| Objective Separate Lactic acid bacteria which produce EPS in homemade yak milk(yogurt, pull) from Qinghai pastoral herdsmen, to get the population phylogenetic tree of EPS Lactic acid bacteria in Qinghai region, identify high-yielding EPS of lactic acid bacteria, and to study the characteristics of high EPS production of lactic acid bacteria, providing theoretical basis for the late stage development and application of EPS Lactic acid bacteria in dairy industry. Methods MRS plate method was used to isolate the lactic acid bacteria, combined with morphological, and biochemical screening experiments, 16 s rDNA sequencing, NCBI BLAST data base comparison, BioEdit software fast sequence. The adjacency method(Neighbor- Joining) was used to establish the evolutionary tree, and related species of lactic acid bacteria were determined; The Phenol-Sulfuric acid method was used to quantitatively analysis the production of polysaccharide; AGAR diffusion method(Papamaloni et al., 2003) was used for the test of inhibiting the growth of bacteria. PH combined with a particular enzyme in vitro experiment was used to mimic survival experiment of gastrointestinal. Probiotic properties were measured by using in gavage modeling, and cytokines measured by ELISA. Results 86 pure strains were isolated from 57 samples, the biochemical tests in combination with the analysis of the 16 s rDNA identified 52 strain Lactic acid bacteria, belongs to Five species, namely, the Streptococcus thermophiles(33 strains), Enterococcus faecalis(6 strains), Brevibacillus laterosporus(4 strains), Lactobacillus casei(5 strains), Lactobacillus acidophilus(4 strains). All strains had a weak inhibition of the growth of pathogenic bacteria. In the experiment mimic the gastrointestinal survival environment, survival rates were different at different times 2χ =247.87,P<0.01. The median of survival rates(Quartiles, %): grow in simulated gastric juice at 3h is 92.70(91.75,93.54), grow in simulated gastric liquid at 3h is 88.54(87.99,89.18, grow in simulated gastric liquid at 6h is 84.36(83.21,86.28), grow in simulated gastric liquid at 12 h is 76.02(74.42,77.21), grow in simulated gastric liquid at 24 h is 65.43(64.32,66.94), survival rates decreased as the time increased. Viscera index: in the antioxidant properties study, heart, liver and kidney three viscera index had no difference, differences between spleen index were significantly different(P<0.01), pairwise comparison found that there were difference between the aging group spleen index and the rest groups, and higher than other groups. Apart from the aging group, the other four groups had no difference; in the immune properties study, the EPS treatment group and normal group after experiment had a significant difference on weight(t = 10.19, P<0.01); the weight of the EPS treatment group was greater than the normal group, both groups showed no difference in the organs(heart, liver, kidney, spleen and thymus). There were significant differences between groups on MDA factor(F= 50.97, P < 0.01), pairwise comparisons found that normal group and each treatment group were differences, the aging group was different from other groups; the EPS low dose group was difference from other groups, the EPS high dose group had no difference compared with the positive control group. The CAT factor were different among groups(F= 73.42, P< 0.01), all treatment groups were different compared with normal group; the EPS low dose group had no difference compared with the aging group, the EPS high dose group and the positive control group had no difference; Significant differences existed between the EPS low dose group and EPS high dose group, EPS low dose group and positive control group. Differences existed between the EPS group and the normal group on serum immune factors(IL- 10-12, 1 l, IFN-γ); The EPS treatment group had a higher immune factor expression than that of normal group. Conclusion Main advantages of lactic acid bacteria in Qinghai 57 yak traditional fermented milk is streptococcus thermophilic, followed by Enterococcus faecalis and Lactobacillus casei; Lactic acid bacteria in traditional fermented yak milk from Pastoral areas in Qinghai produce EPS is relatively low; The inhibition of Lactic acid bacteria to the four kinds of pathogenic bacteria is relatively weak, with the strongest inhibitory effect on E. coli; Weak ability to resist gastric acid growth. Survival rates differences in different simulated intestinal environment. Survival rates among different strains are not identical. EPS produced by G10-3-3 has no side effect; EPS produced by G10-3-3 can increase the production of SOD and CAT, and reduce the MDA production, with antioxidant properties; EPS produced by G10-3-3 enables the high expression of IL 10, IL- 12, IFN-γ high, and has a certain role in immune regulation. |
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