Screening Of Strain With High Ethyl Lactate Esterase Activity And Its Application In Laobaigan Wine

Ethyl lactate is an important flavor substance in Laobaigan-flavor liquor,and together with ethyl acetate,it constitutes the main aroma of Laobaigan liquor.With the advancement of the mechanization and clean production of liquor in recent years,the lactic acid bacteria in the brewing environment and the content of ethyl lactate in the wine have decreased,especially the content of ethyl lactate and total ester in Laobaigan Sancha wine very low,and its corresponding the quality of the base wine is poor.In this study,strains with higher activities of saccharification enzymes,proteases and esterases were screened from Laobaigan Daqu and applied to the production experiment of Laobaigan Sancha wine,in order to improve the content of ethyl lactate in Sancha wine and the quality of Sancha base wine;and by the method of genetic engineering breeding,a strain with high yield of ethyl lactate esterase was constructed,which laid the foundation for the development and application of ethyl lactate esterase for winemaking.The main findings are as follows:(1)Screening of strains with high enzyme activity.Two strains of Aspergillus niger,T2 and T16,with high brewing enzyme activity were screened out from Laobaigan-flavor Daqu.Among them,T2 catalyzed the synthesis of ethyl lactate with the highest content of 1706.60 mg/L,and T16 had the highest saccharification enzyme activity of 2438.65 u/g.(2)Analysis of enzymatic properties of Aspergillus niger lipase and optimization of ethyl lactate synthesis conditions.The lipase gene excavated from Aspergillus niger T2 was heterologously expressed in Escherichia coli,and the lipases tab U and tab I were obtained,with specific enzyme activities of 48.67 U/mg and 54.34 U/mg,respectively.The enzymatic properties of lipase tab U and tab I were analyzed,and it was concluded that the optimal substrates of lipase tab U and tab I were p-nitrophenol acetate with Km values ??of 0.79 mmol/L and 0.85 mmol/L,respectively.The Vmax values ??were 71.32 mmol/(m L·h)and 63.75 mmol/(m L·h),respectively;the optimum temperature of lipase tab U and tab I were both 40 ℃,and the optimum p H was 9 and 8,respectively;In the system with lactic acid concentration of 1.17mol/L and ethanol concentration of 8.190 mol/L as solvent,the contents of ethyl lactate catalyzed by lipase tab U and tab I were 8.08 mg/L and 13.75 mg/L,respectively.The conditions for synthesis of ethyl lactate by lipase tab I were further optimized.After optimization,under the conditions of acid-alcohol ratio of 1:3.75,temperature of 40 ℃ and reaction time of 12 h,the synthesis amount of ethyl lactate reached210.24 mg/L;In the ethanol system with water as solvent,lactic acid concentration of5 g/L and volume fraction of 20%,when the p H of the reaction solution was adjusted to 6,the content of ethyl lactate catalyzed by lipase tab U was 7.96 mg/L,When the liquid p H was 8,the content of lipase tab I catalyzed the synthesis of ethyl lactate was5.12 mg/L.Under this condition,both lipase tab U and tab I showed the ability to catalyze synthesis in the aqueous phase.This study lays the foundation for the development and application of high-yield ethyl lactate esterase.(3)The application of high enzyme activity strains in Laobaigan Sancha wine.The strain with higher brewing enzyme activity was applied to the production experiment of Laobaigan Sancha wine,and it was determined that the best saccharifying agent was bran koji T16,the best yeast was △por,and the optimum addition amount of acid protease was 4 u/g fermented grains,the optimum ratio of grain addition is 1:6,the optimum steaming time is 15 minutes,and 10 days is the optimum fermentation time.Under this fermentation condition,the alcohol content in the fermented grains was 7.5 %v/v,11.26 mg/L of isoamyl acetate appeared in the fermented grains,the content of ethyl acetate increased by 32.12 times,the content of ethyl lactate increased 1.97 times,total ester content increased 2.3 times.