Study On The Interaction Of Dominant Microorganisms In Brewing Process Of Baiyunbian Liquor

Bacillus bacteria,such as Bacillus amyloliquefaciens and Bacillus licheniformis widely exists in each wine making process,and can produce a variety of flavor substances,have important contributions of liquor aroma.Saccharomyces cerevisiae is the main microorganisms in alcoholic fermentation,and it is widely present in the koji-making stage,the accumulation fermentation stage and the mid-fermentation stage.They all play an important role in the process of brewing Baiyunbian wine.It is of great significance to study the interaction between them and to improve the brewing process of Baiyunbian wine and to guide production practices.In this study,7 strains of Bacillus sp.(BYB1?BYB7)from Baiyunbian Distillery were identified and preliminary research was conducted.One strain of B.amyloliquefaciens,one strain of B.licheniformis were selected.And one strain of S.cerevisiae(BYS1).The interaction of these three strains was studied.This study mainly has the following contents and results:(1)Identification of Bacillus.Seven strains of Bacillus sp.were screened in the laboratory.16S rDNA,gyrA gene and gyrB gene sequences were analyzed,the identified strains BYB2,BYB4,and BYB5 were B.amyloliquefaciens,and BYB1,BYB6,and BYB7 were B.licheniformis,BYB3 is Bacillus sonorensis.(2)Study on the antibacterial activity of Bacillus.The inhibitory effect of Bacillus on the plates of Escherichia coli,Staphylococcus aureus,Micrococcus wallis,S.cerevisiae was investigated using a filter paper-mixed plate method.B.amyloliquefaciens was found to have inhibitory effects on the above four indicator bacteria.B.licheniformis and B.sonorensis can inhibit Sta.aureus and Mic.staphylococcus,do not inhibit Esc.coli and S.cerevisiae;and three strains of B.licheniformis have inhibitory effects on Sta.aureus and Mic.staphylococcus.The big difference is that the inhibition ability is BYB7>BYB1>BYB6 from strong to weak.It shows that in the aspect of antibacterial activity,Bacillus species are very different,and there are also certain differences in B.licheniformis.(3)Selection of B.amyloliquefaciens and B.licheniformis representative strains.Under liquid co-cultivation conditions,the effects of B.amyloliquefaciens and B.licheniformis on the ethanol production of S.cerevisiae were investigated,and the influence of B.licheniformis on the biomass of S.cerevisiae was further explored.It was found that B.amyloliquefaciens can strongly inhibit S.cerevisiae.The degree of inhibition between the three strains of B.amyloliquefaciens was not significantly different.The strains BYB2 and BYB5 were slightly stronger than BYB4.B.licheniformis can inhibit S.cerevisiae relatively mildly.Three strains of B.licheniformis have different effects on S.cerevisiae,ranging from strong to weak as BYB7>BYB1>BYB6.Select B.amyloliquefaciens BYB2 and B.licheniformis BYB7,which have strong inhibitory ability against S.cerevisiae,as the representative,and conduct follow-up studies.(4)Study on the interaction between B.amyloliquefaciens BYB2,B.licheniformis BYB7,and S.cerevisiae BYS1.For the first time,the paper explores the interactions between the dominant microbes of white liquor under a variety of conditions.Seven combinations were established(3 in pure cultures,3 in mixed cultures,and 1 in mixed cultures)and under 3 conditions(liquid shaker,liquid standing seal,solid standing seal)In culture,the interactions among the bacteria were explored using the concentration of viable bacteria,residual glucose,and ethanol production as indexes.It was found that with the change of culture conditions,the viability of each bacteria has greatly changed.When B.amyloliquefaciens is the dominant strain when cultured on a liquid shaker,in the combination of B.amyloliquefaciens,B.licheniformis could not be detected during the whole culture period,and the viable cell count of S.cerevisiae was of the order of 1×105 from the early stage of culture.It fell to the late 1×102 order of magnitude.B.amyloliquefaciens is still a dominant species when the liquid is kept standing and sealed,but the inhibition by B.licheniforrmis and S.cerevisiae is significantly reduced;in the combination of B.amyloliquefaciens,B.licheniformis can be detected in the early culture period.The number of viable cells was on the order of 1×107,and no B.licheniformis was detected in the middle and late stages.The order of magnitude of S.cerevisiae fluctuates from 1×105 to 1×107.When the solids were left standing and cultured,B.licheniformis became the dominant species.In the mixed culture,the viable counts of B.licheniformis were all above 1×108;the number B.amyloliquefaciens in the combination with B.licheniformis It is always 1 to 2 orders of magnitude lower than B.licheniformis.The mid-stage biomass of S.ceravisiae is between 1×107 to 1×108 orders of magnitude,and it is reduced to 106 orders of magnitude in the later period.In solid static culture,due to the steric hindrance effect,all three species of bacteria can exist in large amounts,and solid fermentation has the best biodiversity.(5)Qualitative analysis of solid fermentation product GC-MS.Qualitative analysis of 7 combinations of solid fermentation products by GC-MS revealed that each of the three cultures had its own unique peak spectrum.B.amyloliquefaciens BYB2 produced acetoin,butanediol,tetramethyl pyrazine,etc.B.licheniformis BYB7 can produce various flavors such as vinyl guaiacol,acetoin,4-ethylguaiacol,tetramethylpyrazine,2,3-dihydrobenzofuran,etc.Yeast produces high yields of phenylethyl alcohol and isoamyl alcohol.In the three-bacteria mixed fermentation,B.licheniformis contributed the most to the flavor.This study has conducted a comparatively comprehensive study of Bacillus identification,Bacillus bacteriostatic activity,and the interaction between B.amyloliquefaciens,B.licheniformis,and S.cerevisiae and their effects on the production of flavor substances.Brewing has laid the foundation.