Investigation On Microflora Composition And Antitumor Activity Of Tibetan Kefir Grains

Tibetan kefir grain is a kind of natural starter for fermented milk in Tibet, China, which is composed of various microorganisms. The microflora compositions and antitumor activities of Tibetan kefir grains from Tibet and surrounding areas were investigated in this thesis and they were TK-ZJUJO1from Lhasa, TK-ZJUJ02from Chengtu, TK-ZJUJ03from Urumchi and TK-ZJUJ04from Sining.The culturable microflora in TK-ZJUJ01-04were isolated and identified via phenotypic and genotypic methods. There were4species of microorganisms from TK-ZJUJ0l:Bacillus subtilis, Lactococcus lactis, Kluyveromyces marxianus, Saccharomyces cerevisiae;4species from TK-ZJUJ02:Lactobacillus kefiri, Pichia kudriavzevii, Kluyveromyces marxianus, Kazachstania unispora;6species from TK-ZJUJ03:Leuconostoc lactis, Lactococcus lactis, Lactobacillus plantarum, Kazachstania unispora, Kluyveromyces marxianus, Kazachstania exigua; and4species from TK-ZJUJ04:Lactobacillus plantarum, Acetobacter fabarum, Kazachstania unispora, Pichia guilliermondi. Yeasts like Pichia kudriavzevii and Pichia guilliermondii isolated in this study were the first time reported in Tibetan kefir grains.Bacterial diversity of TK-ZJUJ01-04was investigated by culture-independent metagenomic and high-throughput sequencing methods, and comprehensive taxonomic profiling was done.11genera were found in all the4samples, and they were Lactococcus, Lactobacillus, Acetobacter, Shewanella, Leuconostoc, Pseudomonas, Streptococcus, Acinetobacter, Pelomonas, Dysgonomonas and Weissella. Shewanella, Acinetobacter, Pelomonas, Dysgonomonas, Weissella and Pseudomonas were the first time found in Tibetan kefir grains. a-Diversity analysis showed that they varied in bacterial richness and diversity while β-diversity showed that the variation of genera composition among sampling sites was small. Principal component analysis showed that Lactobacillus, Lactococcus and Acetobacter were the main genera which caused the diversity.A new grain, TK-ZJUJ06, was obtained via compositional cultivation of TK-ZJUJ 01-04. Investigation on microflora composition revealed that bacterial diversity of TK-ZJUJ06declined. There was a stable composition of prevalent bacteria in TK-ZJUJ06, including Lactobacillus, Lactococcus and Acetobacter and reduction of dubious bacteria, and only one species of yeasts was isolated, which was Saccharomyce cerevisiae. Fermentability determination demonstrated that TK-ZJUJ06had a good performance on fermentation and volatiles production, and was used for further study.The anti-proliferative effect of cell-free fraction of Tibetan kefir fermented by TK-ZJUJ06on human gastric cancer cell lines SGC7901in vitro was investigated. The3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay manifested a dose-dependent antiproliferative effect, and when the concentration of Tibetan kefir cell-free fraction was8mg mL-1, the cancer cells viability was22%, and a distinct anti-proliferation of SGC7901cells was observed. Flow cytometric analysis indicated that SGC7901cells treated with a cell-free fraction of Tibetan kefir were inhibited in the G1/S phase, and RT-PCR analysis indicated that both early (50.41%) and late apoptosis (9.89%) were induced through up-regulation of bax (from3.30±0.19to14.25±0.31) and down-regulation of bcl-2(from9.50±0.23to4.72±0.11).Response surface analysis was used to optimize culture conditions for biomass production of TK-ZJUJ06. Results showed that the optimal culture conditions were skim milk concentration,41.6%; temperature,30.05℃; inoculation amount,1.86%; time,20h, at which the growth rate of TK-ZJUJ06was14.33%,39.4%more than initial rate (10.28%). Comparison of preservation conditions confirmed that both freeze-dried method and thermally-dried method were suitable for the preservation of Tibetan kefir grains, and the viability of microorganisms was more than60%with each of the methods.