Distribution And Application Of Rib Protein In Lactic Acid Bacteria And Its Antioxidant Activity

The adhesion to animal gastrointestinal tract is the prerequisite and foundation for lactobacillus to colonize the host and produce various good effects. However, few study of probiotics adhesion mechanisms were reported at present. In this study, Surface protein Rib of Lactobacillus reuteris ATCC 55730 was researched by lactic acid bacterial isolation, identifiaction and application in pickle, genetic recombination, protein purification, polyclonal antibody preparation and protein interaction to clarify the interaction between the probiotics and lactic acid bacteria and host,provide theoretic bases for the production of healthy pickle and improvement of their flavor; provide theoretical basis for probiotics reforming and improving. For these purposes, we performed the following research works.First,we put the Lactobacillus reuteris 55730 as a reference to contrast and blast the Rib protein of Lactobacillus reuteri from NCBI protein database, which showed that the protein contains 1786 amino acid residues, and was about 190 kDa, Then partial rib gene was cloned from this Lactobacillus reuteri and expressed in E.coli. The best expression conditions is initial bacteria OD4900.9, add IPTG 0.02 mM, induced overnight in 25℃.Second, to detect the protein’s distribution of other lactic acid bacteria especially those from nature, three lactic acid bacteria (two Lactobacillus plantarums ZN002, ZN007 and one Leuconostoc mesenteroides ZN011) with antioxidant activity were isolated and identified from homemade pickle of chili. These strains were used in pickle fermentation. The fermented pickle broth showed higher antioxidant activities than the automatic fermented pickle. Although there was no significant difference (P<0.05) in the DPPH free radical clearance, reducing power, hydroxyl radicals and ABTs radical scavenging rate of naturally fermented pickle was significantly lower than the pickle with added lactic acid bacteria. The scavenging rate of hydroxyl free of the two bacterial adding groups showed the most significant increase, with two times higher than that in the natural group which was 28.41%. Preliminary taste and flavor evaluation of the pickle also showed that the pickles with higher antioxidant activity showed better taste and flavor. Accordingly, GC-MS analysis showed that the inoculated bacteria provided more abundant flavor contents like alkanes and esters into the pickle, and the ratio of their total percentage in measuring peak area were 27%-30%higher than that of automatic fermented pickle.Finally, a optimum purification methods (NTI columella, the denature conditions with 8M urea)for the purification of the recombinant Rib protein was work out after multiple tests.1.5 mg purified recombinant protein were obtained after affinity chromatography combine molecular sieve with ultrafiltration technology. The polyclonal antibody were prepared by use rib as antigen in immunization of mousses. The polyclonal antibody is tittered to be 1:2000 by ELASA. Rib protein was detected in lactic acid bacteria with Western blot analyses. The result showed that Rib protein was also detected in other three Lactobacillus reuteris and two Lactobacillus plantarums ZN002 and ZN007, but not detected in Leuconostoc mesenteroides ZN011, representing the wide distribution of Rib protein in the microbe.The distribution of Rib protein in the microbial world illustrates the universal function of Rib in natural probiotics especially Lactobacillus reuteris and lactobacillus plantarum in adherence to human and animal gastrointestinal tract.