Analysis Of Prokaryotic Microbial Community And Screening And Application Of High Amylase Producing Strain In Medium-temperature Daqu

Medium-temperature Daqu,as an important saccharifying and fermenting agent in Chinese strong-flavor Baijiu(CSFB)production,contains abundant microbes and various microbial enzymes,which play a key role in CSFB production,such as activities of saccharifying,fermentation and flavoring.Futhermore,amylase is the main contributor to the saccharification of Daqu,which is an important enzyme for the hydrolysis of grain.The activity of amylase system is one of the important factors affecting the utilization rate of raw materials and the yield of CSFB.In this study,high-throughput sequencing technology combined with culturable technology were used to analyze prokaryotic microbial communities of medium-temperature Daqu with fermentation time for 0 days,3 days,9 days,13 days and 30 days,respectively.A high-yielding amylase strain DQ47 was separated,and its growth and enzyme production conditions were optimized.By making into liquid koji and Fuqu two forms,DQ47 was used in brewing simulation of Xiaoqu Baijiu.The main results are as follows:(1)High-throughput sequencing technology was used to analyze the change of prokaryotic microorganism community structure,function and phenotype during the fermentation for theproduction of medium-temperature Daqu.The results showed that the dominant bacterial phyla in Daqu were Firmicutes,Proteobacteria,Bacteroidetes,Actinomyces and so on;the dominant bacterial genera were Welchiella,Staphylococcus,Escherichia,Lactobacillus and so on.Daqu samples could be clustered into three categories,0-day and 3-day samples(early stage),9-day and 13-day samples(medium term),and 30-day samples(end period).The growth and reproduction ability of microorganisms in the early stage and end period was higher than that in the mid-fermentation stage,and the utilization rate of the starch and protein in the raw materials was higher by the flora in the mid-fermentation stage.Gram-negative facultative anaerobic bacteria were present in the early stage of fermentation,gram-positive aerobic bacteria were present in the middle stage,and gram-positive anaerobic bacteria were present in the late stage,and the flora with mobile elements was maintained throughout the fermentation.During the process,it showed a trend of first increase and then decrease.In the whole fermentation process,the average relative abundance of Bacillus was relatively low,not belonging to the dominant genus,but its relative abundance gradually increased during the fermentation process,especially in the late fermentation,the relative abundance reached the highest0.94%.(2)A total of 137 strains of bacteria were screened from medium-temperature Daqu samples by culturable methods,belonging to 11 genera,including Bacillus,Staphylococcus,Paenibacillus,Myroides,Pseudomonas,Proteus,Kosakonia,etc.Among them,the maximum number of strains belonging to the genus Bacillus was 111.The hydrolysis circle method was used for primary screening,and the fermentation was re-screened to obtain a high-yielding amylase Bacillus subtilis DQ47,which was used as the starting strain for subsequent experiments.(3)The enzyme fermentation cycle of strain DQ47 was determined to be 8 d by microbial fermentation test.Single factor experiment and response surface experiment were used to optimize the fermentation conditions of DQ47,and the optimal factors was obtained.The optimal conditions were soluble starch 60 g/L,yeast powder 37 g/L,fermentation temperature 39?,liquid volume 34%,rotate speed 175 r/min,initial p H 6,and inoculation amount 3%.The optimized amylase activity was8158.23 U/m L,which was 14.75 times of that before optimization.The amylase was isolated and purified and its enzymatic properties were studied.The highest recovery rate of amylase was obtained at 80%salt-out degree.The purification ratio of amylase protein was 3.74 and the recovery rate was19.74%.The relative molecular weight of the amylase was 61.2 KDa after electrophoresis.The optimal reaction temperature was 65?and the optimal reaction p H was 6.The effects of metal ions and EDTA on the activity of amylase showed that 1 mmol/L of Zn²⁺,Mg²⁺,Mn²⁺could activate amylase,while Na⁺,K⁺,Ca²⁺,Fe²⁺,EDTA could inhibit amylase.(4)In the simulated brewing of Xiaoqu Baijiu,DQ47 was made into bacterial Fuqu and its optimal fermentation conditions were determined through single factor experiments and orthogonal optimization experiments.The optimal factors were as follows:fermentation time was 72 h,bran to pea was 4:1,fermentation temperature was 40?water content was 50%,inoculation amount was15%,and raw material addition amount was 5 g.Three validation tests were carried out according to the optimal conditions,and the final enzyme activity was(58479.23±120.45)U/g.The effects of Fuqu and fermentation liquid,which were made by adding amylase produced by DQ47,to the brewing of Xiaoqu Baijiu were tested.The physicochemical properties of fermented grains in the fermentation process were determined,including temperature,water content,p H,total acid,total ester,reducing sugar and starch.The results showed that the experimental group was better than the control group.The alcohol content,liquor yield,total acid and total ester of the experimental group were higher than that of the control group.The liquor yield of the experimental group by adding bacterial fuqu and fermentation liquid were 7.4%and 2.7%higher than that of the control group,respectively.In addition,the contents of total acid and total ester and flavor substances of the experimental group were also increased.In this study,the microbial community of medium-temperature daqu in Henan province was analyzed,and a high-yielding amylase strain DQ47 was separated.By studying the fermentation conditions and application of its amylase production,we hope to provide theoretical and practical basis for improving the utilization of starch materials and liquor yield in the process of liquor making.