Structural characterization of polar melanoidins deriving from Maillard reaction intermediates - A model approach.

Chemical conversions of reducing sugars and amino compounds induce the formation of heterogenous, high-molecular-weight colorants ('melanoidins') with widely unknown chemical structures. Model experiments of reactive intermediates have proven to be suitable for unravelling the formation mechanisms of colored reaction products. Here, the active methylene norfuraneol was selected and incubated individually as well as in combination with glyoxal, glycolaldehyde, and acetaldehyde at elevated temperatures. Photometric and chromatographic methods as well as mass spectrometry were used to analyze the colored reaction products and reveal the reactivity of different carbonyls regarding the formation of heterogenous oligomers. Aqueous solutions of norfuraneol and glyoxal exceeded the color formation of all other model reaction systems and it could be shown that the initial reactants as well as their degradation products were incorporated into the colorants. The colored oligomers described herein were composed of carbohydrate-based intermediates of the Maillard reaction and defined as melanoidin precursors or pre-melanoidins.

Formation of melanoidins - Aldol reactions of heterocyclic and short-chain Maillard intermediates.

In the course of the Maillard reaction, reducing sugars and amino compounds are converted to colorants, whose chemical structures are still mostly unknown. Active methylene compounds like norfuraneol that can initiate aldol condensation reactions are considered as key intermediates in this reaction. The aim of the present study was to characterize color formation of norfuraneol with different carbonyl compounds and to identify the underlying mechanisms of the reaction. Norfuraneol was incubated with methylglyoxal or diacetyl at elevated temperatures and the resulting reaction mixtures were analyzed by means of high-resolution mass spectrometry. It was demonstrated that aldol reactions lead to the formation of heterogeneous carbohydrate-based oligomers, which are likely to contribute to the elevated browning observed in the reaction mixtures. Furthermore, redox reactions were identified as another important part of the reaction, resulting in an increasing number of double bonds in the detected reaction products.

Melanoidins Formed by Heterocyclic Maillard Reaction Intermediates via Aldol Reaction and Michael Addition.

Thermal treatment of food leads to the formation of melanoidins by reactions of carbohydrates with free amino acids or proteins in the late stage of Maillard reaction. The aim of this study was the identification of reaction mechanisms responsible for the formation of melanoidins involving active methylenes with the heterocyclic structure and the structural characterization of the resulting products. For this purpose, norfuraneol was incubated with the aldehydes pyrrole-2-aldehyde or furfural at 125 °C. With the help of high-resolution mass spectrometry, the structural composition of oligomers of up to fifteen C5-units could be described for the first time. Aldol and Michael reactions could be identified as crucial steps for the formation of new C-C-bonds. With increasing heating time, the elimination of water from the products was facilitated, and oxidation reactions of integrated reductone structures lead to the expansion of conjugated double-bond systems responsible for the color formation of the samples.

Formation of Reactive Intermediates, Color, and Antioxidant Activity in the Maillard Reaction of Maltose in Comparison to d-Glucose.

In this study, the Maillard reaction of maltose and d-glucose in the presence of l-alanine was investigated in aqueous solution at 130 °C and pH 5. The reactivity of both carbohydrates was compared in regards of their degradation, browning, and antioxidant activity. In order to identify relevant differences in the reaction pathways, the concentrations of selected intermediates such as 1,2-dicarbonyl compounds, furans, furanones, and pyranones were determined. It was found, that the degradation of maltose predominantly yields 1,2-dicarbonyls that still carry a glucosyl moiety and thus subsequent reactions to HMF, furfural, and 2-acetylfuran are favored due to the elimination of d-glucose, which is an excellent leaving group in aqueous solution. Consequently, higher amounts of these heterocycles are formed from maltose. 3-deoxyglucosone and 3-deoxygalactosone represent the only relevant C6-1,2-dicarbonyls in maltose incubations and are produced in nearly equimolar amounts during the first 60 min of heating as byproducts of the HMF formation.

Browning Potential of C6-α-Dicarbonyl Compounds under Maillard Conditions.

In this work, the three major C6-α-dicarbonyl compounds glucosone (GLUC), 1-deoxyglucosone (1-DG), and 3-deoxyglucosone (3-DG) were synthesized and examined under Maillard conditions (aqueous solutions with the addition of l-alanine at 130 °C and pH 5/8). For the first time, the resulting color formation, antioxidant activity, and generation of short-chained α-dicarbonyls were investigated and compared to incubations of d-glucose and d-fructose. An additive effect on the formation of color, an antagonistic effect on the generation of α-dicarbonyl compounds, and a synergistic effect on the antioxidant activity could be observed for the 1-DG/GLUC combination. Despite their common degradation products, different extinctions could be measured, with 3-DG showing the strongest color formation, followed by GLUC and 1-DG. The analyzed α-dicarbonyl compounds have no direct impact on the formation of color but are precursors for most of the colored compounds. The main difference between the three substances is their ability to form different heterocyclic degradation products, such as pyranones (1-DG), furanones (1-DG), furans (GLUC and 3-DG), and the corresponding N-heterocycles in the presence of amino components. This seems to be the main reason for their varying browning potential and antioxidant activity.

