Effect Of Bioaugmented Inoculation On Physichemical Characteristics And Microbiota Dynamics During Daqu Fermentation Using Autochthonous Bacillus Strains

Chinese liquor has been consumed in China for thousands of years as a unique fermented alcohol in China.Daqu as an important source of substances,microorganisms and enzymes in the brewing process of Chinese liquor,which directly determines the flavor and quality of the final product.In order to improve the quality of Daqu,In this study,the quality of Baiyunbian Daqu was improved by bioaugmentation of Bacillus,and the properties,microbial communities and volatile substances of Daqu after inoculation were determined to study the changes after bioaugmentation.The main content and results of the paper were as follows:1.Screening of functional strains of Bacillus based on enzymatic properties.From 12 strains of Bacillus preserved in the laboratory,a high-yielding protease activity Bacillus licheniformis 9 JCB3-1-1（164.83 U/m L）and a high temperature and acid-resistant amylase activity Bacillus amyloliquefaciens 50 were screened out CRB7-1-1N（700.3U/m L）.The physiological properties of the two strains were determined,and the growth curves of the two strains were drawn.And through different solid medium formulas,a solid inoculum suitable for culturing Bacillus was screened out,and the solid inoculum of 2 strains of Bacillus was prepared with this formula,and the viable counts were 1.5×10¹⁰CFU/g（9 JCB3-1-1）,2.0×10¹⁰CFU/g（50 CRB7-1-1N）.2.Daqu experiment of inoculation of Bacillus functional bacterial agent.After inoculation,the liquefaction power and saccharification power of Daqu were significantly increased,and the protease activity of Daqu was increased by nearly 2times.The microbial community structure and volatile substances in Daqu after inoculation were determined by amplicon sequencing technology and HS-SPME-GC-MS.The results of volatile substances showed that the overall content of acids,esters and aldehydes in Daqu increased after inoculation,and tetramethylpyrazine,a sauce-flavored substance in liquor,increased from 350.022mg/kg to 525.820 mg/kg.The results of amplicon sequencing showed that the content of Virgibacillus,Scopulibacillus,Metschnikowiaceae and other bacterial genera increased significantly after inoculation,while the content of Aspergillus decreased significantly after inoculation.3.Bacillus functional bacterial agent factory expanded inoculation experiment.The fermentation capacity and protease activity of Daqu were significantly improved after factory inoculation.The microbial species and functions of Daqu were studied by metagenomic technology.The results showed that the relative content of Saccharopolyspora in Daqu decreased significantly after inoculation,and the relative content of Rasamsonia,Aspergillus,Talaromyces,Kroppenstedtia,Bacillus,etc.increased after inoculation.Based on the KEGG database,the functional genes of the factory-enhanced Daqu metabolism were annotated,and the results showed that the functional genes related to the utilization of raw materials,such as Amino acid metabolism,Energy metabolism,and Carbohydrate metabolism,accounted for was significantly higher than that of the control group.In terms of volatile substances,the flavor substances that were significantly improved after inoculation were isobutyric acid,isovaleric acid and ethyl acetate,and the content of tetramethylpyrazine increased from 18.341 mg/kg to 138.038 mg/kg.In summary,in this paper,based on high-throughput sequencing technology,the microbial community structure and functional genes in Daqu after Bacillus biofortification were studied.The excellent changes of Daqu microbial community and volatile substances are of great significance for exploring the reasons for the differences after biofortification at the micro level,as well as stabilizing and improving the flavor and quality of Baijiu Daqu.