Biodiversity And Identification Of Dominant Microorganisms In Tibetan Kefir Fermentation Milk

“Tibetan mushroom” is one strain of kefir grains, Tibetan kefir is widelyspreeded in the families, but due to the existence of security risks in the Tibetankefir made by the origin starter—“Tibetan mushroom”, the industrial productionscale of Tibetan kefir is limited. In this study, on the basis of previous research,we detected the changes of microorganisms and free amino acids during thefermentation process, we studied on the microbial structure and the chang law ofmicrobial diversity in Tibetan kefir, in addition, we isolated and identified thedominant microorganisms and analysised the REP-PCR classification of thedominant microorganisms.(1) The results showed during the fermentation process, the pH value wasranged from6.65(0h) to4.51(24h), the titratable acidity value was ranged from17°T(0h) to100°T(24h).As while, during the fermentation process of1-24h, thedominance ordering of the microorganisms was Lactobacillus＞Lactococcus＞yeast. The number of Lactobacillus, Lactococcus and yeast were8.10Log cfu/mL、6.07Log cfu/mL、6.35Log cfu/mL. In addition, the free amino acidanalysis results showed the most dominant free amino acids in the end Tibetankefir were Glu, Lys and Pro. Compared to the starting point, the content of someamino acids in the end Tibetan kefir were rised, some were dropped. Inconclusion, the content of17kinds of free amino acids was ranged from104.53±0.34mg/L to113.12±1.40mg/L during the fermentation process.(2) PCR-DGGE results showed that there were Lactobacillus helveticus,Lactobacillus kefiranofaciens, Lactobacillus kefiri, Streptococcus thermophilus,Lactobacillus paracasei, Microbacterium testaceum, Saccharomyces cerevisiae,Kazachstania unispora, Meyerozyma guilliermondii∕Pichia quilliermondii in theTibetan kefir. Lactobacillus kefiranofaciens、Lactobacillus kefiri were the mostdominant and second dominant bacteria, Lactobacillus kefiri was stable duringthe subculture process, but Lactobacillus kefiranofaciens was not stable.Kazachstania unispora and Saccharomyces cerevisiae were the most dominantand second dominant yeast, Kazachstania unispora was stable during thesubculture process, but Saccharomyces cerevisiae was not stable.(3) The results of traditional microorganism separation technique、16SrDNAsequencing technique and REP-PCR analysis showed that we isolated29strainsof Lactobacillus,25strains of Lactococcus,3strains of Microbacterium and22strains of yeast.REP-PCR results showed there were11different fingerprintings of bacillus,10different fingerprintings of micrococci and2differentfingerprintings of yeast.In conclusion, the fermentation characteristics of the“Tibetan mushroom”we used in this study were good, and the microbial community composition ofthe Tibetan kefir was complex, the structure of the bacteria and yeast in theorigion fermented milk was stable, but unstable during the subculture process.We obtained57strains of bacteria and22strains of yeast in combination ofnon-culture technique—PCR-DGGE、the traditional microorganism separationtechnique and16SrDNA sequencing technique, the REP-PCR results showedthere were21different fingerprintings of bacteria and2different fingerprintingsof yeast. The purpose of this study is provide the basic materials for the in-depthstudy of “Tibetan mushroom” and the development of Tibetan kefir complexstater with stable and secure property.