Study On The Diversity And Fermentation Characteristics Of Core Fungi In The Fermented Grains Of Maotai-flavor Liquo

Maotai-flavor liquor is a product of solid fermentation with multiple strains.It is important to analyze the important functional microorganisms and their metabolic characteristics in the fermentation process to regulate the fermentation of Maotai-flavor liquor.In this study,the fungal community structure and diversity of fermented grains in pit of Maotai-flavor liquor at 0,5,10,21,35 days were analyzed based on high-throughput amplicon sequencing technology.The contents of volatile flavor compounds in five samples of fermented grains were determined by headspace solid-phase microextraction-gas chromatography-mass spectrometry(HS-SPME-GC-MS).By using multivariate statistical analysis method,the succession of fungus community and volatile flavor substances in the fermentation process of Maotai-flavor liquor was revealed,the core fungal flora was found,and the influence of physical and chemical factors on the core fungal flora was analyzed.The core fungi were isolated and screened by culturable method,and the fermentation characteristics of single and mixed core fungi were studied.The main fundings of the study are as follows:(1)Analysis of fungal community diversity,flavor compounds and physicochemical factors of fermented grains in pit.During the whole fermentation,the contents of moisture,alcohol and acidity in fermented grains increased,while the contents of starch and reducing sugar decreased significantly.Fungi communities in fermented grains were analyzed by high-throughput amplicon sequencing.The top 15 predominate fungi genera were unclassified Dipodascaceae,Thermoascus,Candida,Aspergillus,Kodamaea,Meyerozyma,unclassified Aspergillaceae,unclassified Fungi,Zygosaccharomyces,Wickerhamomyces,Cephalotrichum,Penicillium,Saccharomyces,Issatchenkia and Pichia.A total of 86 volatile flavors were detected in five fermented grains samples at different fermentation times,including 16 kinds of alcohols,38 kinds of esters,12 kinds of acids,4 kinds of aldehydes,8 kinds of ketones,4 kinds of phenols and 4 kinds of other compounds.There were differences in the diversity and contents of volatile flavor substances in fermented grains.Twelve flavor compounds,including ethyl lactate,diethyl succinate,ethyl phenylacetate,ethyl acetate,ethyl caproate,ethyl linoleate,isovaleric acid,caproic acid,furfural,2,4-di-tert-butylphenol,ethyl palmitate and caprylic acid,were the main aroma compounds leading to differences in fermented grains with different fermentation times.The distribution and abundance of fermented grains,width degree,microbial interaction,and production capacity of synthesized flavor compounds were analyzed from multiple perspectives.The results showed that Thermoascus,Aspergillus,Zygosaccharomyces and unclassified Aspergillaceae were the core fungi genera of fermented grains.Aspergillus and Zygosaccharomyces showed positive correlation with starch and reducing sugar content.There was significant positive correlation between Thermoascus and acidity.Thermoascus and unclassified Dipodascaceae were positively correlated with ethanol content.The abundance of these 4 fungi in fermented grains was very high and they were widely distributed.They not only had strong interaction with other fungi in the community,but also had tight correlation with the volatiles of fermented grains.They were the core fungal flora of fermented grains,and played an important role in the fermentation of Maotai-flavor liquor.(2)Isolation,purification and identification of core fungi.Nine strains of mold and eight strains of yeast were identified from the fermented grains in the pit.They are Aspergillus niger,Aspergillus fumigatus,Aspergillus flavus,Aspergillus oryzae,Monascus purpureus,Monascus ruber,Penicillium paneum,Penicillium coprophilum,Paecilomyces variotii,Zygosaccharomyces pseudobailii,Zygosaccharomyces bailii,Pichia manshurica,Pichia kudriavzevii,Saccharomyces cerevisiae,Aureobasidium pullulans,Meyerozyma guilliermondii,Filobasidium magnum.Among the fungal strains,Aspergillus was the most frequently isolated.Among the yeast strains,Zygosaccharomyces was the most frequently isolated.(3)Detection of fermentation characteristics of core fungi.The fermentation characteristics and stress tolerance assays showed that Aspergillus niger m6 had a fast growth rate,low spore production capacity and high enzyme production capacity,and had a certain acid production capacity.At the same time,it showed high tolerance to acid(p H2.0),temperature(40℃)and ethanol(10%).Zygosaccharomyces pseudobailii J71 had high growth rate,sugar consumption and ethanol production.It showed high tolerance to sugar(60%),acid(p H3.0)and ethanol(16%).In the mixed fermentation system with Saccharomyces cerevisiae J73,when the addition ratio of Aspergillus niger m6 is low(SA1:0.1),the total contents of alcohols,esters,acids,aldehydes and ketones were higher than those of Saccharomyces cerevisiae J73.In the mixed fermentation system with Saccharomyces cerevisiae J73,when Zygosaccharomyces pseudobailii J71 was added in high proportion(SZ1:10),the synthesis of ethanol,phenyl ethanol,isobutanol,isoamyl alcohol,phenylethyl acetate,propyl nonolactone,ethyl phenylacetate,acetic acid,isobutyric acid,acetal,3-hydroxy-2-butyl ketone and other important aroma substances in Maotai-flavor liquor was enhanced.Their content in SZ1:10 was 1.5-20 times higher than that of the pure system of Saccharomyces cerevisiae J73.This study revealed the core functional fungal flora in the in fermented grains,and conducted the isolation of the core fungal flora and the fermentation characteristics study,which laid the foundation for the construction of the synthetic microbial community in the brewing process of Maotai-flavor liquor.