In Vitro Activation Of Immune Cells By Lactic Acid Bacteria Surface Surface And Its Allergic Alleviation Effect

Lactic acid bacteria is a widely distributed bacterial flora in human and animals'digestive and genital tract, from which many strains have been isolated and applied in the production of probiotics and fermented food. These strains showed unique physiological effects on maintaining the normal function of the gastrointestinal tract, inhibiting the invasion of pathogenic bacteria and preventing preventing diarrhea and food allergy. More than 200 kinds of proteins are found on the surface of lactic acid bacteria, which play an important role in the interaction with the host, and some of the proteins were found to be the important material basis for host adhesion and immune regulation, but the function of the most proteins is unclear. In-depth research and scientific evaluation on the function of the lactic acid bacterial surface proteins are of great significance in improving the beneficial function of lactic acid bacteria and other related products, as well as in screening new strains and function factors. In this paper, the intestinal epithelial cells, macrophages, spleen cells and sensitized mast cells were used as models to study the function of the lactic acid bacterial surface proteins on adhesion, inducing proliferation and relieving allergy effect, with a view to systematically study the function of surface protein and provide the basis for its application.Surface proteins of Lactobacillus plantarum LpYZU09, Lactobacillus casei LcYZU02, Lactobacillus rhamnosus LrGG, Lactobacillus fermentium LfYZU15 and Pediococcus pentosaceus PpYZU32 were extracted using the method of lithium chloride (5molL-1) combined with guanidine hydrochloride. The effects of surface proteins on the adherence inhibition of the intestinal epithelial cell (IEC-6), the macrophage (MC) and spleen cells (SC) from mice were tested in vitro. The surface proteins had significant inhibition effects on the adhesion of bacteria to the tested immunocytes, and inhibition effects were strain and cell-dependant. The surface protein from Lactobacillus rhamnosus LrGG showed the strongest inhibiting effects on the adherence of 5 strains to the macrophage, with inhibition rate varying between 38.7% and 76.0%. The results suggested that surface proteins play important rolls on the adherence of lactic acid bacteria to the intestinal epithelial cells, macrophages and spleen cells.The effects of inducing proliferation on the intestinal epithelial cell (IEC-6), the macrophage (MC) and spleen cells (SC) from mice were tested in vitro by the method of MTT. The surface protein induced proliferation on the different cells was significantly strain-and dose-dependent. The lactic acid bacterial surface proteins induced proliferation indexes were varied from 0.05 to 0.35 for IEC-6, from 0.05 to 0.42 for MC and from 0.02 to 0.40 for SC,respectively. The induced proliferation index by surface protein of LrGG was significantly higher than those of the other four strains.The results suggested that surface proteins play important rolls on the inducing proliferation between lactic acid bacteria and the intestinal epithelial cells, macrophages and spleen cells.The sensitized mast cells were obtained by the method of intraperitoneal injection of BALB/c mice with OVA sensitized serum, and the allergic cell model in vitro was established to test the inhibiting effects of different surface protein on the mast cell degranulation. The result showed that the level of IgE in OVA sensitized serum of mice was up to 4.58 ?g/mL at the 14th day, the purity of mast cell was 87.60%, and the surface protein(45 ?g/mL) had no effect on the proliferation of mast cells. The removal rate of OVA (1.0 mg/mL) induced sensitization mast cell was 71.7 ±7.6%, significantly higher than control group (PBS induced group,19.3±4.9%). The lactic acid bacterial surface proteins showed inhibiting effect on the specific activation of mast cells, with inhibition rate varying between 7.5%and 35.4%, but the effects showed obvious differences between strains. The degranulation inhibition rate of surface proteins from Lactobacillus plantarum LpYZU09, Lactobacillus rhamnosus LrGG and Lactobacillus fermentium LfYZU15 varied from 22.3%to 35.4%, which were significantly higher than those from Lactobacillus casei LcYZU02 and Pediococcus pentosaceus PpYZU32 (7.5-15.8%). No obvious dose effect was observed on the extent of alleviating mast cells degranulation in the range of protein dose, and all groups with different dose of surface protein can significantly inhibit the degranulation of mast cell mediated by IgE (P<0.01). The result indicted that the immune activated mast cells were extremely sensitive to the lactic acid bacterial surface proteins.The histamine release in the process of surface protein and mast cells in vitro was determined by the method of high performance liquid chromatography(HPLC). The results showed that the histamine content in the mast cells reaction system, after the intervention by different surface proteins, varied between 71.5 ?g/L and 104.2 ?g/L, which significantly changed when compared with the control group (PBS group,107.9 ?g/L). The different strains and the doses of surface proteins inhibited histamine production, with inhibition rate varying between 3.4%and 33.7%, which correlated to the degranulation remission degree. The mitigative effects of histamine releasing with surface proteins on the immune activated mast cells were obiviously strain-dependant. The surface proteins which had a strong capability for alleviating histamine productions were SPLp?SPLr and SPLf (Inhibition rate varied between 22.2%and 33.7%). SPLp (0.45 ?g/mL) showed extremely significant effects on inhibiting the histamine releasing (P<0.01), with the inhibition rate up to 33.7%. Histamine release could be significantly inhibited by SPLr and SPLf (P<0.05), because histamine contents in these SP induced systems were significantly lower than that in the control group (10 ?g/mL BSA group, 102.8±5.2 ?g/L). In the tested dose range for the same strain, no obvious dose effect was observed in the inhibition rate. Histamine release in the mast cells can be inhibited by Pediococcus pentosaceus PpYZU32 surface protein (SPPp) and Lactobacillus casei LcYZU02 surface protein (SPLc) (Inhibition rate was between 3.4%and 8.3%), but the effects were not significant compared to the control group (PBS group and BSA group). The results showed that there were significant inhibition effects on the histamine production of mast cell among different lactic acid bacteria strains surface proteins.