Screening Of Functional Yeast In Luzhou-flavor Liquor And Its Application In Sichuan Xiaoqu

Xiaoqu liquor is one of the distilled liquors in China.It has a high sales volume and is popular with consumers.However,Xiaoqu liquor has been facing the shortcomings of weak taste and unclear brewing mechanism,which is not conducive to the upgrading and reform of products.Due to the unique technology of natural inoculation and continuous grains fermentation in Luzhou-flavor liquor,there are many functional microorganisms in pits.Most of these functional microorganisms have not been effectively utilized and developed.In this study,a series of studies have been carried out on these defects.The results are as follows:1.A Saccharomycopsis fibuligera yeast code 20C-128 was screened by pure culture and fermentation of 46 yeasts from 13 species in Luzhou-flavor liquor grains,and the flavor substances in fermentation broth were detected.The content of ethyl acetate,total esters and flavor substances were taken as indexes.After 7 days fermentation in sorghum juice medium,the yield of ethyl acetate reached 992.1894 mg/L,total ester yield reached 1395.1757 mg/L,and various esters could be metabolized.Subsequently,the strain was compared with industrial Saccharomycopsis fibuligera in performance,and found that the strain had more carbon metabolism;although the tolerance of the strain was similar to that of industrial Saccharomycopsis fibuligera,the strain had higher ethyl acetate content and more abundant esters.2.A Saccharomyces cerevisiae,code 30A-136,was screened by Duchenne tubule gas production screening and re-screening under stress conditions,and its fermentation performance was compared with that of industrial Saccharomyces cerevisiae.Firstly,the ethanol content in fermentation broth was determined.It was found that the yield of 30A-136 ethanol was relatively low,but there was no significant difference between 30A-136 ethanol and industrial Saccharomyces cerevisiae（P > 0.05）.Secondly,the fermentation rate of 30A-136 was faster,and the ability of reducing sugar to ethanol was significantly better than that of industrial Saccharomyces cerevisiae.Finally,the volatile flavor compounds in the fermentation broth of the two liquors were compared.It was found that 30A-136 had higher yields of ethyl decanoate and ethyl octanoate than industrial Saccharomyces cerevisiae,and had potential application in Xiaoqu liquor.3.The screened functional yeasts（Saccharomyces cerevisiae,Saccharomycopsis fibuligera）were mixed fermented to study the interaction and fermentation characteristics between the two strains.The results showed that Saccharomyces cerevisiae had inhibitory effect on Saccharomycopsis fibuligera,which was reflected in the reduction of the number of Saccharomycopsis fibuligera in the mixed culture of the two strains.The results showed that the inhibition of Saccharomyces cerevisiae was the inhibition of Saccharomycopsis fibuligera by pure culture broth of Saccharomyces cerevisiae,while the effect of Saccharomycopsis fibuligera on Saccharomyces cerevisiae was more complex.Saccharomycopsis fibuligera pure culture broth had little effect on the growth of Saccharomyces cerevisiae,but mixed culture with equal inoculation volume had little effect on Saccharomycopsis cerevisiae growth.CES cerevisiae showed growth promotion and growth inhibition respectively.The two strains had higher ethyl acetate content and total ester content in mixed culture than in pure culture,which indicated that mixed culture could promote more esters production than pure culture.4.On the basis of laboratory scale,two strains of functional yeast were made into pure bran koji and added with a certain amount of commercial koji（0.6%）for culture and fermentation（enhanced inoculation）.The control group was commercial koji without functional yeast（enhanced inoculation）.The Physicochemical characteristics,flavor substances and microbial community structure of Xiaoqu fermented grains during fermentation were compared,and the interaction between Physicochemical indexes and microbial community was established.It was found that the change trend of flavor substances in fermented grains was similar between intensified inoculation and non-intensified inoculation.At the end of fermentation,the contents of total alcohol and esters were higher than those of non-intensified inoculation,but the contents of volatile acids,aldehydes and ketones,heterocycles and terpenes in fermented grains without intensified inoculation were higher than those of intensified inoculation.Enhanced inoculation resulted in the rapid accumulation of ethanol and reducing sugar in the initial stage of fermentation,and the activities of glucoamylase and acid protease were significantly higher than those of non-enhanced fermentation（P < 0.05）.In order to explore the effect of intensive inoculation on bacterial community of fermented grains,the traditional isolation method combined with Illumina Miseq was used to analyze the microbial community structure of two kinds of fermented grains.PCOA and cluster analysis were used to compare the differences of community structure between two kinds of fermented grains.The results showed that after intensive inoculation,the number of culturable bacteria in Xiaoqu fermented grains decreased,and intensive inoculation and non-intensive inoculation fermented grains were fermented for 2,4,8 days.There are some differences in the bacterial community of fermented grains.The correlation between microorganisms and Physical andchemical indexes,flavor substances in the fermentation process of two kinds of Xiaoqu was studied.The correlation analysis was used to analyze the Physical and chemical indexes,flavor substances and microbial community structure in the fermentation process of two kinds of Xiaoqu.The results showed that the correlation between intensified inoculation and non-intensified inoculation was more significant in ethanol and acidity.Similarly,Saccharomyces cerevisiae,Pseudomonas,Herminiimonas had strong correlation with ethanol;Lactobacillus,Weissella had strong correlation with acidity;Rhizopus stolonifer,Lactoillus,Clostridium_sensu_stricto₁,Terrisobacter were more consistent in the correlation between glucoamylase and reducing sugar,and acid protease.On the other hand,there are some differences between intensified inoculation and non-intensified inoculation,and no correlation between the same strains was found.There are also some differences in the correlation of flavor substances.Bacillus,Pantoea,Acinetobacte are the core microorganisms in Xiaoqu fermented grains,which are related to the main flavor substances in Xiaoqu fermented grains.However,there are many kinds of microorganisms related to the main flavor substances in enhanced inoculation,and there are some differences between them.After correlation analysis,Saccharomycopsis fibuligera,Clostridium_sensu_stricto₁,Rikenellaceae_RC9_gut_group,Prevotellaceae_UCG-003,Terrisporobacter are the main core microorganisms,which are positively correlated with the main flavor substances in Xiaoqu.It is worth further studying their functions.It was also found that Pseudomonas,with the highest relative abundance,had weak correlation with volatile flavor substances in the process of intensified and non-intensified fermentation.It was speculated that Pseudomonas contributed less to Xiaoqu liquor flavor in the process of brewing.In order to further study the mechanism of Xiaoqu liquor brewing process,the interaction relationship between microorganisms in Xiaoqu fermented grains was predicted through the change of microbial community structure.The results showed that the correlation between most bacteria was weak by intensified inoculation,and that some bacteria were negatively correlated by intensified inoculation of Saccharomyces cerevisiae;Pseudomonas was negatively correlated with most microorganisms in Xiaoqu fermented grains.Microorganisms are negatively correlated.Pseudomonas may have adverse effects on Xiaoqu liquor production in combination with the correlation of flavor substances.At last,distillation of distilled wine grains with and without intensified inoculation was carried out,and the flavor of distilled wine was compared.It was found that the flavor of distilled wine was significantly increased except for ethyl acetate and a few other esters.Most of the other flavor substances had not been significantly changed,which indicated that intensified inoculation could not change the environment of Xiaoqu liquor brewing,and the technology or strain mixing process should be adjusted to increase the flavor of distilled wine...