The Formation Mechanism Of Ethyl Carbamate During The Vinification Process

Ethyl carbamate(EC), a kind of genotoxic carcinogen which has great threat to human health, is mainly found as a natural trace constituent in wine and other alcoholic beverages. Therefore, the measures to reduce EC in fermented alcoholic beverages were become the most urgent problem.In this article, the precursor and formation mechanism of EC were analyzed systematically during the vinification process.High phase liquid chromatography(HPLC) has been developed for simultaneous determination of 20 kinds of amino acids content in wine. It was used to analyze the types and content of 20 kinds of amino acids in six different varieties of grape juice from the different wine-producing areas.The results showed that different varieties of grape juice contained the same kinds of amino acids,but the content of amino acids was differed with grape varieties.The composition of chemically defined must was determined according to the results of measurement.The chemically defined must was used for fermentation. The content of amino acids were tested in different time periods to establish the experiment of amino acids deficiency and determined the effect of missing amino acids on the formation of EC.The analytical method of HPLC-FLD and GC-MS were developed to test the content of urea and EC in eight kinds of chemically defined must in brewing process.The results showed that the content of urea and EC generated in chemically defined must missing other amino acids were same to complete medium fermentation in brewing process,and the trace amounts of urea and EC were produced in chemically defined must only lack of arginine during the brewing process. Thus it could draw that arginine played a very important role in the formation process of urea and EC.By increasing the concentration of arginine in the fermentation process,there was correlation between the amount of arginine and EC, and the correlation coefficient reached to 92.68%. It was concluded that urea was identified as the major precursor of EC through combining with the arginine metabolic pathways of yeast.Isotope 15N was used to mark the two nitrogen atoms of arginine guanidine, and it came to a conclusion that the nitrogen atoms took part in the formation way of EC. Thus, The formation mechanism of EC was confirmed definitely.The effect of temperature, pH, and ethanol on urea alcoholysis reaction was studied using Box-Behnken experimental design. The apparent kinetics model of the urea react with ethanol to form EC was developed based on the reaction mechanism. Results indicate that the high temperature, high ethanol content and low pH value are conducive to the formation of EC. The predicted value of the EC fits well with the measured values in urea-ethanol-water system, but fits poorly in the real wine samples. In fact, the predicted values are only 66.4% to 86.5% of the measured values. It suggests that the urea is the main precursor of EC, but not the only one in red wine from the point of kinetics.Using Real-time fluorescence quantitative PCR technique to detect and analysis the related genes expression of arginine and urea metabolism by adding the missing nitrogen source (DAP) in different time during the fermentation process. The results showed that the additional treatment in different time had a different influence on the expression of related genes. Adding DAP at the time of nitrogen deficiency in 48 h could relieve the stagnation of fermentation properly, as well as could limit the formation of the EC, so that less EC could be produced during the vinification process.