Abstract Methylglyoxal and glyceraldehyde were used to demonstrate the involvement of retro-aldol degrada- tion products of carbohydrates in browning (A420), ...
Eur Food Res Technol (1999) 209 : 261–265
Q Springer-Verlag 1999
ORIGINAL PAPER
Bettina Cämmerer 7 Bronislaw L. Wedzicha Lothar W. Kroh
Nonenzymatic browning reactions of retro-aldol degradation products of carbohydrates
Received: 9 October 1998 / Revised version: 15 December 1998
Abstract Methylglyoxal and glyceraldehyde were used to demonstrate the involvement of retro-aldol degradation products of carbohydrates in browning (A420), the formation of hydroxymethylfurfural (HMF), and the extent to which the mechanism of browning proceeds by homolytic or heterolytic pathways in the caramelization and Maillard reactions of mono- and disaccharides. The amino component of Maillard systems is required for the retro-aldol cleavage of D-glucose. The shortchain a-dicarbonyl compounds so formed appear to dominate not only the formation of brown products but also the mechanism of the browning of D-glucose under these conditions, even if only small amounts are formed. Evidence is presented to link the formation of free radicals in the browning of glucose to reactions of the retro-aldol products. A novel reaction pathway is suggested for the formation of HMF from glyceraldehyde.
rangement products (ARPs) or deoxyhexosuloses [2–5]. They react easily with each other or with other retro-aldol, carbonyl, or amino compounds in addition and condensation reactions. Because of their high reactivity, and their possible formation from the reactants and intermediates of the Maillard reaction, they should contribute significantly to the formation of brown products in sugar-amino acid reactions. In order to evaluate the participation of C3 sugar degradation products in the nonenzymatic browning of mono- and disaccharides, we chose two typical retroaldol products, glyceraldehyde and methylglyoxal [6], and investigated their influence on browning (A420), and the elemental composition of the brown products, the formation of 5-hydroxymethylfurfural (HMF), and on the type of browning reaction mechanism.
Key words Retro-aldol 7 Nonenzymatic browning 7 Carbohydrates
Materials and methods
Introduction Depending on the reaction conditions, monosaccharides undergo retro-aldol reactions in the early stages of the Maillard reaction. Oxidative and dicarbonyl cleavages result in the formation of C2, C3, and C4 a-hydroxycarbonyl and dicarbonyl compounds [1]. It is well known that most of these short-chain products are considerably more reactive towards browning than other Maillard reaction intermediates such as Amadori rear-
B. Cämmerer 7 L.W. Kroh (Y) Institut für Lebensmittelchemie, Technische Universität Berlin, Gustav-Meyer-Allee 25, D-13355 Berlin, Germany B.L. Wedzicha Procter Department of Food Science, University of Leeds, Leeds LS2 9JT, UK
Glyceraldehyde, D-glucose, D-fructose, maltose and chloroform were obtained from Merck (Darmstadt, Germany), glycine and methylglyoxal from Fluka (Buchs, Switzerland), diphenylpicrylhydrazinyl radical (DPPH) from Sigma, FeCl3 from Reanal, and 2-phenanthroline from Chemapol. All chemicals were of analytical grade. Dialysis tubing (nominal molecular weight cut-off 12–14 kDa) was purchased from Spectrum Medical Industries, Houston, USA. Fructosylglycine, an ARP, was prepared as described [7]. Reaction mixtures contained the carbonyl compound (0.1 M) being investigated in the absence and presence of glycine (0.1 or 0.2 M); the initial pH (;5) was not adjusted. The aqueous solutions were heated under reflux for up to 10 h. The development of colour was measured at 420 nm using a Pharmacia UVICON spectrophotometer. HMF was determined by reverse-phase HPLC using a Spherisorb ODS2 column (2!250 mm!4.6 mm) at 55 7C, eluting with 0.01 N hexanesulphonic acid/methanol (77 : 23 v/v) at 0.7 ml/min and detecting HMF in the eluent at 280 nm. For melanoidin isolation, as described [8], the heated reaction mixture was dialysed against distilled water (10!12 h) in dialysis tubing. The absence of low molecular weight components was verified by GP-HPLC. After dialysis the nondialysable fraction was freeze dried.
