The Dominant,Stable Bacterial Species And Its Distinct Morphotypes In Response To Its Distribution In Tibetan Kefir Grains

Tibetan kefir grains(TKGs), the so-called Tibetan mushroom, a kind of natural starter for fermented milk in Tibet, China, host various microorganisms of lactic acid bacteria, yeasts, and occasionally acetic acid bacteria in a polysaccharide/protein matrix.In present study, the methods of PCR-DGGE、clone-library、metagenomic data analysis、real-time quantity PCR、scanning electron microscopy(SEM),SYBRGreen I staining 、fluorescence in situ hybridization(FISH) methods were used to study the isolation of the genome DNA of microorganisms in Tibetan kefir grians、diversity of bacteria and yeast、the dynamic change of microorganism and the morphology and distrubition of Lb.kefiranofaciens and Lb.kefiri,which may pave the way for further study of the specific symbiotic associations between yeast and bacteria in TKGs.In order toisolate genome DNA from microbial in Tibetan kefir grains,two kits(TIANGEN kit and QIANGEN kit) were used. Howeverthe experiment failed. After making modificationby adding lysozyme and liticase to do genome DNA isolation,both kits could success. Also the gel picture showed that the genome DNA got by TIANGEN kit were betterthan that got by QIANGEN kitand the gene copy number of both bacteria and yeastquantified by real-time q PCR was higer than that got by QIANGEN kittoo.So the TIANGEN kit was appropriate for genome DNA isolation from Tibetan kefir grains. In another side, TIANGEN kit was cheaper than QIANGEN kit thatreducing the experiment cost.The q PCR mothod was used to study the number of microorganism in Tibetan kefir grains. The TKGs samples were taken to do experiments at 1th week, 4thweek,8th week and 12 thweek dependentanly. The number of bacteria and yeast in TKGs was almost the same between the 4 samples.The diversityof bacteria in TKGs was investigated based on DGGE, cloning library and Metagenomic data analysis of the 16 S r RNA gene. The results of these experiment showed that the diversity of bacteria was relatively low with one dominant phylμm-Firmicutes(80%). Of it, the dominant species was the Lb.kefiranofaciens. The diversity of the four samples(N6、N8、S6、S8)had almost no difference between each other. The Lb.kefiranofaciens was the dominnat species no matter it was cultured naturally or sterility, and was stable in TKGs after cultured for 10 months later.The Lb.kefiranofaciens and Lb. kefiri were isolated and identified sucssesfully by pure culturing and PCR methods. The Lb.kefiranofaciens was long-rod bacteria, 1.0~10μm in diameter; the Lb.kefiriwas short-rod bacteria 0.5~2μm in diameter based on SEM results. The SYBRGreen I staining verified it, meanwhile, it was found that the Lb.kefiranofaciens incubated for 7 days was longer than that incubated for 5 days,butas to the Lb.kefiri, it had no change. It was found that the Lb.kefiranofaciens had two different morphologys in TKGs based on FISH mehord. Lb.kefiranofaciens was rod outside compared with curing inside.In order to distroy the structure of Tibetan kefir grains, the VB2 and AMB reagents were added duing the TKGs culturing. During the VB2 experiment, the symbiotic relationship of bacteria and yeast still exist, also the number of them was no less than TKGs cultured naturally under SYBRGreen I staining, FISH and q PCR experiments. As to the AMB experiment, the yeast decreased significantly, the net structure of Tibetan kefir grains was looser than the natural one. But not all the yeast was killed, the symbionts were not dispersed completely. As to the diversity of yeasts in TKGs, the DGGE method was used to study the 9 samples, the results was the same as that done before by cloning library, q PCR methods. Three kinds of yeast species existed in TKGs samples,and they are dominant.While the other yeastspecies were not found in TKGs samples.On the whole, obvious differences were not observed between the naturally cultured and asepticlly cultured TKGswith respect to the diversity of bacteria.Lb.kefiranofaciens was the dominant species and stablely exeisted in TKGs no matter culturing methods or cuturing time changed. This impliedthat the TKGs was also stableat a different angle. It’s interesting that the Lb.kefiranofaciens had different morphologies in TKGs, long-rod and short-rod outside and curing inside. However,under pure culture conditions, the Lb.kefiranofaciensjust was rod bacteria.This phenomenon may be caused by the growing conditions. The nutrition was enough and distributedequally in pure culturing condition and only Lb.kefiranofaciens was in it,nevertheless, the environment was different to the organism outside and inside in TKGs,the organism outside was easy to touch the milk, organism inside could access nutrientshardly. TKGs represented one of the unusual microbial symbiotic systems in nature, which possibly resulted from a long-term symbiotic interaction among the microbial community colonizing the grains. The diversity and distribution of yeast species mainly depend on the culture milk and conditions. All these findings pave the way for further study of the specific symbiotic associations in TKGs and furthermore give insights into how the grain structure formed.