Immune Effect And Molecular Mechanism Of Peptidoglycan Derived From Marine Source Lactobacillus Acidophilus

In this article, we explore the fast separation method of lactobacillus acidophilus (L,acidophilus) derived from marine plankton intestine through the combination of molecularidentification technology and traditional identification technology, peptidoglycan of L, acidophiluswas extracted and modified with phosphate, with the purpose of improving the solubility andimmune regulation activities, Finally the structure and effect of peptidoglycan on cell immunefactors in macrophage were analyzed. The main results were as follows:1. Separation and identification of L, acidophilus derived from marine source28suspected strains of lactic acid bacteria were isolated from intestinal tracts of fish inmodified MRS. By physiology biochemistry and molecular biology identification, two excellent L,acidophilus named D1and B3were identified from28suspected strains. According to DPPHscavenging experiments, the higher clearance rate strain D1, which was selected from the twopurposes strains, would be used to further research. The results indicated that D1is an excellent L,acidophilus, which had a high antioxidant capacity and resistance of neomycin, ceftriaxone,amikacin, gentamicin.2. Extraction and enzymatic preparation of L, acidophilus peptidoglycanD1was used as type strain, peptidoglycan was extracted by TCA method; then to increase thesolubility. Mixed solution of mutanolysin and lysozyme was selected by response surface methodaccording to the changes of surface structure and antioxidant ability. The results obtained that theoptimal enzyme hydrolysis condition of L, acidophilus peptidoglycan [Mutanolysin/Lysozyme(μg:mg)=20/1, pH=6.0, Temperature=41℃, Time=16h], and found that the modifiedpeptidoglycan’s superoxide anion resisitance and SOD activity was better than normalpeptidoglycan.3. Phosphorylated modification and structure analysis of L, acidophilus derived from marinesourceThe peptidoglycan was phosphorylated with mixed phosphate solution (STPP:STMP=5:2,pH=6.0), and then the structure of phosphorylated peptidoglycan was detected by HPLC, H1-NMR,FT-IR, SEM and MS. The results indicated that phosphorylated peptidoglycan’s hydrophilicity andadhesion were strengthened and hydroxyl replaced by phosphoric acid root.4. Research on immune effect of L, acidophilus derived from marine sourceRaw264.7macrophages was selected in the experiment, LPS (0.1μg/ml), peptidoglycan (50μg/ml), different concentrations of phosphorylated peptidoglycan (25g/ml,50μg/ml,100μg/ml)were added to cultured cells, after18h, Elisa kit was used to detecting cytokines of IL-1α，TNF-αand GM-CSF, lysosome fluorescent probe method was used to detecting secretion of lysosome inmacrophages. According to the result of SPSS analysis, phosphorylated peptidoglycan couldsignificantly increase the secretion of IL-1α, TNF-α, GM-CSF, lysosome, and its secretive situationhas a certain correlation with the concentration of phosphorylation peptidoglycan.