Characteristics Of Chinese Rice Wine Fermented By Kluyveromyces Marxianus And Construction Of Xylose Metabolic Engineered Strains

In this study,the fermentation performance of HY32 at different temperatures and aeration conditions was detected.The results showed that the ethanol content of the rice wine fermented by HY32 was 7.13% vol at 37 ?,which was 1.2 times higher than that of 28 ?,and there were also a significant increase in ethyl acetate ? isobutyl alcohol ? isoamyl alcohol ? ?-phenylethanol and acetoine contents.However,the concentrations of acetic acid,propionic acid,butyric acid,isovaleric acid,valeric acid and caproic acid obviously reduced.There was no significant difference in the ethanol content between shaking(3.29% vol)and static fermentation(3.47% vol).The concentration of ethyl acetate reached up to 1369.69 mg / L in shaking fermentation,which was 9.7 times higher than that of anaerobic fermentation.In shaking fermentation,the generation of other volatile compounds,such as isobutyl alcohol,isoamyl alcohol,acetoine,acetic acid and caproic acid were also improved.Whereas,the concentrations of acetaldehyde,propanol,propionic acid,butyric acid and valeric acid decreased.The co-fermentation of K.marxianus HY32 and S.cerevisiae TRADY were performed with different inoculation ratios,and the optimal mixing ratio of 1:1was obtained.In the mixed fermentation of 1:1,the dynamic fermentative parameters of yeasts viable biomass,sugar,and the productions of ethanol,ethyl acetate and ?-phenylethanol were determined.The results showed that the K.marxianus HY32 counts were similar to the S.cerevisiae TRADY populations at the beginning of fermentation.However,in the middle and late fermentation period,the growth of HY32 was inhibited and TRADY was dominant.As fermentation proceeded,the ethanol content increased while the sugar content decreased gradually.The production of ethyl acetate mainly occurred at the first 3 days in the co-fermentation.In the later fermentation stages,the content of ethyl acetate remained steady.In the whole mixed fermentation process,the content of ?-phenylethanol showed an increasing trend.At the end of fermentation,volatile compounds were measured by GC–FID.The ethanol content of mixed fermentation was 13.89 % vol.Compared with the S.cerevisiae TRADY single-strain fermentation,the concentrations of ethyl acetate,acetic acid and acetaldehyde of mixed fermentation increased from 21.43 mg/L,284.09 mg/L,90.43 mg/L to 52.79 mg/L,371.90 mg/L and 101.38 mg/L,respectively.And the content of ethanol,total sugar,total acids,and ?-phenylethanol and p H-value all achieved the occupation standard.Moreover,mixed fermentation generated two flavor compounds: ethyl caproate(2.11 mg/L)and 2,3-butanediol(24.12 mg/L).In general,mixed fermentation increased the total acids,esters,aldehydes content and reduced the higher alcohols content,the flavor and the aroma of the rice wine significantly improved.Principal component analysis was carried out on the concentration of flavor compounds in order to establish relationships between flavor and microbial variables.Statistical analyses were performed with SPSS 17.0 software.The results showed that isovaleric acid,isoamyl alcohol,?-phenylethanol,ethyl oenanthate,ethyl phenylacetate,2,3-butanediol(meso),ethyl lactate,acetaldehyde and aldehyde acetal in mixed fermentation wine sample were mainly produced by TRADY;ethyl acetate,caproic acid,butyric acid,isobutyl alcohol,isobutyric acid and acetic acid were mainly produced by HY32;2,3-butanediol(L),valeric acid,ethyl caproate and acetoine were mainly produced by mixed cultures.Kluyveromyces marxianus BM2-5 was used as the parent strain to construct the engineered strain that could utilize xylose efficiently.The yeast BM2-5 expression vector p PIC6-xyl2 was constructed by cloning xylitol dehydrogenase gene xyl2,which originated from pichia stipitis and placed under the inducible promoter GALl of the vector.The plasmids containing xyl2 gene were transformed into Kluyveromyces marxianus BM2-5 by electroporation.Flask fermentation tests showed that there was no significant difference in xylose consumption rate and ethanol production between recombinant strain and wild-type strain.