Microbal Community Structure Dynamics Of Pu-Er Tea Fermenting

"Pile fermentation" is the key step during Pu-er tea manufancture, in which quantity of microorganisms reaches much higher level than any one else, because of the importance of microbial vital movement and metabolics. The traditional process of Pu-er tea fermentation is easily influenced by human factors and environmental change, long production period, unstable quality. To improve manufacturing techniques, it is fundermental to perform basic researches on microbial community structure and diversity in the traditional pile fermentation. Fungal growth is considered to be the key stage in the manufacture of Pu-er tea, when complex biochemical changes take place producing its characteristic aroma and flavour. And bacteria play a subsidiary role.We chose the Pu-er tea fermentation samples as our study object using PCR-DGGE method, and preliminarily analyzed the changes of fungal and bacterial community in the process of pile fermentation. Compared the four DNA extraction methods, including enzyme lysis, kit method, ultrasonic lysis and denaturant lysis. This paper investigated the influence of Touchdown PCR and Nested PCR on the diversity of PCR products. At the same time, the optimal conditions for DGGE were found. And based on this conclusion, the fungal and bacterial community structure were researched by analyzing NS31/Glol region of the18S rRNA gene and variable region V3of16S rRNA gene. DGGE dominant bands were cut and sequenced, in order to obtain the information of microbial community structure dynamics. We looked forward to paving the way for artificial inoculation, guaran-ting product quality, and ensuring the standardized and industrial production.The main results are as follows:(1) This study had established PCR-DGGE systerm for analyzing the microbial community structure of Pu-er fermentation. The result showed that a favorable effect could be obtained using with denaturant lysis. Nested PCR was used to amplify NS31/Glol regi-on of the18S rRNA gene and variable region V3of16S rRNA gene, and the PCR products were specificities and ample. We also optimized the electrophoresis time and the concentration gradient of DGGE respectively by timetravel method and perpendicular DGGE. It revealed that, concentration gradient between45%-65%and the electrophoresis time for12h could make separation effect be best for fungi. Moreover, concentration gradient between35%-65%and the electrophoresis time for12h could make separation effect be best for bacterial.(2) We studied the change of fungi in the course of Pu-er tea fermentation, according to the optimization method of PCR-DGGE. We found that the dominant microorganisms in the raw material was Saccharomyces Cerevisiue, and maintained the dominant position throughout. Along with the fermentation, Aspergillus niger growed gradually, the relative number increased significantly at second and third mixture, then declined relatively. After first mixture, Penicillium glabrum increased as the dominant microbe.(3) We found that the change of dominating bacteria community structure by PCR-DGGE. The dominant bacterium included Lactobacillus, Bacillus, Thermoactinomyces, Erwinia, Pseudomonas sp., Enterobacter sp., Enterobacter aerogenes and Microbacterium arborescens. The dominant bacterium of raw and fermented tea in early advantage, including Enterobacter aerogenes, Pseudomonas sp., Erwinia sp., Bacillus thuringiensis, Microbacterium arborescens, Enterobacter sp., Lactobacillus brevis, Lactobacillus plantarum and Bacillus subtilis. With the heat release of microbial metabolism, the tea temperature gradually increased, heat-resistant micro-organisms breeded rapidly or abund-antly, such as Bacillus coagulans, Bacillus thermoamylovorans and Thermoactinomyces. Bacterial species were significantly reduced at post-fermentation, Thermoactinomyces sp., Lactobacillus plantarum and Bacillus subtilis were detected from the final fermentation sampes.