Antioxidant Properties of Heterocyclic Intermediates of the Maillard Reaction and Structurally Related Compounds.

It is well established that a wide range of reductones is formed in the course of the Maillard reaction and that these substances contribute to the oxidative stability of food. The aim of this study was to analyze 12 important heterocyclic intermediates with and without reductone structure as well as structurally related substances under equal conditions to compare their antioxidant properties in detail. For this purpose, five methods were selected including photometrical methods such as the trolox equivalent antioxidant capacity assay and an electron paramagnetic resonance spectroscopic method. Reductones with furan-3-one structure and 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one were reducing in all assays, whereas isomaltol and maltol did not react in assays based on the reduction of metal ions because of their complexing abilities. The introduction of protecting groups to the free hydroxyl functions of selected reductones could nearly eliminate their reducing abilities. In addition, the oxidation products of the different reductive heterocycles were compared after treatment with iodine. Mainly short-chained organic acids such as lactic, glycolic, and glyceric acid are formed as result of the degradation, which indicates 1,3-dicarbonyl cleavage reactions of corresponding tricarbonyl compounds as intermediates of the oxidation.

Antioxidant capacity of 1-deoxy-D-erythro-hexo-2,3-diulose and D-arabino-hexo-2-ulose.

The antioxidant capacity of two 1,2-dicarbonyl compounds, 1-deoxy-d-erythro-hexo-2,3-diulose (1-deoxyglucosone) and d-arabino-hexo-2-ulose (d-glucosone), was investigated. Both compounds are key intermediates of the Maillard reaction, and both possess a reductone-like structure. The reductive potential of the reductones was measured with the trolox equivalent antioxidant capacity (TEAC) assay and the Folin-Ciocalteu reagent (FCR) assay. Their antioxidant capacity set them apart from their precursors and other typical Maillard reaction products. Using electron paramagnetic resonance (EPR) spectroscopy, the special radical scavenging behavior of 1-deoxyglucosone and d-glucosone was measured. Both exhibited a slow, but constant, scavenging ability over the course of several hours, even days. It was postulated that this characteristic behavior is caused by the isomeric composition and the transformation to the particular antioxidant form. Reaction mixtures of 1-deoxyglucosone showed a correlation between the decrease of antioxidant properties and the decomposition of 1-deoxyglucosone.

Reactivity of thermally treated α-dicarbonyl compounds.

The degradation reaction of thermally treated 3-deoxy-d-erythro-hexos-2-ulose and methylglyoxal, both key intermediates in Maillard chemistry, was investigated. Different analytical strategies were accomplished to cover the broad range of formed products and their different chemical behavior. These involved HPLC-DAD and accordingly LC/MS analysis of the quinoxaline derivates, GC/MS analysis of the acetylated quinoxalines, and GC-FID analysis of the decyl ester of acetic acid. As a main degradation product of 3-deoxy-d-erythro-hexos-2-ulose, 5-(hydroxymethyl)furfural could be identified. At alkaline pH values, 3-deoxy-d-erythro-hexos-2-ulose generated various acids but no colored products. In contrast, thermal treatment of methylglyoxal yielded high molecular weight, brownish products. A dimer of methylglyoxal, first precursor for aldol-based polymerization of methylglyoxal, could be clearly identified by GC/MS.

Milieu dependence of isomeric composition of 1-deoxy-D-erythro-hexo-2,3-diulose in aqueous solution determined by high-resolution NMR spectroscopy.

In this study, high-resolution (1)H (600.03 MHz) and (13)C NMR spectroscopy (150.89 MHz) were used to elucidate the structures of the identifiable isomers of a complex mixture of equilibrating 1-deoxy-D-erythro-hexo-2,3-diulose (1-DG) (1) isomers in aqueous solution. Approximately 15 isomers were formed from the investigated deoxy-ketohexose, and the equilibrium of the isomers is dependent on the pH values and temperature. In contrast to non-hydrated isomers, hydrated 1-DG (1) isomers were exclusively formed in aqueous solution. Their relative concentration increased when the pH was increased to more basic conditions. An increase in solution temperature led to the elimination of water and an increase in the relative concentration of the non-hydrated 1-DG (1) isomers. The observed influence of different conditions on the isomeric composition was explained for all isomers. Also, these observations provided information for structure elucidation. Furthermore, the reaction behaviour of 1-DG (1) under basic conditions at high temperatures (up to 347.5K) was interpreted and described.