Analysis Of The Stability Of Jiuyao Quality And The Change Pattern Of Microorganisms And Metabolites In The Production Process

Jiuyao is a unique saccharification starter for winemaking in our country.It provides microorganisms in the production of traditional handmade huangjiu,and has the dual function of saccharification and fermentation.In the production of traditional Jiuyao,the old Jiuyao inoculation technology passed down from generation to generation is used to keep the high-quality strains.However,in the actual production process,there are problems such as production season constraints,high labor intensity,low efficiency and high breakage rate.At present,there are few studies on the stability of Jiuyao,the succession of the microbial community structure of Jiuyao and its correlation with the dynamic changes of environmental factors,which seriously restricts the development of modernization of Jiuyao production.The driving force of structural succession is of great significance for promoting the modernization of Jiuyao and the quality improvement of huangjiu.The main conclusions of this study are as follows:（1）The stability of Jiuyao in different years and wineries was explored.For the microbial community structure of Jiuyao in different years,the results showed that there was no significant difference between the microorganisms between any two years（P>0.05）.The bacterial species Pediococcus pentosaceus and Weissella cibaria and the fungi species Saccharomycopsis fibuligera were the dominant species.For the physicochemical indicators of Jiuyao in different years,there was no significant difference between the three years（P>0.05）.For metabolites of different year,the Jiuyao metabolites in 2019 were more similar to the 2020,while 2018 was different from the other two year.The analysis of the main metabolites showed that the substances with higher content in the three years were relatively consistent,and the main difference was the content ofα-linolenic acid and ricinoleic acid.The results showed that there was significant difference in the bacterial and fungi microbial community structures between different wineries（P<0.05）.The bacteria were mainly Pediococcus pentosaceus,Weissella cibaria,Weissella confusa,Sodalis Other,Enterobacter Other,Klebsiella Other,etc.The fungi were mainly Saccharomycopsis fibuligera,Rhizopus rhizopus,Rhizopus microsporus,Wickerhamomyces anomalus,Candida tropicalis,Issatchenkia orientalis,Candida glabrata,Saccharomyces Other,etc.There were certain differences in the physicochemical indicators and metabolites of Jiuyao from different wineries.（2）In the fermentation process of Jiuyao,W.cibaria and P.pentosaceus were always the dominant microorganisms in bacteria community,and the sum of their relative content accounted for about 70%of the total content.S.fibuligera was the dominant microorganism among fungi.In the main fermentation process of Jiuyao,the content firstly decreased and then increased with the extension of fermentation time,and finally tended to be stable,increasing from about 30%to about 80%at first.The followed fungi are R.arrhizus,Mucor indicus,S.cerevisiae and so on.The abundance of R.arrhizus and M.indicus increased during pre-fermentation and decreased during post-fermentation.S.cerevisiae began to increase during pre-fermentation,and other yeasts increased in later fermentation.During the fermentation process,the physicochemical indicators had obvious changes.The trend of physicochemical indicators in different years is basically the same.The water content dropped from 41%to about 13.17%;The activity of acid protease could reach up to 125 U·g^-1;the acidity could reach up to 22 g·kg^-1.Through redundant analysis,moisture content was found to be the primary driver of the main microbial succession.Top drivers remained consistent for both years.Inoculation of aged Jiuyao affected the initial colonization of Jiuyao microorganisms.On the other hand,environmental filtration further affected the microbial colonization,so that the abundance of the dominant microorganisms in Jiuyao gradually increased and remained stable.Therefore,the stability of the Jiuyao production environment was one of the main reasons affecting the stability of Jiuyao passage.（3）There was a big difference in the relative contents of the epidermal and the core microorganisms of Jiuyao.Among the bacteria,P.pentosaceus and W.cibaria were most significantly different.P.pentosaceus was more distributed in the epidermis and W.cibaria was more distributed in the core.Among the fungi,S.fibuligera and S.cerevisiae differed the most.S.fibuligera was more distributed in the epidermis and S.cerevisiae was more distributed in the core.Different changes were observed in the physicochemical environment of the epidermis and core of the Jiuyao during the fermentation process.Except for the moisture content,other physicochemical indicators of the Jiuyao epidermis were slightly higher than the core.Through redundant analysis,humidity was the main driver of succession of microbial communities in the epidermis of Jiuyao during fermentation.Temperature was the main driver of succession of microbial communities in the core of Jiuyao during fermentation.The difference of physicochemical indicators inside and outside the microenvironment of Jiuyao is the reason for its spatial heterogeneity.With the change of fermentation time,the epidermis and core metabolites of Jiuyao showed significant differences and the number of different metabolites of Jiuyao increased significantly.Compared with the same time point,the microbial metabolism of Jiuyao epidermis was more active than the core.Among the metabolic pathways predicted by function,lipid metabolism,amino acid metabolism,sugar biosynthesis and metabolism,carbohydrate metabolism,cofactor and vitamin metabolism were the most active.Through traditional screening methods,71 strains of microorganisms were isolated from Jiuyao,including Wickerhamomyces anomalus,S.fibuligera,S.cerevisiae and P.pentosaceus.The results of functional analysis showed that S.fibuligera had the highest amylase activity;some S.fibuligera and S.cerevisiae had higher acid protease activity,and these microorganisms were considered as potential enzyme-producing strains in Jiuyao.To sum up,this paper takes Jiuyao as the research object,and uses high-throughput sequencing,metabolomics,physiochemical factor tracking and other methods to study the stability of Jiuyao,and the microbial community succession and metabolite changes in the production process.The main influencing factors and metabolic pathways of microbial community succession in the process of Jiuyao production were analyzed,the reasons for the stability of Jiuyao passage were analyzed,and the core functional dominant microorganisms in Jiuyao were finally screened.