Analysis Of Microbial Diversity In Zhejiang Rosy Rice Vinegar By PCR-DGGE

Denaturing gradient gel electrophoresis (DGGE) is one of the most commonly used molecular methods in analysis of microbe diversities. It is based on the separation of polymerase chain reaction (PCR) amplicons of the same size but different sequences. Bands from members of the same species showed the same migration distance in denaturing gel, hence supporting the identificative value of the method. DGGE are now routinely used in many microbiological laboratories worldwide as molecular tools to compare the diversity of microbial communities and to monitor population dynamics.The dynamics and diversity of the microbial community of Zhejiang rosy rice vinegar was studied by PCR-DGGE. The total DNA of bacteria in Zhejiang rosy rice vinegar was directly extracted without culture. CTAB method and kits method were compared. The results showed that the CTAB method was better then that of kits. The V3 regions of 16S ribosomal DNA and 18S ribosomal DNA were then amplified with universal primers F357-GC/R518 and F1427-GC/R1616 and the squences used for DGGE analysis. The addition of a 40 bp GC clamp to one of the PCR primers insured that the fragment would remain partial double-strand, and the region screened was in the lowest melting domain. Different DNA samples from the beginning of fermentation process to the end were analyzed.In the DGGE profiles, the brighter bands represent the dominant bacteria. The sequences of the reclaimed specific bands were obtained. Comparison of V3 regions of 16S ribosomal DNA sequence similarity revealed that the dominating microbial species were Acetobacter pasteurianus , Lactobacillus delbrueckii subsp. Bulgaricus, Gluconobacter oxydans, Leuconostoc mesenteroides subsp. Mesenteroides, Lactobacillus reuteri, Lactobacillus casei, Lactobacillus acidophilus NCFM andFinally, phylogenetic tree of these bands was made. Of which, Acetobacter pasteurianus and Lactobacillus delbrueckii subsp. Bulgaricu occurred along the whole fermentation process, while Gluconobacter oxydans was firstly identified in the Rose vinegar, just occurred in the last phase.And a dramatic shifts in the microbial community structure between fahua phase and fermentation phase was indicated. Only Lb.bulgaricus, G.oxydans and A.pasteurianus dominated in the last phase of the fermentation. The dynamic changes in fungal community during the fermentation of Zhejiang Rosy Rice Vinegar were showed in the DGGE profiles. The period from day 4 to day 38 was characterized by 4 bands with high intensity, and the following days showed a decrease in the intensity of these bands. All fungal bands was disappeared in the last phase of the fermentation. DNA sequence of these bands couldn't be detected because of special fragment.