| Lactobacillus is one kind of important probiotics,which can improve microenvironment of intestine and enhance immune function in human or animal’s bodies.Surface layer protein(S-layer protein)is one kind of bioactive molecule attached in the outset of bacteria cell.Main function of S-layer protein that has been reported includes:keeping and protecting cell shape,acting as adhesion factor,inhibiting pathogenic bacteria from adhering to intestinal epithelial cell(IEC),molecular sieves and so on,which has received much more attention in recent years.Only part of S-layer proteins of lactobacillus have been confirmed to have such function till now,while the majority still needs further research.The main methods and results of our research were as follows:1.Optimal conditions to extract S-layer proteins from Lactobacillus acidophilus CICC 6074 that analyzed through SDS-PAGE were 5 mol/L acid LiCl(pH 2),37 ℃and 30 minutes.SephadexG-75 chromatography was used to purify crude S-layer protein and LC-MS-MS was used to identify the pure protein.Results showed that the molecular mass and isoelectric point(pI)of S-layer protein was 46.5_kDa and 9.59 respectively.2.The second part of this study was to investigate the viability of L.acidophilus CICC 6074 with or without S-layer proteins in simulated gastric juice and intestinal juice.Results showed that the survival rate of L.acidophilus with S-layer proteins decreased by 18.5%and 26.8%respectively after treated with gastric juice and intestinal juice for 4h,while that of L.acidophilus without S-layer proteins fell by 40.1%and 26.9%respectively,which proved that complete L.acidophilus had a good gastrointestinal tolerance and S-layer protein was existent to the benefit of higher viability of L.acidophilus in gastric juice but lack of significant influence as in intestinal juice.To investigate the antienzyme action of S-layer protein,we compared the change of protein contents extracted from equivalent L.before and after treatment with simulated gastric juice and intestinal juice through SDS-PAGE.S-layer protein content decreased but still with much left after treatment with digestive enzymes(pepsin and trypsin),which showed S-layer protein had a certain ability of antienzyme and was helpful for the lactobacillus to exert beneficial action to bodies.3.To study the anti-inflammatory capacity of L.acidophilus S-layer protein in IEC,we established UC model in mice.Compared with the model group rats,the indexes of L.acidophilus with or without removing S-layer protein intervention group changed as follows:IEC apoptosis rate decreased by about 37.0%and 54.5%,the expression quantity of the cell apoptosis key enzymes Caspase-3 declined by 21.8%and 49.0%,while Caspase-8 level decreased by 23.8%and 44.4%respectively;cytokines in rats serum as TNF-a level fell by 9.3%and 20.9%,IL-6 level fell by 7.9%and 11.1%,and IL 8 level fell by 6.9%and 18.0%respectively.Results showed that efficacy of L.acidophilus with S-layer protein was more obvious than that without S-layer protein in regulating the intestinal immune(P<0.01).4.The immunoregulation action of S-layer protein was evaluated in macrophage RAW 264.7 cells.Results showed L.acidophilus S-layer protein could increase the phagocytic activity,induce the production of NO,and adjust the secretion of TNF-a,IL-6 and IL-8 to exert the immunomodulatory effects,and each index was concerned with the content of proteins.This project studied the basic character of acidophilus S-layer protein,optimized the extraction condition and did research on L.acidophilus tolerance to simulated gastric and intestinal juice mediated by S-layer protein.Then mainly study its role in relieving UC in mice and its action on immunoregulation to macrophage RAW264.7 in vitro.The target of this study was to clarify the mechanism of action on inhibiting UC and immunoregulation taken by L.acidophilus S-layer protein and provide theory evidence to its further use in food,medicine or other industries... |
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