Analysis Of The Microbial Ecology In The Strong Aromatic Chinese Spirits Cellar

Microorganisms in the spirits cellar are collected by PBS buffer, then DNA is extracted after the cell were broken with beads. On the base of molecular taxonomy, the 16S rDNA gene library of prokaryotic microorganisms in the cellar is constructed by polymerase chain reaction(PCR), recombinant plasmid construction and cloning. The full length of 16S rDNA genic sequence of prokaryotes is sequenced with the chain-termination, and the phylogenetic tree is constructed by comparing with the 16S rDNA sequence of the similar microorganism in the GenBank. The results indicated that these microorganisms which prevail on in the cellar during the first half part of the fermentation are not present because of bad environment when fermenting for 60 days, but some others can survive and grow regardless of cellar environmental conditions. Distribution of these particular microorganisms as follow : There are some archeaes and bacteria in the top layer of the cellar when fermenting for 60 days. And these bacteria are clustered into 6 branches in the phylogenetic tree , they are high G + C mol% gram positives Corynebacteria, low G + C mol% gram positives Paenibacillaceae, low G + C mol% gram positives Clostridiaceae, low G + C mol% gram positives Lactobacteria, gram negatives Bacteroidete and TM7 phylum. Among these bacteria, Lactobacteria is about 36%. The archeaes in the top layer belong to Melhanumicrobiaceae class. they are clustered into 2 branches. One is Methanoculleus genus, the other is Methanospirillum genus. The former is 80% or so among the archeaes of the toplayer. As for the middle layer, the only bacteria distribute in the center of the cellar when fermenting for 60 days, and these bacteria are clustered into 4 branches in the phylogenetic tree, they are high G+C mol% gram positives Actinobacteria, low G+C mol% gram positives Lactobacillales, gram positives Fusobacteria and gram negatives Proteobacteria and Proteobacteria. The rate of Protebacteria is about 80% among these bacteria in the cellar. In the bottom layer of the cellar , there is only a sort of archeae which belongs to the Methanoculleus.On the base of the microorganism analysis of the cellar, further survey the ecological factors that influence on the microbial growth, metabolism and distribution in the cellar. Such as temperature, pH, sugar, total sour and water activity etc. Experimental result shows that the ecological factors vary according to the activity of the microorganisms. When fermenting for 7 days, temperature in top layer drops from 27 to 23, and it rises from 16 to 23 in the middle layer, however temperature in the bottom does not change , it still maintains at 22. In each layer of the cellar , temperature soars to about 33 during 7 day~14 day , and it keeps at 33 till the prime fermentation finishes. After prime fermentation, the temperature will descent slowly. The pH in the cellar is about 3.5 during the fermentation. But the sour concentration rises slowly in each layer. The sour of the bottom is always higher than that of middle layer and top layer. When fermentation is over, the concentration of sour climbs to the top level, about 35 , in every layer. The starch always declines except that of bottom, and it falls fast during the prime fermentation. When the prime fermentation is over, the concentration of starch in top layer drops from 28% to about 20%, and that in the middle layer fail from 35% to about 23%, but it hold the beginning level of 17% during the whole fermentation because of no resolved polysaccharides in the bottom layer. The reduce sugar waves in the course of fermentation. In the former stage of the prime fermentation, it goes up to the top level. Then it falls fast to 2.5% and slightly rises. The top level of reduce sugar in the top layer, middle layer and bottom layer are respectively about 8.6%, 6.2% and 9.5%. The concentration of alcohol is one of important limiting factor against lives of microorganisms. It can respectively reaches about 5.0%, 5.5%,and 5.3% in the top layer, middle layer and bottom layer when the...