262 The apparatus used for GPC was a Kontron HPLC system (Kontron Instruments, Neufahrn, Germany) with a 325-gradient pump and a programmable photodiode-array detector (DAD) controlled by data management system 450-DAD. Separations were performed on two Nucleogel columns (Nucleogel aqua-OH 40, 7.7 mm!300 mm, Macherey-Nagel, Düren, Germany) and the mobile phase was pure water. The flow rate was set at 2.0 ml/min and the DAD detector was allowed to use the maximum wavelength. The contribution of homolytic and heterolytic mechanisms in the nonenzymatic browning reaction was investigated by means of DPPH and 2-phenanthroline as described previously [9]. Samples were withdrawn at timed intervals from the browning reaction mixture (see above), cooled rapidly and mixed with DPPH reagent or with Fe 3c/2-phenanthroline reagent. Absorbance was measured at 520 nm (DPPH) or at 505 nm (Fe 2c/2-phenanthroline complex). Extinction coefficients were determined by means of standard solutions.
Results and discussion Browning and melanoidin formation The kinetics of the browning (A420) of glyceraldehyde show no induction phase, in contrast to the browning of D-glucose [10]. Under both caramelization and Maillard reaction conditions, glyceraldehyde and methylglyoxal gave much higher absorbances at 420 nm than D-glucose (Fig. 1). The higher rate of browning of methylglyoxal (ca. 10!) compared with glyceraldehyde (Fig. 1) could be explained by the higher carbonyl reactivity of the former because of the presence of an activated methylene group in the molecule. In the presence of glycine (Maillard reaction), both carbonyl compounds showed significantly more browning than in caramelization; the absorbance of the methylglyoxal reaction increased about twice as quickly as in the glyceraldehyde reaction. In the presence of an amino acid, glyceraldehyde should lead to methylglyoxal via b-
Fig. 1 Formation of coloured products (measured at 420 nm) during the caramelization and Maillard reactions of glyceraldehyde (GA), methylglyoxal (MG), and D-glucose (Glc). Reaction conditions: [carbonylic reactants] p0.1 M; [glycine] p0, 0.1 or 0.2 M; reflux. Solid line: caramelization; broken line: Maillard reaction
Scheme 1
elimination (see Scheme 1). This subsequently forms coloured products faster than glyceraldehyde itself. The formation of brown products from D-glucose alone or in glucose/glycine mixtures was negligible on the same time scale (Fig. 1). Browning was only detectable after increasing the concentration of the amino acid to give a molar ratio of 2 : 1 (glycine : D-glucose). Davies et al. [10] have shown that the rate of browning in the D-glucose-glycine reaction depends on [glycine] 2 and so accounts for the sensitivity of colour formation as the amino acid concentration is increased. Also, it is possible that, at the higher amino acid concentration, retro-aldol reactions are favoured, and the observed browning could be due to further reaction of the cleavage products. The isolated non-dialysable (high molecular weight) fraction of glyceraldehyde/glycine and methylglyoxal/ glycine melanoidins possesses the same retention time in GPC-HPLC and similar UV-Vis spectra as that of the D-glucose/glycine melanoidin formed under the same reaction conditions. Therefore it can be assumed that the molecular weights of all three melanoidins are in the same range. Nevertheless, the microanalysis data of the melanoidins formed with C3 carbonyl components differ significantly from the data of the D-glucose/glycine and maltose/glycine melanoidins (Table 1). The melanoidin formed from D-glucose/glycine contained about 1.3 mol carbonyl compound per mol amino acid (Table 1). In contrast, 2.3–2.9 mol carbonyl compound were incorporated in melanoidins built up from C3 carbonyl compounds as the starting reactant. This means that, in all three melanoidins, the microanalysis data reflect an incorporation of six C-atoms of carbonyl compound per N-atom of amino acid. One way in which this could take place from glyceraldehyde or methylglyoxal is the combination of two molecules of the C3 compound to form a reactive melanoidin precursor. For maltose/glycine melanoidin a different reaction pathway has to be assumed because of the lower nitrogen content (Table 1). Under the reaction conditions used, fragmentation reactions of maltose are restrained in favour of a “peeling-off reaction” [3]. Maltose reacts
263 Table 1 Comparison of microanalysis data (%) of nondialysable melanoidins formed from several carbonyl compounds/glycine (1 : 1)
Carbonyl compound
C (%)
H (%)
N (%)
O (%)
aa
Empirical formula
Glyceraldehyde Methylglyoxal D-Glucose Maltose
55.47 58.03 51.77 46.68
5.46 6.78 5.76 6.19
7.63 7.18 6.47 5.17
31.44 28.00 36.01 41.96
2.30 2.93 1.26 0.78
C8H10NO4 C9H13NO3 C9H13NO5 C11H17NO7
a Number of moles carbonyl compound (starting material) incorporated into the polymer per mole of amino acid [17]
