Research Of Reducing EC In Niulanshan Erguotou By Microbial Of Fermented Grains-Screening Of Producing Urea-degrading Enzyme Microbes And Researching Characteristics

A large number of liquor is produced and consumed in our country,with the improvement of living standards,people increasingly concern about the safety of liquor.The study found that ethyl carbamate(referred EC),generated during the production of liquor,was carcinogen.EC was classified to 2A carcinogen list by the IARC.Studies reported,in liquor many factors could affect content of EC,such as temperature,light,ingredients,storage period,distillation techniques,distillation time,kinds of koji(microbial species)and so on.EC in liquor mainly comes from the reaction of urea and ethanol.Since urea-degrading enzyme can break down EC’s precursor-urea,so our study is around koji microorganisms which can produce urea-degrading enzyme.That is,screen out microorganisms which can produce urea-degrading enzyme from fermented grains,and research their ability to decompose urea.Thus reduce the generation of EC.Eventually,we successfully separated 14 strains which produce urea-degrading enzyme from Niulanshan Erguotou fermented grains and koji,their arithmetic mean of urea-degrading enzyme vitality are 0.427,0.149,0.082,0.252,0.782,0.199,0.222,0.189,1.056,0.326,0.309,0.333,0.755 and 0.420μmol/min.The 9th bacteria have the largest ability to produce urea-degrading enzyme,its enzyme activity is 1.056μmol/min.The shape of the colonies is nearly round,smooth,milky white and opaque.We found it belongs to Gram-positive bacteria by microscopic examination.The optimum acidity for cell growth is 0.8;the optimum temperature is 30℃;ventilation benefits cell growth;adding organic nitrogen may promote the growth of bacterial cells,it can also speed up the bacteria secrete metabolites;in the case of organic nitrogen exist,the addition amount of urea has little effect on cell growth;urea-degrading enzyme may be accumulated mostly when temperature around 42℃and acidity lower than 2.0;the urea-degrading enzyme is extracellular enzymes and it is producted simultaneously with cell growth.The study founds that time,temperature and acidity have little influence on the decomposition of urea under normal production conditions,little of the urea could be entrained into the finished wine in distilling;adding the sieved strain(urea-degrading enzyme)to a medium which contains urea,the level of urea is reduced significantly.By reducing EC’s precursor,we can initially infer that urea-degrading enzyme can reduce the synthesis of EC.We developed a method for the determination of ethyl carbamate in liquor by gas chromatography-mass spectrometry for detecting the residual amount of EC.EC of samples was adsorbed by alkaline diatomite SPE cartridges,and eluted by n-hexane,and eluted by ethyl acetate/ethyl ether.The eluate was dehydrated by anhydrous sodium sulfate before being concentrated by blowing nitrogen.The results show that the method had a good linear on standard curve,the linear range from 10μg/L to 1,000μg/L of EC,the correlation coefficient is R~2=0.9968,the method’s detection limit is 20μg/kg,the average recoveries were 78.3%to 101.8%when added standard substance between100μg/kg and 1,000μg/kg.This method was simple,sensitive and accurate.It is an practical method to analysis EC in liquor.