pOglucose during caramelization (I60 "C) and B, Formation o$ AHG during caramelization (160 "C). a-Dglucose. 0; Bo-glucose. o
Maillard reaction
The absorbance at 280 nm and 420 nm and the formation of Amadori cornpound and HMF appear to be slightly higher in the case of a-Dglucose (Figures 5 and 6). This could be caused by an easier formation of the P-glucosylamine from a-Dglucose as a precursor for the Amadori compound. The P-anomer, solved in n-octanol, shows a higher browning activity in comparison with a-glucose.' Without a solvent, however, during the observed time a-&glucose browns slightly more compared to the P-configurated carbohydrate. Also HMF was formed more from the a-anomer, whereas the Amadori rearrangement compound shows this behaviour only in the increasing phase (Figure 6).
176
Abundi
The Maillard Reaction in Foods and Medicine
a-D-g'ucose 180 ' C I 6 0 min
ce
240000
200000
C
200000 160000
120000
B 14 %
80000
A
Abundance
'i
p -D -g lu c o s e 1 8 0 OC / 6 0 m i n C
'rc
160000
1200001
D 16 %
40000
80000
D 6 %
-
40000
i
0 6 8 1 0 1 2 2 4 6 8 1 0 1 2 time [min] time [min] Figure 5.Browning intensity of a- and $-&glucose with alanine (135 "C)at 280 nm (A, 1:200 dilution) and 420 nm(B, 1:50 dilution). a-&glucose. *; $-&glucose. o
2
4
Kinerics ond Analyricol Aspects 800
177
7 .A
5
600
-
a 400
-
0
x
s 0
e (P
0
E
=aa 0 0
200
t
L
OO
2
4
6
time [min] time [min] Figure 6. Fonnotion oJHMF (A) ondfiuctose olonine (B) during the Moillord reoction at 135 "C. a+ glucose, *; Po-glucose, o
Acknowledgements The authors sincerely thank the Deutsche Forschungsgemeinschafl Grand No. Kr 1452/2-1 for financial support, further we would like to thank Renate Brandenburger for technical assistance and Anke Hollnagel for scientific discussion.
References 1. L. W. Kroh and G. Westphal, Die Maillard-Reaktion in Lebensmitteln, Miffeilungsbluff der Chemischen Gesellschajlder DDR, 1986,35,73-80. 2. L. W. Kroh, W. Jalyschko and J. Hbeler, Non-volatile reaction products by heat-
3. 4.
5. 6. 7.
induced degradation of a-glucans. Part I: Analysis of oligomeric maltodextrins and anhydro-sugars, SfurcWSfdrke,1996,48,426-433. R. Schallenberger and G. G.Birch, Sugar Chemistry, The Avi Publishing Company. Inc., Westport, Connecticut, 1975. T. L. Lowary and G. N. Richards, Mechanism in pyrolysis of polysaccharides: Cellobiitol as a model for cellulose. Carbolyu?. Res., 1990, 198, 79-89. I. Ciucanu and F. Kerek, A simple and rapid method for the permethylation of carbohydrates, Carbohyuk Res., 1984, 131,209-217. J.G. Jun and G.R. Gray, A new catalyst for reductive cleavage of methylated glycans, Carbohy&. Res., 1987,163,247-261. G. Westphal and L. W.Kroh, Nuhrung, 1985,29,757-764.
Browning Reaction J6rg Haseler, Beate Beyerlein and Lothar W. Kroh TECHNISCHEUNIVERSITATBERLIN - INSTITUT FUR LEBENSMITTELCHEMIE GUSTAV-MEYER-ALLEE 25, D-13355 BERLIN, GERMANY Summary
The Linetics of the nonenzymatic browning d o n , especially caramelization. was influenced by the configuration of D-~~IJCOSC. Spearophotometric analysis suggests a higher reactivity of P-~glucose in comparison with a-D-glucosc during caramelization. More 5-hydroxymethyl-2-furaldehyde(HMF) and 2furaldehyde (FF) were obtained from b-~-glucose than from a-pglucose. In addition to the 3deoxyglucosonc pathway, an alternative mechanism via two C3-fragments was proposed. The formation of 1 , 6 - a n h y d r o - ~ - ~ - gand l ~ glucobioses was independent of the configuration of glucose because it paaes through a glycosyl cation. Due to steric preference, the 14-linked glucobioses isomaltose and gentiobiose an favoured. Under Maillard reaction canditions, the formation of HMF was also influenced by the configuration. At the start of the d o n the Amadori compound was formed slightly easier from a-r>-glucose.
