Screening Of Lactobacillus Paracasei In Koumiss And Study Of Its Effect On Intestinal Mucosal Barrier And Blood Immunity In Mice

The aim of this research was to study the serum-immunity of Lactobacillus paracasei subsp.tolerans,a protein-producing substance is selected from sour milk,which against vaccination in mice with pathogenic E.coli Os-induced diarrhea.The peripheral blood immunity and intestinal mucosal barrier mechanism provide a theoretical basis for further research and development of probiotic micro-ecological preparations,medical care and functional foods.The study is divided into the following three trials:Experiment 1:The agar diffusion oxford cup method,organic acid exclusion test,hydrogen peroxide action and protease sensitivity test were used to screen and rescreen 54 isolated lactic acid bacteria,and then the strains were identified by microbial morphology,physiological and biochemical tests and 16S rDNA sequence analysis,and the optimal ammonium sulfate precipitation concentration of the strains was determined by salting out method.The results showed that only one of the 54 strains of lactic acid bacteria isolated had a strong antibacterial activity metabolite.The cell-free fermentation supernatant of the strain was sensitive to proteinase K and pepsin,and the antibacterial activity was completely lost,thereby determining that the bacteriostatic substance is a protein.16S rDNA sequence analysis indicated that it belonged to Lactobacillus paracasei subsp.tolerans.The optimum ammonium sulfate precipitation concentration of the strain was 70%.Experiment 2:A mouse diarrhea model was prepared by injecting a pathogenic E.coli O8 suspension and intervention was carried out using Lactobacillus paracasei.70 SPF Kunming mice weighing(20 ± 2)g were randomly divided into 7 groups,10 units in each group.5 groups were intragastrically administered with 0.3 mL of viable lactic acid bacteria suspension of 1×106,1×108,1×1010 cfu/mL,0.1 g/mL of Nisin and 0.15 g/kg of ciprofloxacin hydrochloride.The negative control group received 0.3 mL normal saline without treatment meanwhile the normal control group was intragastrically administered with 0.3 mL of normal saline.The test period is 7 days.The cytokines,humoral immunity,antioxidant index and endocrine hormones in serum were detected by ELISA test.The immunomodulatory effects of Lactobacillus paracasei were evaluated by flow cytometry to detect cellular immune function in peripheral blood of mice.The results showed that comparing with the negative control groups:(1)Different doses of lactic acid bacteria are able to promote the growth performance of mice(P<0.05).(2)The high dose group of lactic acid bacteria which significantly increase the spleen index of mice(P<0.05).The thymus index of the mice showed an upward trend,but the difference was not significant(P>0.05).(3)The lactic acid bacteria group significantly increased the anti-inflammatory cytokines IL-4,IL-10,TGF-? and decreased the pro-inflammatory cytokines IL-1?,IL-6,TNF-?,IL-17,IL-23,IFN-?.The high dose group is optimal.(4)The lactic acid bacteria group increased the content of IgA,IgG and IgM.Meanwhile,the high dose group increased by 53.42%,33.64%and 36.37%,respectively.(5)The lactic acid bacteria group increased the ratio of CD 19+,CD3+,CD4+and CD4+/CD8+but decreased the ratio of CD8+.(6)The lactic acid bacteria group significantly increased the activity of CAT,T-AOC,SOD and GSH-Px but significantly decreased the MDA content(P<0.05).The high dose group had the best effect.(7)The high-dose lactic acid bacteria group significantly decreased GC(P<0.05)and ACTH content(P<0.01)while significantly increased T3,T4 and GH content(P<0.05).It can be seen that lactic acid bacteria is able to promote the growth performance and immune organ development of mice by regulating the immune and antioxidant indexes to improve the immunity of mice.In addition,this bacteria has ability to regulate endocrine hormones,thereby maintaining the relative stability of the internal environment and keeping the body in a healthy state.Experiment 3:Real-time fluorescent quantitative PCR was used to detect TJ-related proteins and cytokines in ileal mucosa.After HE staining,small intestinal tissues was observed.The slgA content and MPO activity in NO,DAO,D-lactic acid,endotoxin and ileum tissues of mice were detected by ELISA.Illumina HiSeq high-throughput sequencing technology was used to detect the flora structure in mouse cecal contents.The results showed that:(1)Compared with the negative control group:the length of small intestine villi in the lactic acid bacteria group was significantly increased(P<0.05).The V/C value was increased and the small intestine villus shortening and morphological damage in the lactic acid bacteria group were restored.The effect in the high dose group was obvious.DAO,D-lactic acid and endotoxin in the serum of the lactic acid bacteria group were significantly decreased(P<0.05).The expression levels of OCCLUDIN,CLAUDIN-1,JAM-A,Z0-1,Z0-2 and Z0-3 in the lactic acid bacteria group were significantly increased(P<0.05).(2)Compared with the negative control group:the NO content,MPO gene expression and MPO content in the lactic acid bacteria group were significantly lower(P<0.05).The slgA of the lactic acid bacteria group was significantly increased(P<0.05)while the expression levels of COX-2,IL-6 and TNF-? were decreased.(3)At the level of the phylum,the dominant intestinal flora of the mouse was the Bacteroidetes and Firmicutes.Antibiotics reduced the abundance of Firmicutes in the intestine of mice,accounting the proportion was 7.82%.After the intervention of lactic acid bacteria,the Firmicutes recovered to the normal level.The proportion of Firmicutes in the low,medium and high dose groups of lactic acid bacteria was 45.56%,66.38%,80.92%,responsibly.Therefore,it can be seen that with the increase of the dose of lactic acid bacteria,the Bacteroidetes is reduced while the Firmicutes were increased.The abundance of Proteobacteria in the normal group was 7.8%and that in the negative control group was 24.04%.The other groups were lower than the normal groups.The differential species in the high dose group of lactic acid bacteria were Lactobacillus,Lactobacillus and Bacillus.The difference species in the middle dose group of lactic acid bacteria was Trichophyton,There were 42 OTUs in the high-dose lactic acid bacteria group and 41 OTUs in the normal group.The antibiotic group had the least specific OTUs which can be seen that the intervention of lactic acid bacteria can increase the diversity of intestinal flora in diarrhea mice and the treatment with antibiotics leads to a decrease in intestinal flora diversity.Lactic acid bacteria increase the normal flora and beneficial bacteria in the intestine,thereby maintaining the intestinal micro-ecological balance.