Study On The Bacterial Diversity And Function Of Maotaixiang Daqu Using The Combination Of Third-generation Sequencing Technology And Culture Method

In situ cultivation has been widely used in the isolation and screening of environmental microorganisms,but so far,it has not been reported that was used in the food field,especially in the screening of brewing microorganisms.This study is the first time to use the in situ cultivation technology(Ichip),which is a new culture method,to isolate and screen the microbe of the Moutai-flavor wine.Bacteria play an essential role in Moutai-flavor Daqu.The study of bacterial diversity can provide a theoretical reference for related systematic research and functional analysis.Therefore,in the thesis,the bacterial community structure in the simples was analyzed by using the third generation high-throughput sequencing technique(culture-free method)with five different storage periods of Moutai-flavor Daqu;at the same time,the bacteria in the simples were isolated and screened by in situ culture and traditional culture,and related functional analysis of the cultured bacteria was carried out;finally,the main differences in the bacterial flora diversity in the Daqu samples obtained by the cultivable method and the culture-free method were analyzed.The main findings are as follows:(1)Using Pac Bio SMRT’s third-generation high-throughput sequencing technology,a total of 192 species of bacteria belonging to 18 phyla and 167 genera were detected in5 Moutai-flavor high temperature Daqu samples.At the species level,they are Kroppenstedtia sanguinis,Enterobacter cloacae,Lentibacillus massiliensis,Kroppenstedtia eburnea,Lactococcus garvieae,Sebaldella termitidis,Staphylococcus gallinarum and Thermoactinomyces Daqus.Furthermore,the bacterial flora(relative content>1.00%)in 5 Daqu samples with different storage periods was analyzed.It was found that the bacterial groups of the 5 Daqu samples showed certain differences,but the main strains were similar.(2)A total of 679 strains of bacteria were isolated by traditional cultivation method and in situ cultivation method.After the initial screening,392 strains were finally selected for molecular biological identification.After identification,they can be attributed to 2phyla,17 genera and 46 species of bacteria.6 genera and 18 species were obtained by the traditional cultivation,and 16 genera and 36 species were obtained by the in situ cultivation.There are five common bacterial genera and eight common bacterial species between them.By comparing the bacterial structure diversity obtained by the two culture methods,it is found that 10 more genera and 18 more species were cultured by in situ cultivation method than which by the traditional cultivation method.It can be seen that the bacterial structure diversity obtained by the in situ cultivation method is more abundant than that of the traditional cultivation method.By comparing the structural changes of the bacterial flora structure of 5 Moutai-flavor high-temperature Daqu samples with different storage periods during 3-8 months of storage,it is found that Bacillus and Enterobacter are the core bacterial genera that can be cultured in Daqu samples.Bacillus amyloliquefaciens,Bacillus velezensis,Bacillus megaterium,Bacillus subtilis,and Bacillus licheniformis are the core bacterial species that can be cultured in Daqu samples.The cultivable bacterial flora of the five Daqu samples with different storage periods differs in composition,but maintains a certain similarity and stability in the main dominant groups.Among them,the Bacillus genus accounts for the vast majority of the total number of bacteria to promote fermentation and aroma production during the winemaking process.(3)By comparing the diversity of bacteria in Daqu with the culture-free method and the cultivable method,it was found that the high-temperature actinomycetes detected by the culture-free method were not detected by both cultivable method,although the relative abundance in the samples.From the five Daqu samples with different storage periods,few strains can be detected by the culture-free method and the cultivable method at the same time.On the one hand,most of the strains detected by the culture-free method cannot be separated by the culture method.On the other hand,some strains can be isolated by the cultivable method,but they are not detected by the culture-free method.(4)Three research methods were used to analyze the bacterial diversity of 5 Moutaiflavor Daqu samples with different storage periods.It was found that the bacterial groups of the 5 Daqu samples all had high diversity,and the core flora showed a certain degree of stability.There are differences in species composition and abundance among samples.