Screening Of Bacteriocin-producing Strains And Functional Studies On Its Bacteriocin,Bacillocin TK1

Bacteriocin is a bioactive peptide or protein encoded by ribosome during metabolism of bacteria.Compared with antibiotics,bacteriocin has the advantages of being targeted,safe and not easy to cause resistance.Lactic acid bacteria,as the most important probiotic source,can improve the intestinal barrier function of the host.Presently,few attention is being focused on the beneficial properties of bacteriocins secreted by lactic acid bacteria,especially on intestinal barrier and inflammatory regulation.The purpose of this study is to screen bacteria producing bacteriocin from 12 strains of lactic acid bacteria isolated and preserved in our laboratory,and the bacteriocin was extracted and highly purified.The effect of bacteriocin on IPEC-J2 cells barrier and inflammation regulation was studied thro?gh in vitro experiments.The major content and results are listed as follows:Trial 1 Screening of Bacteriocin-producing strainsPathogenic strains of Staphylococcus aureus ATCC25923,Enterotoxigenic Escherichia coli(ETEC)K88ac,Salmonella choleraesuis C78-1,Listeria monocytogenes 1598,Clostridium perfringens type C5 were used as indicators,The best bacteriostatic strain TC23 was selected from 12 lactobacillus strains by the Oxford cup method.After excluding the interference of organic acid and hydrogen peroxide,the fermentation supernatant of TC23 still has antibacterial activity against ETEC K88 ac,and the antibacterial activity disappeared after the treatment with protease.TC23 was identified as Bacillus coagulans by cell morphology observation,physiological and biochemical identification and 16 SrDNA sequence analysis,and it was named Bacillus coagulans TK1.Trial 2 Isolation and purification of bacteriocinBacillus coagulans TK1 was inoculated in MRS medium for 18 h at 37 °C,and bacteriocin in culture supernatant was extracted with ammonium sulfate of 80% saturation,followed the bacteriocin was purified by SP Sepharose Fast Flow cation exchange chromatography and Sephadex G50 gel filtration.Tricine-SDS-PAGE showed that molecular weight of the purified bacteriocin was about 4.6~10kDa.The biological characteristics of bacteriocin were studied.It was found that bacteriocin was heat stable and had good stability in the range of pH 4-8.Bacteriocin was analyzed by LC-MS/MS.Its molecular weight was 11207 Da and its N-terminal sequence was GGAGMNPIDYDR.It was identified as a novel bacteriocin by BLAST comparison and named Bacillocin TK1.Trial 3 The effect of Bacillocin TK1 on IPEC-J2 Cells? Adhesion test.IPEC-J2 cells which were cultured for 10 d were co-cultured with Bacillocin TK1 and(enterotoxigenic E.coli,ETEC)K88ac and it was revealed that Bacillocin TK1 could significantly inhibit the adhesion of K88 ac.In order to test whether the adding order of Bacillocin TK1 had an effect on the inhibition of K88 ac adhesion,an experiment had been done and the process is as following: IPEC-J2 were co-incubated with Bacillocin TK1 and K88ac,or IPEC-J2 were co-incubated with Bacillocin TK1 and then K88 ac,or IPEC-J2 were co-incubated with K88 ac and then Bacillocin TK1,respectively,to detect the adhesion number of K88 ac.The results showed that the IPEC-J2 were incubated with Bacillocin TK1 in advance,and the inhibition effect of K88 ac adhesion cells was the best.? The regulation effects of Bacillocin TK1 on inflammatory cytokines released by IPEC-J2 cells.First,IPEC-J2 cells were pre-cultured with Bacillocin TK1 for 3h and then following co-incubation with K88 ac,then tested the expression of inflammatory cytokines.The results showed that Bacillocin TK1 could relieve the increase of IL-8 and TNF-? and the decrease of TGF-?1caused by K88 ac,and promote the expression of IL-10 in IPEC-J2.? The Effect of Bacillocin TK1 on IPEC-J2 Cells barrier.IPEC-J2 cells were cultured for 10 d to form a complete single layer,which coulid be used in the following experiment.First,IPEC-J2 cells were pre-cultured with Bacillocin TK1 for 18 h and then following co-incubation with K88 ac,the transepithelial electrical resistance was measured after 3,6,9,18 h.The results showed that Bacillocin TK1 could protect the membrane integrity of the IPEC-J2 cells,and relieve the membrane structure destruction of IPEC-J2 cells caused by K88 ac.Finally,Western Blot was used to detect the effects of Bacillocin TK1 on the expressions of OCLN,CLDN1 and ZO-1 in k88ac-induced IPEC-J2 cells.The results showed that Bacillocin TK1 could significantly relieve the decrease of three tight junction proteins in k88 ac induced cells.