The Metabolism Mechanism And Inhibition Method Of Ethvl Carbamate Formation During Traditional Chinese Rice Wine Fermentation

Ethyl carbamate(EC)is a probable carcinogen that has been found mainly as a by-product of fermentation,especially in alcoholic beverages,such as grape wine,Japanese sake and Chinese yellow rice wine,etc.In 2007,the International Agency for Research on Cancer identified urethane as 2A carcinogen,which forced the international community paid a lot of attention in recent years.Recently,foreign countries have limited the maximum acceptable level of EC in beverages,and take a variety of ways to control the content of EC in alcoholic beverages.Although the biological basis and metabolic regulation of ethyl carbamate in grape wine and Japanese sake have been reported a lot,the metabolism mechanism of ethyl carbamate in Chinese traditional rice wine brewing process is seldom studied.Therefore,it is of great theoretical and practical significance to carry out the study on it.The main findings of this thesis are as follows:1.The inhibition of EC formation by adding exogenous inhibitors-0.01 mol/L ornithine and 150 U urease in the fermentation process have reduced EC content to 40%,in the mean time,increased the total sugar and total acid concentration,promoted the total amount of amino acids and the difference of flavor compounds could be ignored.2.The Saccharomyces cerevisiae ZJU was isolated from the complex Chinese starter system,using it to substute Chinese starter could reduce the EC concentration significantly by 85.6%,the content of total amino acids was significantly increased.But the kinds and contents of volatile flavor substances were decreased,indicating that the mixture of all the microbes in the traditional Chinese starter contributed to form the unique flavors of rice wine.3.Using the Roche 454 GS FLX + second-generation sequencing technology,the bacterial diversity in rice wine fermentation process was determined.A total of 228 microbes were isolated,among which the Enterobacteriaceae and Pantoea were abundant.The question sounded an alarm to rice wine safety issues.4.The differential expression of genes between single Saccharomyces cerevisiae ZJU and mixed fermentation with Lactobacillus brevis was studied by RNA-seq sequencing.In the whole metabolic process,there were 57 significant difference genes,and the functional analysis of the differentially expressed genes showed that after mixture with Lactoacillus brevis the flavors were more intense,the degradation process of sugar and fatty acids accelerated,the secondary metabolites increased but ethanol content decreased.It was also found that BTN2 is the key regulator of arginine metabolism in S.cerevisiae during yellow rice wine fermentation.5.In the process of traditional rice wine fermentation,the formation of urethane was negatively correlated with OTCase.After ultrasonication,ammonium sulfate fractionation and column chromatography,pure OTCase was obtained and its enzymological properties were studied.The optimum temperature of OTCase is 55? and quite stable under different temperature condition.The optimum pH is 4.0,also stable to a wide range of pH,indicating that OTCase is an acid enzyme.The enzyme is inhibited by many metal ions,indicating that OTCase is non-metallic enzyme.For high concentrations of salt and low concentrations of ethanol,OTCase shows tolerance to some extent.