| Henan is an important liquor producing province and has a strong culture about liquor.Most liquors produced are Luzhou-flavor liquors in Henan.As the saying goes,"the old cellar produces good wine",the quality of the microbial flora in the pit mud has a decisive influence on the quality of the wine.Part of the production enterprises of Yujiu are facing the problem of insufficient aroma of liquor,especially the problem that the main aroma is not prominent.This topic selects two liquor factories in Henan that are basically close to each other in terms of production technology and raw materials.Gas chromatography is used to analyze the content of the main volatile aroma substances of the base wine produced by new and old pits,and Illumina-Miseq high-throughput sequencing technology is used to analyze the microbial community structure of the new and old pit mud and functional strains,including caproic acid bacteria,butyric acid bacteria and ester-producing yeasts,etc.Then the key parameters for compounding of functional strains were studied through single-factor experiment and response surface design,and the formula of pit mud curing liquid was designed,and then the pit mud curing liquid was prepared and applied to the simulated brewing of liquor.The effect of the curing liquid is evaluated from the sensory,physical and chemical indicators of the pit mud and the content of the main volatile aroma substances of the liquor.This subject aims to increase the content of main flavor components in liquor and improve the quality of liquor.The preliminary experimental results are as follows:(1)Gas chromatography was used to detect the main aroma substances of the base wine produced in the new and old pits of a winery in Zhumadian,Henan,and the microbial community structure of the pit mud was analyzed.Results manifested that the base liquor from the old cellar contains higher content of flavoring substances,such as ethyl caproate,ethyl acetate,and fatty alcohols than that of the new cellar.The content of Bacilli(P ? 0.01),Lactobacillus sp.(P ? 0.01)in the new cellar mud were significantly higher than that of the old cellar mud.Clostridia(44.44%),Caproiciproducens(31.45%),Proteiniphilum(28.12%),and Hydrogenospora(16.08%)were predominant in bacterial community in the old cellar mud,and Limnochordia(P ? 0.05),Hydrogenospora(P ? 0.05)in the old cellar mud were significantly higher than that of the new cellar mud.The analysis of bacterial community revealed that both the diversity and the abundance of bacterial community related to liquor flavour compounds were higher in the old cellar mud compared to that of the new mud;Methanomicrobia(P?0.05),unclassified?k??norank?d??Archaea(P?0.01),and Methanosarcina(P?0.05)of the new pit mud are significantly higher than those of the old pit mud,while the Methanobacteria(P?0.05),Methanobacterium(P?0.01)is significantly lower than the old pit mud;Analyzing the fungal microbial community structure,in addition to the dominant fungal phyla(content >1%)shared with the new cellar mud,including Eurotiomycetes and Tremellomycetes,also added Saccharomycetes,Sordariomycetes,Intramacronucleata,and Norank?p??Mucoromycota in the old cellar mud.At the genus level,in addition to the dominant fungal genera shared with the new cellar mud(content >1%)in the old cellar mud,Pichia and Candida sp.were added,and Candida sp.is the key to the synthesis of ethyl caproate and ethyl acetate.The main aroma substances of the base wine of another winery in Luohe were tested,and the high-throughput sequencing technology analyzed the changes in the bacterial community composition of the new cellar and old cellar in the upper,middle and lower layers of the cellar wall,and it was found that the base wine produced in the old cellar had higher contents of ethyl caproate,ethyl lactate and ethyl acetate,but less ethyl butyrate;Lactobacillus showed an increasing trend from the bottom to the top of the cellar wall in both new and old cellars,and Caproiciproducens showed an increasing trend from the top to the bottom of the cellar wall in both new and old cellars.The microbial community composition of the cellar wall was calculated by summing the microorganisms in the upper,middle and lower layers of the cellar wall,and it can be inferred that the abundance of Caproiciproducens was higher in the old cellar mud and the abundance of Lactobacillus was lower,which is consistent with the analysis of Zhumadian Winery.(2)A hexanoic acid-producing strain called JS-4 was isolated by using ethanol sodium acetate medium and facultative anaerobic method,identified by molecular biology as Aneurinibacillus,and a butanoic acid-producing strain called DS-5 was isolated by using ethanol sodium acetate medium and facultative anaerobic method,identified by molecular biology as Paenibacillus.The saponification reflux method was used to screen out a higher ester-producing yeast with a total ester content of 3.02 g/L,which was identified by molecular biology as Debaryomyces.The acid production performance test of JS-4 strain showed that the best JS-4 strain inoculation amount was 10%,the initial p H was 7,the ethanol content was 2%,the fermentation time was 7 days,and the fermentation temperature was 37?.The acid production performance of the butyric acid bacteria was tested,and it was found that the optimal inoculation amount for butyric acid production was 10%,the initial p H value was 7,the fermentation time was 5 days,and the fermentation temperature was 37?.Butyric acid bacteria have a certain ability to produce caproic acid.With the increase of fermentation time,the content of caproic acid gradually increases,and the content of butyric acid first increases and then decreases.(3)The JS-4 and DS-5 strains separated were connected to the caproic acid bacteria liquid medium in different proportions and the inoculum amount was 10%.The results showed that when the ratio between caproic acid bacteria and butyric acid bacteria was 1:2,the hexanoic acid content reached the highest value of 4.37 g/L.Then,the ternary bacteria of caproic acid bacteria,butyric acid bacteria and yeast were combined,the fermentation conditions of caproic acid production were optimized through single factor and response surface method.The analysis showed that the optimal fermentation temperature was 33.36?,and the yeast inoculation amount was 12.99%,the amount of glucose added is 2.61%.The test is carried out under this test condition,the content of caproic acid after optimization is3.42 g/L.(4)The pit mud functional bacteria liquid is applied to the simulated liquor brewing.After fermentation,the sensory and physical and chemical properties of the pit mud are improved.The main aroma substances of distilled liquor were determined by gas chromatography.The results found that the contents of ethyl acetate,ethyl butyrate,ethyl caproate,and ethyl lactate in the liquor produced by the experimental group were higher than those of the control group,increasing by 7.14%,21.03%,11.98%,and 19.18% respectively.And the three major acids and four major esters in the experimental group increased by13.75% compared with the control group,indicating that the use of curing liquid can improve the esterification ability and has a good effect on improving the quality of wine.In short,the good liquor produced by Laojiao is due to the high abundance of the flora related to the aroma enhancement of liquor.The key factor that determines the quality of liquor is the high-quality microbial community structure.The caproic acid-producing JS-4strain,the butyric acid-producing DS-5 strain,and the ester-producing yeast JN3 strain wered isolated from the pit mud,and the curing liquid of pit mud was prepared by ternary bacteria compounding and applied to the simulated brewing of Baijiu.The curing liquid has the effect of curing the pit mud and increasing the content of the main flavor substances of the liquor,which provides a theoretical basis for solving the problem of insufficient aroma and atypical aroma of liquor in liquor enterprises and improving and stabilizing the quality of the liquor. |
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