Formation, Reduction And Immunoassay Of 4-methylimidazole In Maillard Reaction

Maillard reaction is a non-enzymatic browning reaction, which was widely used in food industry. During food processing and storage, Maillard reaction is the main way to give the color, flavor and taste of food. However, Maillard reaction also produces some toxic and harmful substances, such as acrylamide, 4-methylimidazole（4-MI） and advanced glycationend products（AGEs）. Ammonia caramel is mainly via Maillard reaction to produce its color and taste. But the harmful compound 4-MI was formed during the manufacture of caramel color. As a result, there is a huge security risk of caramel in food applications. In this paper, the mechanism of 4-MI formed in Maillard reaction was studied. Moreover, a systemic research was carried out to indicate the effect of the pH value, the divalent cations and the different reducing sugar on the formation of 4-MI. Finally, the monoclonal antibodies against 4-MI were successfully obtained, and the enzyme linked immunosorbent assay and the fluorescent-based immunochromatographic assay were established. The main results and conclusions are as follows:Different reactions between α-dicarbonyl compounds（methylglyoxal（MGO）, glyoxal） and aldehydes（formaldehyde, acetaldehyde） in the presence of ammonium sulfate were performed to investigate the formation of 4-MI. MGO was found to be the primary precursor of 4-MI. Two formation pathways of 4-MI were proposed. One pathway is that two molecular MGO reacts with equivalent ammonia and subduple formaldehyde to form 4-MI. On the other hand, MGO can react with glyoxal and double ammonia to form 4-MI and formic acid. Alternatively, MGO can react with equivalent ammonia to form 4-MI and acetic acid.The value of pH has significant effect on Maillard reaction and its products. The objective of the present study was to detail the change of 4-MI in sulfite and sulfate reactions with different initial pH values. Glucose–ammonium sulfate and glucose–ammonium sulfite reaction systems were anjust to initial pH conditions 4.9, 5.9, 6.9, 8.0 and 8.6 with sodium dihydrogen phosphate–dipotassium phosphate buffer, respectively. Higher concentration of methylglyoxal（MGO） and 4-MI was detected in thermal treated glucose–ammonium sulfite reaction system than that in sulfate system. The SO32- reacting with MGO and other precursors of 4-MI at higher pH conditions prevented 4-MI formation. However, no inhibition of 4-MI was found at lower pH conditions due to higher reactivity of the nucleophilic NH₄⁺ than SO32-. High pH may be useful in suppressing the formation of 4-MI as well as in obtaining preferable colour in class IV caramel.The present study detailed the changes of 4-MI and its precursors in the presence of divalent cations(Ca²⁺, Mg²⁺) in fructose–ammonium hydroxide caramel model system. The content of 4-MI and its precursor MGO was inhibited by divalent cations(Ca²⁺, Mg²⁺). The possible explanation might be that fructose and its Heyenes compound glucosamine interact with divalent cations to form complexes and inhibit the degradation of glucosamine into MGO. Moreover, the changes of fructose, NH₄⁺ and brown intensity in the presence of Ca²⁺ or Mg²⁺ indicated that fructose and glucosamine underwent the reaction of intra-intermolecular polymerization into melanoidins rather than the degradation reaction into aldehydes and ketones. Additionally, the color intensity（A420） was accelerated in the presence of Ca²⁺ or Mg²⁺.It is different that the carbonyl activity of aldose and ketose. Thus, glucose and fructose will have different effects on Maillard reaction products when used as reactant. This paper studied the variation of 4-MI in glucose–ammonia and fructose–ammonia model reactions. The initial products of the intermediate stage of Maillard reaction（i.e., fructosamine and glucosamine） were used as the initial reactant to analyze the variation of MGO. In addition, glucose or fructose was directly used as reactant and the content of MGO was detected. The main results are as follows:（1） In caramel model reaction, the browning intensity（A420） of fructose–ammonia system is greater than that of the glucose–ammonia system, indicating that fructose can promote the Maillard reaction when comparing with glucose;（2） Fructosamine is more active than glucosamine, which can produce more MGO under the same conditions;（3） The content of 4-MI formed in fructose–ammonia system is more than that in glucose–ammonia system, which is mainly due to that more MGO degraded from fructose than that from glucose.The hapten of 4-MI was prepared and identified. The artificial antigens were prepared by conjugating bovine serum albumin（BSA） or ovalbumin（OVA） with the hapten of 4-MI. And monoclonal antibody, evaluated by ic-ELISA, was obtained by immunizing Babl/c mice. After optimizing, a standard curve（y=-0.4885x+0.7002（R2=0.9931）） for ic-ELISA detection on 4-MI was obtained with the linear detection range of 0.64 to 20.48 mg/L. The cross-reactivity（CR） of all the structural analogues of 4-MI was less than 5.62%. The recoveries of 4-MI in caramels detection were ranged from 88.69 to 114.09%, with relative standard deviation（n=3） below 8.07%.A novel fluorescence-based immunochromatographic assay（ICA） for rapid detecting 4-MI is presented in this study. In our work, the conjugates of fluorescent microspheres（FMs） and 4-MI monoclonal antibody were used as probe for ICA. Under optimal conditions, a standard curve of ICA-based detection of 4-MI was developed, linear detection ranged from 0.50 to 32.0 mg/L. The cross-reactivities were observed less than 3.93% by detecting 6 selected structural analogues of 4-MI. The recoveries of 4-MI in caramels detection were ranged from 82.85–102.31%. The coefficient of variation（n=3） was 3.53–9.06%. Quantitative comparison of the established fluorescence-based ICA with high performance liquid chromatography-tandem mass spectrometry（HPLC-MS/MS） and indirect competitive enzyme-linked immunosorbent assay（ic-ElISA） analysis of real caramel colour samples indicated a good correlation among the methods. Therefore, our developed fluorescence-based ICA method shows great reliability and stability in detecting 4-MI.