Study On The Diversity Of Bacterial Resources In Maotai Town Maotai Liquor Brewing Area

Maotai Town is the core producing area of Maotai-flavor liquor,and the longterm liquor brewing has formed a unique microbial ecological structure.The research on the bacterial resources in the brewing process not only deepens the understanding of the scientific problems such as the fermentation mechanism of Maotai-flavor liquor,but also is the premise of realizing its high-quality and high-yield brewing.In this paper,high-throughput sequencing technology and culturable analysis methods were used to study the bacterial flora structure and culturable flora distribution characteristics of brewing environment and Daqu in Maotai town.Combined with simulated fermentation,the characteristics of culturable bacteria were analyzed in detail to further reveal the resource distribution characteristics and fermentation potential of Maotai flavor liquor brewing bacteria in Maotai town.This research provides the scientific theory and technological support for the scientific research and industrial development of Maotai-flavor liquor.The main conclusions are as follows:(1)A total of 23 bacteria phyla were detected from seven environmental rounds in Maotai town by high-throughput sequencing technique,and the numbers of bacteria in 1-7 rounds were 20,18,21,19,18,20 and 20,respectively,of which Proteobacteria,Firmicutes,Bacteroidetes and Actinobacteria were the dominant phyla.At the genus level,a total of 549 bacterial genera were detected,and the 1-7 rounds were 445,397,408,435,431,451 and 400,respectively.A total of 21 bacterial phyla were detected from Daqu samples for brewing production,and the numbers of 1-7 rounds were 17,11,12,13,9,13 and 18,respectively.Of these,Firmicutes,Proteobacteria,Bacteroidetes and Actinobacteria were the dominant phyla.At the genus level,a total of 523 genera were detected,and the 1-7 rounds were 316,248,293,293,201,269 and 397 genera,respectively.After further analysis,Bacillus 、 Lentibacillus 、Oceanobacillus 、 Pantoea 、 Kroppenstedtia 、 Enterobacter 、 Staphylococcus 、unclassified＿f＿＿Enterobacteriaceae 、 unclassified＿o＿＿Bacillales 、 Acetobacter 、Sphingobacterium、Pseudomonas、Pediococcus and Lactobacillus were the common dominant bacterial genera in environmental and Daqu samples.(2)A total of 1370 strains of bacteria were isolated from Maotai liquor brewing environment and Daqu for 1-7 rounds by culturable and molecular identification techniques.Through morphological,molecular,and homologous analysis,they belong to 5 phyla,28 genera and 59 species of bacteria.Among them,44 species from environmental samples and 34 species from Daqu samples.The results of highthroughput sequencing were consistent with the results of dominant bacteria phylum and dominant bacteria.Among them,X1(Bacillus licheniformis),X2(Bacillus sp.),X3(Bacillus subtilis),X4(Bacillus amyloliquefaciens),X5(Bacillus subtilis subsp.subtilis),X7(Bacillus tequilensis),X9(Bacillus megaterium),X12(Bacillus velezensis),X17(Bacillus paralicheniformis),X19(Lysinibacillus fusiformis),X25(Staphylococcus sp.),X26(Staphylococcus saprophyticus),X32(Micrococcus sp.),X54(Alcaligenes faecalis),X57(Bacterium strain)and X58(Uncultured bacterium)were the core dominant bacteria that could be detected in environment and Daqu samples.(3)The results of high-throughput sequencing and culturable results showed that the overall characteristic structure of bacterial flora distribution in Maotai town brewing environment and Maotai flavor Daqu samples can be divided into three groups: 1-2 rounds were grouped into one group,3-5 rounds were clustered into one group,and 6-7 rounds were clustered into one group,which is significantly related to the actual liquor production quality.