Antiviral Activity Of Lactobacillus Amylovorus Metabolites In Regulating The Integrity And Barrier Functions Of IPEC-J2

The purpose of this experiment is to study the food starch Lactobacillus metabolites inhibit the bacteria and anti-porcine transmissible gastroenteritis virus mechanism, While exploring when TGEV infection IPEC-J2, food starch Lactobacillus metabolites influence on cell integrity and barrier function, Provide a theoretical basis for the development of antibacterial and antiviral probiotic formulations.The first part of the study was studied the mechanism action of Lactobacillus amylovorus metabolites antibacterial and antiviral and determine its composition, using organic acids, hydrogen peroxide and protease processing culture supernatant of food starch Lactobacillus, the activity of culture supernatant of Lactobacillus amylovorus against the E. coli, Salmonella and Staphylococcus aureus and the antiviral activity of TGEV infected IPEC-J2detected by the agar diffusion method (Oxford cup method) and MTT. The results showed, culture supernatant of Lactobacillus amylovorus which be treated by protease, antibacterial effects and antiviral activity were significantly reduced; culture supernatant of Lactobacillus amylovorus which be treated by organic acids and catalase, antibacterial effects and antiviral activity were not significant, showed that the antibacterial and antiviral ingredients of culture supernatant of Lactobacillus amylovorus were consistency.The second part of the study was studied when TGEV infected IPEC-J2, Lactobacillus amylovorus played antiviral whether by maintaining or improving cell morphology, proliferation, differentiation and activity. Using vitro microscopic observed cell morphology, MTT detected cell proliferation, AKP activity assay differentiation of cells, ATP enzyme (Na+-K+-ATPase, Ca2+-Mg2+-ATPase) activity assay cell activity. Test group was food starch metabolism pretreatment viral, also set up a control group of normal cells, virus control group, MRS medium treated and Lactobacillus amylovorus metabolites alone treatment groups. The results showed, changes of cell morphology were:the size of cell were uniform, tightly packed, irregular round, transparent cytoplasm, no particles in control group of normal cells; slow cell growth, morphology disappear, shrink or drawing, vacuoles, cell spacing widening, most of the cells broken off in virus control group; MRS medium treated group did not change significantly; cell space narrow, closely packed and cells into groups in ood starch Lactobacillus metabolites alone treatment groups. Morphology of cells in the test group did not change significantly were compared to the virus control group, cell broken off decreased. Cell proliferation were:virus group showed a decreasing trend over the time of cell proliferation; normal cells, group showed an increasing trend over the time of cell proliferation; MRS medium treated and Lactobacillus amylovorus metabolites alone treatment groups with less impact on cell proliferation compared to normal cells; the test group increased more dramatically than the group of viruses on cell proliferation. Cell differentiation was with time of TGEV infection increasing, the AKP in virus control cells was rising. Activity of virus control group was significantly higher than the normal cell control group (P<0.05). MRS medium treated and Lactobacillus amylovorus metabolites alone treatment groups did not significantly affect AKP in cells than normal cell group (P>0.05). Activity of the test group was significantly lower than the virus control group (P<0.05). Situation of cell activity was virus cell control activity was significantly lower than the normal control group (P<0.05). The affection of MRS medium treated and Lactobacillus amylovorus metabolites alone treatment groups was not significant(P>0.05). Activity of the test group was significantly higher than virus control group(P<0.05).The third part of the study was studied the impact of Lactobacillus amylovorus metabolites on cell barrier function. TGEV infection IPEC-J2cells as a model again, focus on exploring food starch by Lactobacillus metabolites protected the barrier function of cells whether by maintain or improved expression mRNA of CLDN-3、KRT8and MUC1, it helps to further understand the mechanisms of probiotics to prevent or treat infection TGEV. The group just as the second part, fluorescence quantitative PCR method to detect the expression changes of mRNA at different time points in CLDN-3, KRT8and MUC1protein. The results showed, mRNA expression of MUC1, CLDN-3KRT8in the normal cells control group did not change significantly with time; MRS culture group did not change significantly compared to the normal cell control group; Lactobacillus amylovorus metabolites alone treatment groups for gene expression of MUC1, CLDN-3and KRT8proteins showed significant time-dependent effect. After be treated12h later, gene expression of MUC1, CLDN-3KRT8in text group did not significant change compared with the virus control, while, after be treated24h,36h and48h later, gene expression of MUC1, CLDN-3KRT8in text group was significantly higher than the control group.