Introduction The constitution and stereochemistry of carbohydrates influences their reactivity in thermolysidhydrothermolysisreactions fimdamentally, for example, the configuration of Dglucose af€ects the kinetics of nonenzymatic browning reactions.' Under given conditions the p-anomer shows higher reactivity. glucose is the main product of the thermolysis of a-glucans.2The individual steps in such reactions are important. Either glucose reacts via transglycosylation to branched maltodextrins or it is an intermediate in the formation of 1,6-anhydro-P-D-glucose(AHG). A glycosyl cation intermediate could exert a considerable influence on the reaction.' The preferred pathway is possibly influenced by stereochemicaleffects. It is known that enolization is a much slower reaction than mutarotation, nevertheless at temperatures above the melting point mutarotation takes place.3 While at 20 "C mutarotation is a matter of hours, at higher temperatures reaction will proceed magnitudes faster. If mutarotation dominates in the initial stage of the browning reaction no difference in the behaviour of the two anomers should be observable. Materials and Methods Reaction mixtures (100 mg) containing glucose or mixtures of glucose-DL-alanine (equimolar) were heated in a closed system at 160 "C or 180 "C for caramelization and in an open system for the Maillard reaction at 135 "C. After heating the samples were dissolved in 2 mL distilled water (filtered if necessary). Spectrophotometric measurements at 280 nm and 420 nm were used for the determination of browning intensity using a W O N 930 spectrophotometer(F'harmacia).
Kinetics and Analytical Aspects
173
Reaction products S-hydroxymethyI-2-fUraldehyde(HMF) and 2-furaldehyde (FF) were detected by HPLC: pump, Phannacia LKB 2248; column, 2 x RP-C 18, Spherisorb ODS2, 250 mm x 4.6 mm, 5 pm; eluent, 0.01 N hexanesulfonic acid-methanol(77:23), flow rate, 0.7 mL/min; detector, UVMS (VWM 2141), A=280 nm; oven temperature, 55 "C; injection volume, 20 pL. Carbohydratesand Amadori compoundswere analyzed by RI-HPLC: pump, Shimadzu LC9 4 column, IEC Aminex HPX 87C; eluent, 0.01 N Ca(NO& flow rate, 0.5 mL/min; detector, Shimadzu RID-64 oven temperature, 80 "C; injection volume, 20 pL. The non-volatile polar &action formed upon ~ g i u c o s ethermolysis was characterized by GC/EI-MS after reductive cleavage'", GC,HP 5890 Series 11 gas-liquid chromatograph; column, SE-54 (0.25 mm x 25 m); temperature, 100 "C for 3 min, 2 "C/min to 250 "C, hold for 10 min; MS,HP 59898; scan, 40-550 amu.
Results and Discussion The chosen compounds allow different reaction pathways and mechanisms during the nonenzymatic browning reaction to be studied. At 280 nm intermediates such as enediols, dicarbonyls and aldehydes were measured while at 420 nm the higher molecular & d o n 8s well as coloured compounds and caramel were detected. The typical products of carbohydrate degradation during caramelition and Maillard reaction are represented by the firan derivatives 5-hydroxymethyl-2-furaldehyde(HMF) and 2-firaldehyde (FF).
time [min]
time [min]
Rflre 1. Browning intens@ of@ and pDgrvcose (I60 "C)at 280 nm (A; diluted 1:200) and 420 nm (B; diluted 130). eD-glucose, *; ~o-glvcose,Q
The potential influence of different configurationsof carbohydrate in the early phase of the Maillard reaction may be highest in the case of glycosylamine and Amadori compounds as typical intermediates. Reactions of the Amadori compound are influenced more by the
The Maillard Reaction in Foods and Medicine
I74
degradation of the carbohydrate than by carbohydrate configuration, therefore differences due to the configuration are rather small in Maillard reaction. Finally, the kinetics of the formation of AHG and the glucobioses were studied in caramelization, since the formation of these products is an important step toward hrther reactions. Similar amounts of AHG were formed, therefore it might be concluded that the mechanism yielding AHG is not influenced by the position of the anomeric hydroxyl group. Caramelization At 160 "C, the j3-D-configuration produces higher browning intensity (Figure 1). This can be explained by the initiating reactions of nonenzymatic browning. Ring opening, enolization, hrther reactions like abstraction of water, oxidative processes and retroaldolreactions were faster in P-&glucose. During the observed time (4 % h) the p-Dglucose shows always a higher absorbance at 420 nm (Figure 1B). It also results in higher formation of hran derivatives (Figure 2). The amount of HMF formed from a-&glucose was approximately half as much as formed from P-&glucose. Furthermore it was interesting that the amount of HMF increases markedly from the start of the reaction, while for the formation of FF an induction phase was observed. These results were consistent with the spectrophotometric data. The higher reactivity of the P-configuration could be explained by the following considerations. In the early phase, configuration and conformation may be decisive. The velocity of ring opening might differ between the a-and p-anomers or, if the velocity of ring opening is equal, the difference might be explained by favoured fragmentation of the ring structure of the two anomers; for example, in the C3-C4 position. 30 I
lBO 140
25
120
J
2 100 0
c
80
3
0
c.