with the amino acid at the reducing group. The participation of C3 carbonyl compounds in the formation of such melanoidins is low. These results are consistent with the suggestion by Cämmerer and Kroh [8] of a general structure for melanoidin polymers which would be influenced by the carbonyl compound only as a result of side chain effects; this has now been confirmed by Yaylayan and Kaminsky [11]. Formation of HMF HMF is an indicator of nonenzymatic browning reactions [12]. Since the compound is produced from hexoses themselves, and is relatively unreactive in the Maillard reaction, it serves as an indicator for both caramelization and the Maillard reaction, and has been used for this purpose as a quality assurance parameter in the food industry. Figure 2 shows a comparison of the amount of HMF formed from different reactants with 3, 6, and 12 Catoms (glyceraldehyde, methylglyoxal, D-glucose, maltose). The highest concentration was observed in the
Fig. 2 Amount of hydroxymethylfurfural (HMF) (mmol per mol of carbonylic reactant) formed during the caramelization and the Maillard reaction of glyceraldehyde (GA), methylglyoxal (MG), D-glucose (Glc), and maltose (Mal). Reaction conditions: [reactants] p0.1 M; reflux. Reaction times 60, 300, and 600 min
caramelization of glyceraldehyde at each reaction time. To explain this observation, isomerization of glyceraldehyde followed by dehydration to methylglyoxal and its condensation with another molecule of glyceraldehyde is suggested in Scheme 1. This route for the formation of HMF from sugar degradation products has, so far, not been reported in detail. Methylglyoxal gave little, if any, HMF (Fig. 2), since it cannot be converted to glyceraldehyde. On the other hand, 5-methylfurfural, whose formation from methylglyoxal is expected, was detected at very low concentration and only after relatively long reaction times. Glucose and maltose gave lower yields of HMF compared with glyceraldehyde (Fig. 2). In the case of D-glucose, the reaction is expected to proceed by the 1,2-enolization pathway followed by b-elimination of water to the 3-deoxyhexosulose as a key intermediate. Maltose gave more HMF than did D-glucose under same reaction conditions. A reason for this observation could be the intramolecular formation of osulose at the reducing end of maltose, followed by elimination of the D-glucose residue by cleavage of the glycosidic bond as described previously [13]. However, in addition to HMF, larger quantities of glyceraldehyde have been detected during the caramelization of maltose [13]. Therefore, both with D-glucose and maltose, HMF is formed by two competing reactions: the retro-aldol cleavage and the conventional sugar dehydration pathway. In the amino-acid-catalysed (Maillard) reaction, Dglucose and maltose formed considerably more HMF than was obtained, on the same time scale, under caramelization conditions (Fig. 2). This may be explained by considering the role of the amino acid, at different stages of the reaction. Simplistically, it catalyses the formation of 3-deoxyhexosulose which, in turn, leads to HMF [4]. The ARP (fructosylglycine) produced about 10 times more HMF than did D-glucose and glycine (not illustrated) under the same conditions. One interpretation could be that the ARP participates in the formation of the 3-deoxyhexosulose and, subsequently, HMF [4]. On the other hand, there is now a growing body of evidence which points to the fact that it is the enaminol precursor of the ARP which is the key intermediate in the formation of 3-deoxyhexosulose [14, 15]. The ARP is the more likely intermediate in the formation of 1- and 4-deoxyhexosulose through 2,3-enolization. These intermediates, in turn, are the species which are liable to retro-aldol cleavage. Under Maillard reac-
264
tion conditions, glyceraldehyde, a possible retro-aldol product of D-glucose, prefers other reaction pathways than the formation of HMF (Fig. 2), for example a direct reaction with amino acid to diimines [1, 5] and/or imidazoles [6]. Therefore, as was the situation in caramelization, the formation of HMF in the Maillard reaction depends on at least two different, competing reaction pathways. Reaction mechanism In the early stages of the Maillard reactions of D-glucose/glycine and maltose/glycine, homolytic and heterolytic mechanisms compete depending on the reaction conditions, particularly the pH. In order to be able to identify whether or not the C3 retro-aldol products contribute to homolytic pathways in the non-enzymatic browning of mono- and disaccharides, the extent to which the reactions of methylglyoxal and glyceraldehyde are controlled by mechanisms involving radicals, in comparison with the D-glucose model system, was investigated. A relatively simple approach to this investigation involves the use of the stable radical DPPH and the use of Fe(II) to detect electron transfer processes as described previously [9]. The results are given in Figs. 3 and 4. In contrast to D-glucose, reactions of methylglyoxal and glyceraldehyde both under caramelization (Fig. 3) and Maillard reaction conditions (Fig. 4), at pH 4–5, are seen to involve free radicals to a large extent (40–60%). The greater contribution of radical reactions in the caramelization of glyceraldehyde, compared with methylglyoxal, could be explained by the formation of enediol structures and/or the higher tendency for enolization in this molecule, which is thought to be essential for the
