| Bacterial Douchi, one of the most popular traditional fermented soybean products in China,used to be made by spontaneous fermentation. It has a long production cycle. It is easily polluted by pathogenic bacteria during post-fermentation because it hasn't explicit bacteria to ferment. The products have unstable quality and safety risks. Therefore, to reveal the bacterial flora during Douchi fermentation and screen high-yield protease producing strains has significance to solve the present problem in fermented soybean. In this thesis, there were two main aspects, which were analysising the consist of bacterial flora in Douchi post-fermentation and screening the high-yield protein producing strains. The results showed that:1. The physical and chemical indicators were detected during the post-fermentation. The results showed that the free nitrogen and total acid content ascended gradually during the post-fermentation period.2. The Douchi cultured at 37℃was studied by denaturing gradient gel electrophoresis (DGGE) at different time. Bands were excised from the DGGE Gel and re-amplified, recovered, and then ligated to pMD18-T cloning vector and transducted into E. coli DH5a. The positive clones were selected randomly for sequence analysis and quantitative analysis. The results showed that many kinds of Uncultured bacteria, Bacillus subtilis and Bacillus amyloliquefaciens were the main strains in the post-fermentation of Douchi. The Enterobacteriaceae bacterium also existed in the post-fermentation.Phylogenetic tree demonstrating that the homoeologies of Uncultured bacteria were higher than cultured bacteria, and therefore, the composition of cultured bacteria was abundant.3. The analysis of the diversity index, evenness and similarity indices of bacterial flora via terminal restriction fragment length polymorphism (T-RFLP) technique during post-fermentation showed that, the diversity index decreased, and then increased, the diversity decreased gradually in the end. There were greater differences in abundance of microbial flora during the post-fermentation of Douchi. The comparison of bacterial sequence length and Ribosomal Database Project II database showed that there were two kinds of uncultured bacteria, one uncultured proteobacterium and eight kinds of cultured bacteria in the different time of post-fermentation. The proportion of Bacillus subtilis was more than 13% and the proportion of uncultured bacterium was more than 1.19% in each sampling time of the whole post-fermentation period. 4. The composition of the bacteria during the post-fermentation of Douchi were analyzed by using microscopy and colony morphology. The results showed that, the different types of bacteria occured in different periods, and the proportion of each specie had significantly changed at different times. Meanwhile, forty five different bacteria were isolated and analyzed by restriction fragment length polymorphism analysis (ARDRA). The six types were checked out from those forty five strains, the 16S rDNA sequences of representative strains of the various types were amplified and aligned. The results showed that, the proportion of Bacillus subtilis was 89%, and Bacillus amyloliquefaciens was 4%. There were Micrococcus luteus and Staphylococcus gallinarum during the post-fermentation of Douchi.5. According to the enzyme activity and the content of free nitrogen of starter of Lobster Sauce, five high-yield protease-producing strains were screened by casein plate and re-screened by starter-making from forty five strains. The five superior strains were used to ferment Lobster Sauce respectively. According to the sensory tests, physical and chemical indexes of five products, the Lobster Sauce fermented by D2 strain had a good flavor and the product was very delicious. By physiological characterization and the sequence analysis of 16S ribosomal DNA genes, the strain D2 was preliminarily identified as Bacillus subtilis. The strain was got from Lobster Sauce and it was safe, so it had further studying and developing value.
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