| Adhesion to the gastrointestinal mucosa is the prerequisite for probiotics to exert their health-promoting activities and the adhered probiotics have been proven to inhibit the entero- pathogens and adjust the balance of gastrointestinal flora.Therefore,the adhesive ability is considered as one of selection criteria for probiotic strains. Studies on the adhesive properties and mechanisms are crucial both for the improvement of microbial eco-system in the gastrointestinal tract and biotherapeutic control of the entero-pathogens. Adopting the enterocyte-like cell Caco-2 as in vitro model, L. plantarum ST-Ⅲ, L.casei LC2W and L.casei BD-Ⅱwhich have been demonstrated health promoting effects in vitro and in vivo were choosen as the test strains.The most adhesive strain was choosen based on its adhesive capability, whose antagonistic effects against the adhesion of entero-pathogens on Caco-2 cells and adhesive mechanisms were also studied.Firstly,the well studied L.rhamnosus GG was selected as a positive control with strong adhesive ability to Caco-2 cells, and the adhesive and cell surface properties of three probiotic strains as well as its primary adhesive mechanisms were investigated. It was shown that ST-Ⅲwas the most adhesive strain among the four tested strains as determined by both microsco- pical examination and plate counting method. When bacterial concentration reached 5.0×108 CFU/mL (the ratio of bacterial cells to Caco-2 cells reached 300:1), the average cells of ST-Ⅲadhered on one Caco-2 cell were 28.43 CFU/cell .There was a good correlation between the adhesive abilty and hydrophobicity of the tested strains, indicating that hydrophobic interaction might be involed in the adhesion of the tested strains on Caco-2 cells. The monosaccharides inhibition assay and the yeast agglutination showed that mannose-specific adhesin was distributed over the surface of ST-Ⅲ.Choosing ST-Ⅲas the studying object, the effects of kinetic and environmental factors impacting the adhesion on Caco-2 cells were studied. The results showed that various factors could affect the adhesion of ST-Ⅲby via influencing the combination of adhesion and receptor. These findings suggested that the nonspecific-hydrophobic and mannose-specific interactions were both involved in the adhesion of ST-Ⅲon Caco-2 cells, among which the specific combination of adhesin and receptor was predominant.Secondly, adopting LGG as a control, exclusion, competition and displacement of E.coli and S.enteritis by ST-Ⅲfor adhesion on Caco-2 cells were studied and the protection of probiotics to Caco-2 cells was studied by the determination of cell memebrane permeability. It was shown that the antagonistic effect of ST-Ⅲagainst the adhesion of entero-pathogens on Caco-2 cells was concentration dependent. Among the three ways via which ST-Ⅲto reduce adhesion of the enteropathogens on Caco-2 cells, the most effective way was exclusion and then competition, with displacement the least effective.These results indicated adding ST-Ⅲin advance could effectively decreased the adhesion and invasion of enteric-pathogens. Cell memebrane permeability assay showed that when probiotics and enteric-pathogens were coincubated with Caco-2 cells, the probiotics could prevent the cells from being damaged by competitive space occupying.Lastly, the mechnisms of the adhesion and the antagonistic effects of ST-Ⅲagainst the adhesion of entero-pathogens were investigated by chemical and enzymatic treatments and extraction of component on the cell surface.The results showed that S-layer protein was involed in the adhesion of ST-Ⅲon Caco-2 cells, which was composed of three proteins with molecular weight among 21-70 kDa.The antagonistic effect of ST-Ⅲagainst the adhesion of E.coli on Caco-2 cells was ascribed to the combination of S-layer protein on ST-Ⅲsurface and the mannose residues on Caco-2 surface, preventing E.coli from combination of the mannose receptor on the mammal cells whereas the inhibition of S.enteritis to adhere to Caco-2 cells was mainly resulted from steric hindrance. |
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