| Lactic acid bacteria (LAB) is an important member of the probiotics. LAB isalso an important part of the body’s innate immune system and is indispensable tomaintain immune homeostasis. The interaction between the LAB with the intestinalimmune system is an important issue to be resolved by the intestinal nutrition. As animportant source of probiotics, LAB play certain helpful action mainly throughrestoring or enhancing intestinal homeostasisthe. LAB is a class of non-pathogenicbacteria, which have adjuvant activity and have potential for use as a mucosalvaccine vector. In food, feed and pharmaceutical industry, LAB are often added tohelp human an animals antagonize pathogenic microorganisms and maintainintestinal health. The value of the use of LAB have received increasing attention.Adhesion to intestinal epithelial cells is an important prerequisite forcolonization of probiotic strains in the gastrointestinal tract, preventing theirimmediate elimination by peristalsis and providing a competitive advantage in thisecosystem and improving the body’s immune system. Therefore, adhesion isregarded as one of the main selection criteria for probiotic strains. The adhesionproperty of LAB is strain-specific and host specificity. Several adhesins have beenidentified, including lipoteichoic acid (LTA), surface layer protein (SLP) andpeptidoglycan. The binding of thses adhesins to specific receptors in the surface ofhost cells results in a complex series of physiological responses.The adhesion of LAB is an important factor involved in theirimmunomodulatory activity. LAB have been found to regulate mucosal immunesystem, protecting the host intestinal health. Strong adhesion of LAB can prolong theinteraction time with the host cell in order to better enhance the body’s immuneresponse. The adhesion and immunomodulatory effects of LAB have significantstrain-specific. In the present study, adhesion of selected Lactobacillus strains to the intestinal mucus was investigated in vitro and in vivo. Stimulation of the hostimmune system via toll-like receptors signaling pathway by the probiotic was alsoanalyzed. The whole research projects include two parts. Part one, assessment of theadhesive properties of probiot, investigation of influence on the adherence ofLactobacillus; identification of adhesion-association protein．Part two, research onthe immunoregulation function of Lactobacillus strains via toll-like receptorssignaling pathway. The main findings are as follows:The surface hydrophobicity and adhesion of nine strains of LAB weresystematically evaluated and the strong adhesive strains were screened. First, fourmethods used to detect hydrophobicity and three methods used to detect adhesionwere evaluated in this study. The results showed that the the methods of BATH andautoaggregation are suitable for the evaluation of cell surface hydrophobicity. All ofthe methods of microscopic examination, plating and fluorescence labeling can beused to determine the adhesion capacity. L. salivarius and L. plantarum werechoosen as adhesive strains by their good surface hydrophobicity and adhesionproperties.Various factors involved in attachment were investigated by in vivo and in vitroexperiments. These factors include bacterial status and cell concentration, growthphase, competition patterns, and surface-layer proteins (SLP). The results indicatedthat live lactobacilli in the mid-log growth phase exhibit maximum adhesion.Moreover, this activity is not significantly influenced at cell concentrations rangingfrom1.0×10~7to1.0×10~9CFU/mL. However, the adhesion is significantly reducedwhen lactobacilli were treated by heating or LiCl.Separation, identification of the SLP of L. salivarius by transmission electronmicroscopy, SDS-PAGE and peptide mass fingerprinting technology. The resultsshowed that SLP of L. salivarius can be clearly observed in the electron microscope.The molecular weight of the SLP is about30KDa determined by SDS-PAGE. ThisSLP consists of15kinds of proteins involved in the adhesion of L. salivarius.Colonization and distribution of the tested Lactobacillus strains in miceintestine by a flow cytometric assay. The results showed that a portion of the testedstrains are able to colonize in different regions of mice intestine. In the primary stage after oral administration, L. salivarius are concentrated in ileum and little in thecolon; L. plantarum are concentrated in jejunum and little in the ileum. In the laterstage after oral administration, L. salivarius are concentrated in jejunum and little inthe duodenum; L. plantarum are as same as the primary stage.Effects of LAB on immune organs, immune cells and immune moleculesdetermined by RT-PCR and ELISA through in vivo and in vitro experiments. Theresults showed that the two tested LAT can increase the spleen index, but only L.salivarius significantly increased the thymus index (P<0.05). The two tested LABcan significantly improve the spleen lymphocyte transformation rate of mice (L.salivarius is best), enhance the energy metabolism level and phagocytic activity ofmacrophages (L. plantarum is best), and Improve the number of CD11c+CD80+double-positive cells (L. salivarius is best). The two tested of LAB significantlyincrease the transcriptional level of TNF-α, TLR-2, TLR-4, TLR-9, IL-10, IFN-α1,IFN-β and TGF-β3and decrease the transcriptional level of IL-1α, IL-8, NF-κB,TGF-β1and TGF-β2. The results of ELISA test showed that the two test LAB haveincrease the expression of Il-10、TLR-2and sIgA (L. salivarius is best) and have noeffect on the expression of IFN-α and NF-κB. IL-12and TNF-αwere not detected. L.salivarius can also significantly increase the expression of IFN-γ.Evaluation of probiotic properties of the tested LAB by in vivo and in vitroexperiments. The results showed that the two tested strains have good resistance tothe environment of the gastrointestinal tract. Their metabolites can inhibit the growthof pathogenic bacteria. The tested LAB bacteria can inhibit the adhesion ofpathogens to intestinal epithelial cells and inhibit the pathogens translocation. Thetested LAB can alleviate the symptoms of mice infected by pathogens. In addition,the tested LAB have other probiotic properties, such as anti-inflammation (IL-8),antioxidative activity, antitumor (p53), cholesterol-and nitrite-lowering ability, andexopolysaccharide (EPS) production.Preliminary evaluation on the safety of the tested LAB by in vivo and in vitroexperiments. The results showed that the tested two LAB strains can increase thebody weight of mice and feed to gain ratio, increase GSH content and reduce MDAcontent in mouse serum. In summary, this research screened two Lactobacillus strains with strong adhesioncapacity. The adhesion mechanism, immunomodulatory effects, and many probioticfunctions were investigated. The two tested strains are complementary as far as thethe immune regulation and functional aspects, suggesting that the two tested strainshave application potential. This research would be helpful to develop immunemodulating foods and products related to lactic acid bacteria，and explain roles ofcommensal microbiota on building and maintaining of host immune homeostasis. |
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