L '0
fu E"
8 ca a
ir" F
20
15
0
60
0
cR 10 a
40
20 -
0
0
50
100
150 200 250 300
I0
time [min] time [min] Figure 2. Formation o$HMF (A) and FF (B) during heoting o$a- and PO-grucose at 160 "C. a-&glucose, 0; @-glucose, o
I75
Kinetics and Analylical Aspects
The formation of AHG appears not to be influenced by the configuration of the starting material. Both graphs show that neither configuration was preferred (Figure 3B). This suggests that the glucosyl cation might be here an imponant intermediate. In contrast, the amount of degraded glucose depended on the configuration of the starting material; the loss of P-glucose was greater than that of a-glucose (Figure 3A). Figure 4 shows the chromatograms of carbohydrate derivatives after reductive cleavage. The proportion of single acetylated derivatives is presented. Therefore the formation of dfierent l i e d glucobioses upon transglycosilation,was not influenced by the configuration of the starting material. 100
-E P)
8o
-0
%
r
0
E
m
60
0
m
E
0 0
\ F
40
m E
20
u
' 0
50
100
150
time [min]
200
250
Figure 3. A , Degradation o$ a-/pOglucose during caramelization (I60 "C) and B, Formation o$ AHG during caramelization (160 "C). a-Dglucose. 0; Bo-glucose. o
Maillard reaction
The absorbance at 280 nm and 420 nm and the formation of Amadori cornpound and HMF appear to be slightly higher in the case of a-Dglucose (Figures 5 and 6). This could be caused by an easier formation of the P-glucosylamine from a-Dglucose as a precursor for the Amadori compound. The P-anomer, solved in n-octanol, shows a higher browning activity in comparison with a-glucose.' Without a solvent, however, during the observed time a-&glucose browns slightly more compared to the P-configurated carbohydrate. Also HMF was formed more from the a-anomer, whereas the Amadori rearrangement compound shows this behaviour only in the increasing phase (Figure 6).
176
Abundi
The Maillard Reaction in Foods and Medicine
a-D-g'ucose 180 ' C I 6 0 min
ce
240000
200000
C
200000 160000
120000
B 14 %
80000
A
Abundance
'i
p -D -g lu c o s e 1 8 0 OC / 6 0 m i n C
'rc
160000
1200001
D 16 %
40000
80000
D 6 %
-
40000
i
0 6 8 1 0 1 2 2 4 6 8 1 0 1 2 time [min] time [min] Figure 5.Browning intensity of a- and $-&glucose with alanine (135 "C)at 280 nm (A, 1:200 dilution) and 420 nm(B, 1:50 dilution). a-&glucose. *; $-&glucose. o
2
4
Kinerics ond Analyricol Aspects 800
177
7 .A
5
600
-
a 400
-
0
x
s 0
e (P
0
E
=aa 0 0
200
t
L
OO
2
4
6
time [min] time [min] Figure 6. Fonnotion oJHMF (A) ondfiuctose olonine (B) during the Moillord reoction at 135 "C. a+ glucose, *; Po-glucose, o
Acknowledgements The authors sincerely thank the Deutsche Forschungsgemeinschafl Grand No. Kr 1452/2-1 for financial support, further we would like to thank Renate Brandenburger for technical assistance and Anke Hollnagel for scientific discussion.
References 1. L. W. Kroh and G. Westphal, Die Maillard-Reaktion in Lebensmitteln, Miffeilungsbluff der Chemischen Gesellschajlder DDR, 1986,35,73-80. 2. L. W. Kroh, W. Jalyschko and J. Hbeler, Non-volatile reaction products by heat-
3. 4.
5. 6. 7.
induced degradation of a-glucans. Part I: Analysis of oligomeric maltodextrins and anhydro-sugars, SfurcWSfdrke,1996,48,426-433. R. Schallenberger and G. G.Birch, Sugar Chemistry, The Avi Publishing Company. Inc., Westport, Connecticut, 1975. T. L. Lowary and G. N. Richards, Mechanism in pyrolysis of polysaccharides: Cellobiitol as a model for cellulose. Carbolyu?. Res., 1990, 198, 79-89. I. Ciucanu and F. Kerek, A simple and rapid method for the permethylation of carbohydrates, Carbohyuk Res., 1984, 131,209-217. J.G. Jun and G.R. Gray, A new catalyst for reductive cleavage of methylated glycans, Carbohy&. Res., 1987,163,247-261. G. Westphal and L. W.Kroh, Nuhrung, 1985,29,757-764.