Urea Metabolic Characteristics Of Yeast Community In Solid-state Fermentation Of Chinese Light-aroma Liquor

Chinese liquor is made by multi-species and spontaneous solid-state fermentation.The fermentation process accumulates complex microbial community.It has theoretical and practical values in brewing mechanism and directional control to study metabolic characteristics of microbial community.Urea is an important intermediate in nitrogen metabolism,and is an important precursor of cancerogen ethyl carbamate(EC).However,microbial community associated with urea metabolism is unclear.It is important to reveal urea formation mechanism which could provide new clues to reduce EC content in Chinese liquor.Chinese light-aroma liquor is one of the most traditional liquor,which made by Qingcha and Xucha manufacture.This study tried to reveal urea metabolic characteristics in Chinese light-aroma liquor with two manufacture processes.We assessed the correlation between yeast and urea,and analyzed the potential main urea-producers.Then,evaluated the effect of environmental factors on yeast community,and investigated the microbial interaction on urea metabolism activities.This study had important theoretical and practical significance to enrich safety theory,and provided the basis for strategies to control potential safety risk in Chinese liquor and improve safety in foods.Below are main research findings:(1)The dynamic profiles of EC and its three potential precursors were analyzed to find main precursor in liquor fermentation.Urea was significantly positive association with EC(R=0.83,P<0.01),while citrulline and cyanide showed no significant association by correlation analysis(P>0.05),indicating urea was an important precursor of EC in Chinese liquor fermentation.Since urea source in liquor making was unclear,urea in sorghum,Daqu,repeated fermented grains(Xucha liquor)and fermentation was analyzed.Urea increased by 10.63 mg·kg-1 and 26.73 mg·kg-1 in Qingcha and Xucha liquor fermentation,which accounted for 41.8% and 41.2% to urea in final fermentation,respectively.This indicated that the fermentation process was one of the most important stages for urea formation.Therefore,it is important to reveal urea formation mechanism to provide the basis for strategies to control EC in liquor fermentation.(2)Correlation analysis was used to evaluate the association between urea and yeast.The result showed there was a linear correlation between urea and yeast population(R=0.44,P<0.01),indicating yeast was a microbial group related to urea metabolism.High-throughput sequencing was used to reveal the dynamic profiles of yeast community in liquor fermentation.Linear discriminative analysis effect size showed that a total of 14 and 16 yeast species relative abundance were much larger in Qingcha and Xucha liquor fermentation,respectively.Environmental factors might result in difference of yeast community in liquor fermentation.Saccharomyces cerevisiae,Pichia fermentans and Hanseniaspora osmophila were positively correlated with urea formation in Qingcha liquor fermentation(P<0.05),and S.cerevisiae,P.fermentans,Pichia manshurica,Saccharomycopsis fibuligera and Wickerhamomyces anomalus were positively correlated with urea formation in Xucha liquor fermentation(P<0.05)by partial least squares regression analysis.(3)To find the key control factors on urea metabolism,the effect of environmental factors on yeast community was evaluated.The different environmental factors were arginine,urea,moisture,reducing sugar and ethanol(P<0.05).Canonical Correlation Analysis(CCA)showed that arginine and urea were the most important environmental factors associated with yeast community succession.In addition,arginine and urea were significantly associated with potential main urea-producers(P.manshurica,W.anomalus and S.cerevisiae)(P<0.01).The higher level of arginine and urea in Xucha liquor fermentation could associate with repeated fermented grains,indicating the manufacture could influence yeast community and the growth of potential main urea-producers.(4)To elucidate urea metabolic characteristics of yeast community,microbial interaction was studied.Single-species fermentation showed that 10 yeast species had the ability to produce urea,with W.anomalus possessing the strongest urea formation activity(14179.98 ?g·L-1·OD-1).The urea production was increased to 1.34-fold and 4.20-fold in co-culture of W.anomalus and S.cerevisiae than their mono-culture at 6 h,respectively.The fold change of arginase gene transcription activity was studied.The results showed that the fold change of arginase gene transcription activity was increased by 1.36-fold in W.anomalus,while decreased by 39.2% in S.cerevisiae at 6 h,indicating that interaction could influence gene transcription activity in yeasts and urea production.