Investigation Of Ethyl Carbamate Degradation In Chinese Rice Wine By Rhodotorula Sp.

Ethyl Carbamate（EC）, an accompany product of fermentation or storage, has beenreported to be a contaminant of various kinds of alcoholic beverages, and no exception ofChinese rice wine. It has been used for human medical purposes in the past, but now it isdisabled because of its toxicity and poor efficacy, and it was classified as a Class2Acarcinogen by IARC in2007. As a result, more and more attention has been paid to the overdose of EC in Chinese rice wine. Since the existing methods can not completely solve theproblem of EC pollution in Chinese rice wine, it is of great importance to explore new waysof EC control in Chinese rice wine.This thesis concerned about the isolation of EC-degrading microorganism, theoptimization of flask fermentation conditions, the preliminary study of urethanase producedby LBMAE-8, and also the preliminary application of free cells and immobilized cells inChinese rice wine. The main results are as follows:1) With EC as the sole carbon source, we isolated an EC-degrading microorganismLBMAE-8from Chinese liquor Daqu, fermented grains and soil through panel separation andflask rescreening. When the initial concentration of EC in the medium was5g·L^-1, after6days incubation, a degradation rate of up to80%can be reached. Physiological andbiochemical properties togrther with26S rDNA sequencing analysis indicates that it belongsto Rhodotorula sp., and it has been preserved in China General Microbiological CultureCollection Center with a number of CGMCC No：5081. Since it came from Chinese liquorDaqu, and Rhodotorula sp. are generally considered safe, therefore, we preliminary view thatit can be applied to the removal of EC in Chinese rice wine.2) Through the single-factor optimization, the optimal conditions for urethanaseaccumulation were determined: glucose20g·L^-1, yeast extract30g·L^-1, MnSO41.4mmol·L^-1,initial pH6.0, temperature30℃, inoculum4.0%,75mL medium in500mL shake flask andculture time42h. Urethanase produced by strain LBMAE-8under the optimal conditions was421.6U per gram of dry cell, which increased by3.52times comparing with the previous.3) Urethanase produced by LBMAE-8was also preliminarily studied. It belongs to atype of constitutive enzyme, which can stoichiometrically decompose EC into ethanol,ammonia and carbon dioxide. Partially purified enzyme was obtained via ion exchangechromatography, hydrophobic interaction chromatography together with gel filtrationchromatography, the specific activity increased by33.1times and the activity recovery ratewas3.0%. The optimial temperature of the enzyme is40℃, and theoptimial pH is neutral,the activity of the enzyme suffered serious losses under acidic conditions, indicating that thefree enzyme is not suitable to be applied directly to actual alcoholic beverages, while wholemicrobial cells, immobilized cells or immobilized enzyme are optional ways, providingcertain reference for follow-up study.4) Through free cells and immobilized cells, we proved the application value of strainLBMAE-8in the degradation of EC in Chinese rice wine. The free cells showed certaintolerance to ethanol and acid. When added to two commercial Chinese rice wine, degradation rates of EC of39.68%and19.01%were achieved. At the same time, theimmobilized cells were also studied, and better tolerance towards ethanol and acid wereachieved. The calcium alginate immobilized cells and chitosan/calcium alginatemicrocapsules （one-step） immobilized cells showed higher activity recovery rates. In thecase of the same enzyme amount, the EC decomposition efficiency increased to50%fromless than5%in one commercial sample when the chitosan/calcium alginate microcapsules（one-step） immobilized cells were used.