| Lactic acid bacteria are a general term for a group of bacteria that can utilize carbohydrates such as glucose and produce a large amount of lactic acid.They are generally recognized as safe and there are increasing evidences indicating that many species of LAB possessed bioactive functions for preventing disease and improving human health.Exopolysaccharides(EPS)are mucopolysaccharides that are synthesized by the microorganisms such as lactic acid bacteria and secreted into the environment,and capsular polysaccharides that adhere to the surface of bacteria.Approximate 30 species of LAB have been reported to produce EPSs.It has been shown that EPS have various potential physiological function and biological activity,including antibacterial,cholesterol-lowering,antioxidation,antitumor and immunoregulatory activity.The chemical structures of EPS affects their physiological functions,those structures of EPSs were investigated usually including the monosaccharide composition,molecular weight(Mw),functional group and sugar linkage pattern.A large number of studies had shown that lactic acid bacteria and their secondary metabolites,EPS,played an important role in regulating body immunity,anti-tumor and optimizing the intestinal micro-ecological environment.Therefore,based on the previous research of the research group,this project studied the structure of EPS from two probiotic bacteria Lactobacillus helveticus LZ-R-5 and Lactobacillus pentosus LZR-17 derived from Tibetan kefir.Then,the in vitro immune activity,in vitro antitumor activity,and intestinal benefit of L.helveticus LZ-R-5 and L.pentosus LZ-R-17 were studied.The main results were as follows:1.Structural characterization of EPS from L.helveticus LZ-R-5 and L.pentosus LZ-R-17.The structure of R-5-EPS and R-17-EPS were further identified by various methods.Scanning electron microscope(SEM)observations showed that R-5-EPS and R-17EPS had similar apparent structure.Their surface were smooth and flaky and there were irregular voids of various sizes.Polysaccharides were connected in filaments or flakes,but there were some differences in the thickness of the fragments,the number of voids and the degree of shrinkage.R-5-EPS was a heteropolysaccharide containing linear repeating units of ?6)-?-D-Galp-(1?3)-?-D-Glcp-(1?3)-?-D-Glcp-(1?3)-?-D-Glcp(1?3)-?-D-Glcp-(1?R-17-EPS was composed of ?2)-?-D-Galp-(1?and ?4)-?-DG1cp-(1? residues.2.Characteristic of L.helveticus LZ-R-5 and L.pentosus LZ-R-17.In sterile PBS buffer,the absolute value of Zeta potential of L.helveticus LZ-R-5 live bacteria was 4.23 mV and the absolute value of Zeta potential of heat-inactivated bacteria was higher.The absolute value of Zeta potential of L.pentosus LZ-R-17 live bacteria was higher than heat-inactivated bacteria.The hydrophobic rate of L.pentosus LZ-R-17 live bacteria was higher than that of heat-inactivated bacteria,reaching 28.45%.L.helveticus LZ-R-5 was basically not hydrophobic.In addition,L.helveticus LZ-R-5 and L.pentosus LZ-R-17 had good self-polymerization.3.Immunomodulatory activity of L.helveticus LZ-R-5 and L.pentosus LZ-R-17.In vitro cell models were used to evaluate the in vitro immune activity of L.helveticus LZ-R-5 and L.pentosus LZ-R-17.The results showed that R-5-EPS,R-17-EPS,L.helveticus LZ-R-5 and L.pentosus LZ-R-17 could effectively promote the proliferation of mouse peritoneal macrophages(RAW264.7).After treatmented of with different concentrations of R-5-EPS and R-17-EPS and L.helveticus LZ-R-5 and L.pentosus LZ-R-17 could significantly improve the phagocytic ability and acid phosphatase activity of RAW264.7 cells(P<0.05).Compared with the blank control,R-5-EPS,R17-EPS(50-400 ?g/mL)and the two strains of Lactobacillus could significantly increase the NO production of RAW264,7 cells(P<0.05).Finally,the ELISA kits were used to determine the cell supernatant after sample treatment.It was found that L.helveticus LZ-R-5 and L.pentosus LZ-R-17 could significantly increase the production of cytokines(TNF-?,IL-6,IL-1? and IL-10,P<0.05).4.Antitumor activity of L.helveticus LZ-R-5 and L.pentosus LZ-R-17.In vitro tumor cell models have found that R-5-EPS,R-17-EPS,L.helveticus LZ-R5 and L.pentosus LZ-R-17 showed remarkable inhibitory effect on human gastric cancer BGC-823 cells.The results showed that L.helveticus LZ-R-5 and L.pentosus LZ-R-17 could significantly increase the activity of lactate dehydrogenase in BGC-823 cells.When BGC-823 cells were treated with L.helveticus LZ-R-5 and L.pentosus LZR-17 and their EPS,nuclei containing condensed-fragmented chromatin and cell shrinkage obviously increased.In addition,flow cytometry analysis found that R-5EPS,R-17-EPS,L.helveticus LZ-R-5 and L.pentosus LZ-R-17 could significantly increase ROS levels and enhance the percentage of apoptotic BGC-823 cells.Annexin V FITC/PI double staining method found that R-5-EPS,R-17-EPS,L.helveticus LZR-5 and L.pentosus LZ-R-17 could inhibit the proliferation of BGC-823 cells by inducing arrest at G2/M phase.Finally,using ELISA kit analysis and detection,it was found that L.helveticus LZ-R-5 and L.pentosus LZ-R-17 and their EPS could increase the activity of the apoptosis enzymes Caspase-3 and Caspase-9(P<0.05).5.Eeffect of L.helveticus LZ-R-5 and L.pentosus LZ-R-17 on intestinal microbial flora.In vitro anaerobic fermentation method was used to study the probiotic function of L.helveticus LZ-R-5 and L.pentosus LZ-R-17.The results showed that R-5-EPS,R17-EPS,L.helveticus LZ-R-5 and L.pentosus LZ-R-17 could reduce the pH of the fermentation broth.In addition,the contents of SCFAs,especially acetic,propionic acids and butyric acid,were significantly increased.At the same time,R-5-EPS,R-17EPS,L.helveticus LZ-R-5 and L.pentosus LZ-R-17 could significantly modulate the composition and abundance of gut microbiota.Thereinto,Megamonas?Bifidobacterium,Lactobacillaceae and Streptococcaceae in genus level,beneficial gut microbiota,were conspicuously enhanced in comparison to Blank.The abundance of the harmful bacteria Escherichia.coli and Lachnospiraceae were reduced.Taken together,L.helveticus LZ-R-5 and L.pentosus LZ-R-17 could act as prebiotics to regulate the intestinal flora and promote the production of SCFAs. |
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