Fig. 3 Contribution of heterolytic mechanism to the overall reaction, and increase in absorbance at 420 nm, in the caramelization of glyceraldehyde (GA), methylglyoxal (MG) and D-glucose (Glc). Reaction conditions: [reactants] p0.1 M; reflux. Solid line: contribution of heterolytic reactions; broken line: A420
Fig. 4 Contribution of heterolytic mechanism to the overall reaction, and increase in absorbance at 420 nm, in the Maillard reaction of glycine with glyceraldehyde (GA), methylglyoxal (MG) and D-glucose (Glc). Reaction conditions: [reactants] p0.1 M; reflux. Solid line: contribution of heterolytic reactions; broken line: A420
formation of radicals [16]. Such C3 radicals should be relatively stable in the weakly acidic medium [16]. After a short induction phase in the Maillard reaction of methylglyoxal, the contribution of radical reactions is approximately 10–15% greater than in caramelization (Figs. 3, 4). This could be explained by the involvement of the amino compound. A reaction product of methylglyoxal and an amine, methylglyoxal diimine, is considered to be a good radical precursor [16]. In the case of the glyceraldehyde reaction, the graphs follow similar curves in the absence (Fig. 3) and presence (Fig. 4) of glycine, and a doubling of the concentration of glycine did not change the result significantly. Therefore, the amino acid had only little effect on the formation of radical precursors in the case of this reactant. In contrast, the reaction of methylglyoxal seems to be more affected by the presence of glycine. In the D-glucose/glycine reaction, a free radical process was identified at pH 4–5, but only after the induction period (approx. 100 min) was over (Fig. 4). Since the caramelization of D-glucose was found to be completely “heterolytic” (Fig. 3), the contribution of free radicals to the Maillard reaction is inferred as being due to the participation of the amino acid. This was confirmed by changing the molar ratio of D-glucose : glycine to 1 : 2 (not illustrated). The higher amino acid concentration shortened the induction phase and the contribution of radical reactions increased. No free radicals have been detected in the browning of the ARP (fructosylglycine) in the presence of an amino acid [16]. Therefore, the effect of the amino acid on the formation of radicals in the Maillard reaction of Dglucose has to be attributed to other reaction intermediates. The fact that there is an induction phase in the onset of a free radical mechanism suggests that radicals may be formed in the later stages of the reaction, for
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instance after a retro-aldol reaction of Maillard reaction products like deoxyosuloses. In the case of the C3 carbonyl compounds examined, as shown in Figs. 3 and 4, there is no direct correlation between the formation of radicals and an effective browning. The browning of methylglyoxal alone, and in presence of amino compounds, was much more intense than that of glyceraldehyde. However, the participation of radical mechanisms in the browning reactions of both compounds differed only to a small extent. This corresponds with investigations of Namiki and Hayashi [16]. Carbonyl compounds which form many radicals also show intense browning [16]. Conclusion Glyceraldehyde is a likely precursor of HMF in the Maillard reaction of D-glucose. On the basis of the homolytic nature of the browning reactions of methylglyoxal and glyceraldehyde, it is suggested that the products of retro-aldol reactions of Maillard intermediates such as the deoxyosuloses are responsible for at least some radical formation in the Maillard reaction of D-glucose. Nondialysable fractions of melanoidins formed from maltose/glycine, D-glucose/glycine, glyceraldehyde/glycine and methylglyoxal/glycine show similar molecular weights. In all cases, six C-atoms of carbonyl compound were incorporated per N-atom of amino acid. Acknowledgements Study carried out with financial support from the Commission of the European Communities, Agriculture and Fisheries (FAIR) specific programme CT96–1080 : “Optimisation of the Maillard reaction: a way to improve quality and safety of thermally processed foods”.
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