Analysis Of The Microbial Communities Of Three Kinds Of Fen-Daqu By PLFAs And Screening Of High Yield Aroma Esters Yeast And Its Application

There are three kinds of Daqu for light fragrant Chinese liquor brewing,which are Qingcha,Hongxin and Houhuo.They are manufactured separately and mixed in brewing.The characterization of Daqu technology are production using materials without sterilization,inoculation from environment and fermentation in open space.So the homogeneity of every pieces of Daqu are very poorly,Daqu from different seasons and batches always have differences.Since Daqu are saccharifing fermentation agent,the quantity of microbes are important index for Daqu quality evaluation.Now the methods are still traditional plate count.Its urgent to find fast and accurate method for detecting the microbes of Daqu.Yeast are very import microbes,especially in light fragrant Chinese liquor brewing,since they are not only related to the yield,but also related to the quality.The mainly flavor component ethyl acetate are mainly produced by yeasts.Study on yeast community and aroma-producing yeast are impending.PLFA(phospholipid fatty acid)was used for analyzing the microbial communities of Daqu.Yeasts were isolated from Daqu and fermented sorghum and identified and diversity analysis.The yeasts producing ethyl acetate were selected,and the culture condition were optimized,and so on.The mainly results were as follows:In order to analyze the microbial community of Daqu for light-fragrant Chinese liquor brewing,the phospholipid fatty acid(PLFA)profiles of Daqu were investigated.Three types of Daqu clearly showed different PLFA features.The PLFA 18:3 w 6c(3,6,9),as one of the biomarkers for fungi,was the highest in the Houhuoqu samples,the lowest in the Hongxinqu samples and moderate with fluctuations in the Qingchaqu samples.The PLFA 18:1 w 9c,another biomarker for fungi,was only detected in the Hongxinqu samples with a steady concentration.The ratio of the fungi to bacteria of Hongxinqu was 11.20,which was significantly higher than that in the Qingchaqu and the Houhuoqu samples.Principal component analysis found that most of the variations in the PLFA patterns were due to the different Daqu types.Variances analysis found that some PLFAs with significant difference had the potential to be used as representative biomarkers for each type of Daqu.All of 48 yeast strains isolated were separated into 5 kinds by colony characterizes on WLN culture medium.Combination of PLFAs and molecular biology methods,also 5 clusters were found,which were Saccharomyces cerevisiae,Issatchenkia orientalis,Cryptococcus uzbekistanensis,Rhodotorula mucilaginosa and Wickerhamomyces anomalus.It were found that 16:1 Cis 9(w7)and 18:2 CIS 9,12/18:0a could be the characteristic PLFA for classification of Saccharomyces yeasts and Non-Saccharomyces yeasts.PLFA fingerprint of pure strains would provide support for culture-free analysis of fermented grains in different periods and Daqu samples.The strain with the highest esterifying capability was selected from 48 isolates of yeast strains,which was identified as Wickerhamomyces anomalus.The optimized culture medium of J2-5 yeast was sorghum saccharification mash.By the orthogonal test,the optimum culture condintions were as followed: the sugar detree was 7 °Bx,temperature was 20℃,p H of cultrue medium was 4 and without ethanol.With the fermentation process continued,the total ester increased.The total ester run up to 7.63g/L when the fermentation time was 20 d,which was three times higher than that before optimization.Mixed the solid fermented grains and the esterified liquid of J2-5,the ester of the alcohol samples was increase 3-4 times compared to the control.The yeast J2-5 were grown well in the bran with moisture content of 50%.When cultured 48 for hours,the quantity of live yeast cells were up to 109 cfu/g bran.In the laboratory application experiment,esters were significantly higher than that of control,when added J2-5 via liquid culture and solid bran.To obtain mutant strains with higher ester yields,the J2-5 was mutated using atmospheric and room temperature plasma(ARTP).The total esters of mutant strains J2-51 and J2-56 were higher than 2.5g/L,significantly higher than that of control and enhanced almost twice.