Effect Of Intestinal Mucosal Immune Barrier Function And Antibacterial Effect On Bovine Pathogenic E. Coli Of Koumiss Microbial Metabolites

Koumiss is a kind of milk beverage with Mongolian specialities, which has a variety of functions for health.It has became the best natural adjuvant therapy milk drinks of treatment for many diseases. More current research is the wild strains in the koumiss and their metabolites,and the overall study on microbial metabolites in koumiss is reported less. The report about the effects of koumiss or metabolites of the wild strains in which on the pathogenic E.coli is only in vitro.The report on the antibacterial activity in vivo,the bacteriostatic mechanism on the pathogenic E.coli,and on intestinal mucosal barrier function in mice has been little. Therefore,this research select Koumiss dope and different ratios (1/4,1/2,1/8 and 1/10) of enrichment bacteria solutions as experiment samples.Extract microbial metabolites by organic solvent ethyl acetate solvent extraction,reveal the inhibitory effect of microbial metabolites on pathogenic E. coli by Oxford Cup and the double dilution method,explore the therapeutic effect of microbial metabolites in koumiss on the pathogenic E.coli by in vivo, and analyze the ingredient of microbial metabolites in koumiss. Explore the antimicrobial effect of microbial metabolites in koumiss on 6 kinds of pathogenic bacteria by in vitro to determine its antimicrobial spectrum. In addition, determine the bacteriostatic mechanism and the effects of microbial metabolites in koumiss on cell surface characteristics of the pathogenic E.coli, describe the effects of microbial metabolites in koumiss on intestinal mucosal barrier function in mice model. The results of experiments are as follows:(1) It can be used to extract Koumiss microbial metabolites by organic solvent ethyl acetate solvent extraction,and the optimal pH is pH 2.(2) The test by Oxford Cup and the double dilution method can prove that, koumiss dope and different ratios (1/4,1/2,1/8 and 1/10) of enrichment bacteria solutions can inhibit the growth of pathogenic E.coli O8,and the effect of koumiss dope is the best.Its diameter of antimicrobial is 28.26mm,and MIC is 0.031mg/mL,and MBC is 0.063mg/mL.The 1/2 dilutions (Non-sterile) and 1/2 dilutions(sterile) are the second best,the diameter of antimicrobial of which respectively are 24.27mm,and 24.48mm.The MIC respectively are 0.055mg/ml,and 0.219g/ml.The MBC respectively are 0.058g/ml,and 0.231g/ml.(3) By in vivo antibacterial experiments, the 80% lethal dose of pathogenic E.coli O8 on mice is between 7.5×1010CFU/m-3.8×1010CFU/mL,OD630 of which are between 1.30-1.35.(4) The protection rate of microbial metabolites in koumiss on mice(infect 80%lethal dose of pathogenic E.coli O8)are up to 62.5%,which are close the rate of ciprofloxacin hydrochloride. Koumiss dope,1/2-W,1/4-W,1/2-M,1/8-M on mice of the best antimicrobial doses were 1.0 mg/mL,0.88 mg/mL,1.03 mg/mL,0.93 mg/mL,1.5 mg/ mL.(5) Koumiss dope,1/2-W and 1/2-M all contain proteins and organic acids.In which,the content of proteins are about 50% of microbial metabolites in koumiss DM(dry matter).At least 12 kinds of amino acids are in microbial metabolites in koumiss,and the contents of glutamic acid,tyrosine and cystine are relativey high.10 kinds of organic acids are in microbial metabolites in koumiss,and their contents are large difference between each other.(6) Microbial metabolites in koumiss can inhibit the 6 kinds of strandard strains of Enterohaemorrhagic Escherichia coli, Salmonella typhimurium, Staphylococcus aureus, Pseudomonas aeruginosa bacteria, Bacilluscereus, Streptococcus agalactiae, and its diameter of antimicrobial distribute between 25-31 mm.The MIC is about 0.03mg/mL,and the MBC is relatively different,which differs from 0.2 mg/mL to 0.5 mg/mL.(7) Koumiss microbial metabolites can inhibit the growth of E.coli O8 through breaking the cell wall,affecting the permeability of the cell membrane and reducing bacterial cell-surface hydrophobicity to increase hydrophilicity, and its funtions are equivalent of ciprofloxacin hydrochloride.(8) Koumiss microbial metabolites can improve immune organs index and phagocytic functions of monocyte-macrophage, and enhance the immunity phagocytosis of mononuclear macrophage and of mice model.The effects of Koumiss dope and 1/2-W are the best.(9) Koumiss microbial metabolites can significantly improve intestinal mucosal immune function in mice. Koumiss microbial metabolites can significantly increase IL-2, IL-10 and IFN-y concentrations in mice blood, significantly reduce the concentration of IL-6 and TNF-a and asignificant enhance the levels of IgA, IgG, IgM.The above results show that, Koumiss microbial metabolites can improve immune function, reduce inflammation, eliminate pathogenic microorganisms by intervention IL-2. IL-10、IFN-γ、IL-6 and TNF-α etal cell factor which synthesis and secretion in animal intestinal mucosal immune barrier of intestinal lymphoid tissue, between the epithelial cells of lymphocytes, distribution of diffusion in the lamina propria lymphocytes and systemic lymphocytes, lymphoid tissue. In addition, not sterilized milk1/2 and sterilized milk1/2 ratio of antibacterial effect of Koumiss enrichment solution of microbial metabolites is close to the Koumiss dope microbial metabolites.