(4)The results of bacterial tolerance show that the optimum growth temperature is 37～40 ℃,but it can tolerate the high temperature of 65 ℃;the optimum p H value for growth is 5.0-5.5,and it can tolerate the acidic environment with p H 3.0;The optimum concentration of growing grape is 5 %,and the tolerable concentration of grape is 40 %;The more suitable concentration of ethanol for growth is 4 %vol,and the tolerable concentration of ethanol is 12 %vol.The tolerance of the strain decreased with the increase of temperature,acidity and ethanol and sugar concentration.In addition,X1(Bacillus licheniformis),X2(Bacillus sp.),X3(Bacillus subtilis),X17(Bacillus paralicheniformis)and X58(Uncultured bacterium)have characteristics of high temperature tolerance.X1(Bacillus licheniformis),X3(Bacillus subtilis),X5(Bacillus subtilis subsp.subtilis),X7(Bacillus tequilensis),X17(Bacillus paralicheniformis),X19(Lysinibacillus fusiformis),X25(Staphylococcus sp.)and X26(Staphylococcus saprophyticus)have high acid tolerance.X25(Staphylococcus sp.)and X26(Staphylococcus saprophyticus)have high ethanol tolerance.X1(Bacillus licheniformis),X2(Bacillus sp.),X5(Bacillus subtilis subsp.subtilis),X25(Staphylococcus sp.)and X58(Uncultured bacterium)have the characteristics of high glucose tolerance.In a word,ethanol inhibited the growth of bacteria more obviously than temperature,acidity and sugar concentration.(5)The results of simulated fermentation showed that 95 % of the fermentation products presented weak sensory aroma after fermentation at 37 ℃ for 2 d,and 96 % of the fermentation products presented obvious sensory aroma after fermentation at 37 ℃ for 2 d and then at 55 ℃ for 5 d,mainly showing fermentation aroma,burnt paste aroma,soy sauce aroma and grain aroma.X1(Bacillus licheniformis),X2(Bacillus sp.),X4(Bacillus amyloliquefaciens),X5(Bacillus subtilis subsp.subtilis),X7(Bacillus tequilensis),X9(Bacillus megaterium),X12(Bacillus velezensis),X32(Micrococcus sp.),X57(Bacterium strain)and X58(Uncultured bacterium)produce soy sauce aroma.While X2(Bacillus sp.),X5(Bacillus subtilis subsp.subtilis),X7(Bacillus tequilensis),X17(Bacillus paralicheniformis)and X32(Micrococcus sp.)showed floral,sweet,and fruity aromas after fermentation.(6)2339 kinds of volatile compounds were detected by GC-MS,including 149 alcohol,145 ketone,160 esters,84 acids,52 sulfides,19 furans,33 pyrazines,537 nitrogen compounds,33 alkanes,303 aromatic compounds and 824 others.The main flavor compounds co-metabolized by dominant strains included 62 alcohols,66 ketones,75 esters,35 acids,18 sulfides,3 furans,17 pyrazine,10 alkanes and 102 aromatic compounds.(7)Combined with the high abundance in high-throughput detection,highfrequency in culturable detection and fermentation metabolic component data,we found that: Bacillus licheniformis,Bacillus sp.,Bacillus subtilis,Bacillus subtilis subsp.subtilis,Bacillus megaterium,Bacillus velezensis,Bacillus paralicheniformis,Lysinibacillus fusiformis,Staphylococcus sp.,and Micrococcus sp.were the characteristic microflora structure of Daqu used in Maotai flavor liquor brewing of Maotai town,and it was suggested to be the microbial index of quality control standard in Daqu producing.Bacillus licheniformis,Bacillus sp.,Bacillus subtilis,Bacillus subtilis subsp.subtilis,Bacillus amyloliquefaciens,Bacillus tequilensis,Bacillus velezensis,Staphylococcus saprophyticus,Micrococcus sp.,Alcaligenes faecalis,Bacterium strain and Uncultured Bacterium were the characteristic flora structure of brewing environment in Maotai town.It is suggested to be the microecological monitoring and evaluation index of brewing environment.