| The spontaneously fermented yak yoghurt in the Qinghai-Tibetan Plateau has lasted for thousands of years. Yak yoghurt is rich in natural lactic acid bacteria (LAB) biodiversity and confers us precious and scarce LAB germplasm resources. Biological genetic variation and adaptation mechanism under the extreme environment is proposed to provide a good special gene pool for screening functional LAB. But nowadays the study of the LAB diversities and its extensive probiotic properties on the whole Qinghai-Tibetan Plateau has not been documented. In order to uncover, exploit, and utilize the high quality LAB resources and the potential functional LAB under the extreme environmental conditions in China, we collected yak yoghurt samples from four major ecology districts of the Qinghai-Tibetan Plateau. The sampling sites with elevations ranged from2612m to4977m, covered17counties and a total of241yak yoghurt samples were collected. Then the advanced technology of high throughput sequencing, metagenomics and metabonomics were applied in this research to uncover the genetic diversity of LAB in yak yoghurt from different ecological districts of the Plateau, and to evaluate the probiotic properties of the alpine LAB from yak yoghurt. The main results are as follows:1. LAB diversities. According to the results of high-throughput sequencing and cultured depended methods, lactic acid bacteria in the spontaneously fermented yak yoghurt are comprised by genuses of Lactobacillus, Streptococcus, Enterococcus, Leuconostoc, Weissella, Lactococcus and Pediococcus, and of which Lactobacillus and Streptococcus are the dominant genuses accounting for98.5%～99.7%of total microbes in yak yogurt. Based on the cultured method, there were17species or subspecies of lactic acid bacteria in yak yoghurt. Lactobacillus genus was mainly comprised by L. plantarum, L. pentosus, L. delbrueckii subsp. Lactis, L.delbrueckii subsp. Bulgaricus, L. Paracasei, L. helveticus, L. brevis and L. fermentum. L. plantarum, L. pentosus, and L. delbrueckii are the dominant lactic acid bacteria in Lactobacillus. In the Steptococcus, only S. thermophilus, S. macedonicus and S. salivarius was detected by using cultured method. From the microbial diversity aspect, yak yoghurt samples collected from alpine coniferous forest district of the east Tibet with a relative mild and humid climate contained the highest biodiverry of microbes, which had29genera. While in the ecological district of alpine rangeland with relative cold climate and high altitude, the yak yoghurt contained the lowest microbial diversity (15microbial genera) among the five ecological districts where the yak yoghurt samples were collected. In addition, little amount of pathogenic bacteria species were also detected in yak yoghurt in genera of Staphylococcus, Steptococcus, Enterococcus, Acinetobacter and Achromobacter.2. LAB with high cholesterol-lowering and probiotic properties. The cholesterol-lowering lactobacillus plantarum strain of S69selected from yak yogurt possessed obvious probiotic properties on Sprague Dawley (SD) rats, which significantly reduced the serum cholesterol, triglyceride, and low density lipoprotein leve of high-fat diet fed rat, and improved leptin and insulin levels in serum of the rats fed a high-fat diet (P<0.05). According to analysis SD rats’ caecum microbial metagenomic sequence, the number of Firmicutes in SD rats’caecum fed with a high-fat diet was significant higher than the rats fed with a normal diet (Relative abundance of16S rRNA gene sequences76.66%vs.96.53%); meanwhile, the number of Bacteroidetes strains in rats’caecum fed with a high-fat diet significantly reduced (20.16%vs.1.6%). The cholesterol-lowering lactobacillus plantarum could obviously reduce the number of Firmicutes microbial (86.29%vs.96.53%), and increase the the number of Bacteroidetes microbial (11.93%vs.1.6%) in rats’caecum. In addition, the cholesterol-lowering lactobacillus plantarum significantly reduced the cecal number of Lachnospiraceae, Erysipelotrichaceae, Streptococcaceae, Clostridiales_XIII_Incertae_Sedis, and increased the number of Ruminococcaceae in rats fed with a high-fat diet. The results showed that the cholesterol-lowering lactobacillus plantarum could improve the intestinal microbial flora of rats when feed with a high-fat diet. According to analysis the metabolome of rats’caecum, the results showed that the rats fed with high-fat diet had some specific metabolites in their caecum, such as fatty acid metabolites, the metabolites related with bile acid synthesis and secretion, urobilin and so on. The content of these metabolites in high fat-diet fed rats caecum were obviously higher than the rats fed with a normal diet. Comparing with the rats fed with a high-fat diet, the special metabolites in the rats’caecum feed with high-fat diet and lactobacillus plantarum were mainly compromised by the metabolites which involved in steroid biosynthesis.3. LAB with high antioxidant and probiotic properties. The free cell extracts of LAB selected from yak yogurt had a stronger composite antioxidant ability, especially expressing strong HO-scavenging activity (all the strains over40%). Most LAB strains expressed strong enzyme activity of leucine arylamidase, valine arylamidase,β-galactosidase,β-glucosidase, especially the Lactobacillus delbrueckii subsp. bulgaricus. All of the selected LAB strains processed the strong resistance ability to staphylococcus aureus, Listeriosis, and strong tolerance ability of the digestive tract environment. The F17strain expressed better comprehensive antioxidant ability and probiotic properties in in vitro experiment, and also had the ability to produce bacteriocin. In vivo trial results showed that F17strain could significantly reduce the D-galactose’ injury to mice brain, and the effect of reducing the injury was better than3mg/one mice/one day dose of vitamin C. Moreover, high antioxidant ability strain F17could significantly increase the B/E ratio in mice intestinal microbial, GSH-Px activity in mice liver and serum, T-SOD activity in mice serum and brain tissue, and reduce the MDA level in mice serum (P<0.05).4. LAB with high production of exopolysaccharide (EPS) and its probiotic propertis. One strain of Leuconostoc lactis H52was selected from Qinghai-Tibet plateau yak yogurt. The strain could produce304.86mg/L EPS under the condition of no optimization. The strain also expressed the strong enzyme activity of leucine arylamidase, valine arylamidase,β-galactosidase, β-glucosidase. H52strain expressed the strongest antibacterial activity as indicated by its strong inhibiting ability to the four pathogenic bacteria(Salmonella typhimuriu, Listeria monocytogenes, Staphylococcus aureus, Escherichia coli) used in the test. H52exhibited the fundamental requirements to be as a probiotic strain based on the in vitro test. According to the in vivo trial, both F17and H52strains could significantly increase cytokines IL-10and IL-12levels in mice serum (P<0.01). F17induced IFN-γ producing in mice serum which was better than H52.The present study is the first report on comprehensively collect and preserve the yak yogurt LAB germplasm resources from the whole Qinghai-Tibetan Plateau. At the same time, the LAB diversity yak yoghurt and the relationship between diversity and ecological environment were revealed in this research. Meanwhile, through the in vitro screening and using the rat model in combination with microbial metagenome and metabolome of cecum, a systematic evaluation of the probiotic properties of LAB isolated from yak yoghurt was conducted. The results of the present study provide important information and scientific data on exploring and utilization of the natural LAB germplasm resource in the spontaneously fermented yak yoghurt from the harsh environment of the Qinghai Tibetan Plateau. |
PDF Full Text Request