Identification Of Bacillus Isolated From Chinese Luzhou-flavor Fermentation Grain And Phylogenetic Analysis Of Complete Sequence Of 16S RDNA

The production of Chinese wine is based on the liquor pit, both the quality and quantity of the wine are determined by all kinds of microorganism especially those inhabiting in the fermenting grain in the pit. Bacterium is one of the three major microorganism colonies during the fermentation course, and the perfume are derived from the esterifying period when Bacillus get a rapid increase becoming the main dominant strain.The fermenting grain for analysis in this study was sampled from an old pit of a famous distillery in Sichuan with well-balanced fermentation. The way of sampling like this: Sample the 0-day fermented grain, 7-day fermented grain, 28-day fermented grain, 49-day fermented grain, 70-day fermented grain, totally 5 samples were gained. First, the traditional identification method was introduced to identify the strains isolated from the fermenting grain, then divided them into several groups based on the identification result; Finally, make sure that the result achieved by the traditional identification method is credible as well as the phylogeny analysis was finished according to the analysis of the 16S rDNA.Phylogeny analysis is showed thereafter: 1. Extraction of Genome DNA of the representative strains; 2. Amplifying complete 16S rDNA by PCR; 3.Sequencing the complete 16S rDNA after some certain procedure such as molecular clone; 4. Homology analysis by Blast and the construction of phylogenetic tree. 69 strains of Bacillus were isolated in accordance with morphological characteristics, then divided them into 5 groups based on the physiological characteristics. And the next procedure of analysis of 16S rDNA confirmed that Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus circulans, Bacillus cereus existed in fermenting grain. Bacillus licheniformis (30.43%) and Bacillus amyloliquefaciens (27.53%) were the largest groups as far as the quantity is concerned, but there were no obvious dominant group; The relationship between Bacillus circulans and Bacillus cereus is close, but other Bacillus is relative far. Totally, bacteria of Bacillus in fermenting grain indicated higher diversity.