A Preliminary Study On The Mechanism Of Bacillus Subtilis-induced Bacteriocin Production By Lactobacillus Paracasei And The Relationship Between The Two Species

Lactobacillus paracasei HD1.7 was isolated from Chinese sauerkraut juice.It produced a kind of bacteriocin called Paracin 1.7 which could inhibit the growth of some Gram positive,negative bacteria and Saccharomyces cerevisiae.Previous studies found that Bacillus subtilis could enhance the production of Paracin 1.7 in L.paracasei HD1.7.However,the mechanism of enhancement production of Paracin 1.7 and ecological relationship are not totally understood.Firstly,effects of carbon sources(glucose,sucrose,maltose,lactose,soluble starch,sorbitol,mannitol)and nitrogen sources(beef extract,peptone,yeast extract,ammonia sulfate,ammonia nitrate,carbamide)on the growth of L.paracasei HD1.7 and B.subtilis were detected in this study to select the best carbon and nitrogen sources.The results showed that both the L.paracasei HD1.7 and B.subtilis grew well in broth where glucose added as the carbon source.The OD_600nm,changing of p H and bacterioccin production in fermentation broth of L.paracasei HD1.7 were maximal during the culture process that were 0.71±0.080?3.47±0.054?71.59±2.343 AU/m L,respectively.The OD_600nm and changing of p H of B.subtilis were also maximal that were 0.615±0.025?5.55±0.030,respectively.Similarly,the growth of L.paracasei HD1.7 and B.subtilis was the best in broth where yeast extract added as nitrogen sources.The OD_600nm,drop range of p H and bacterioccin production in fermentation broth of L.paracasei HD1.7 were maximal during the culture process that were0.64±0.024,3.68±0.035and 72.67±1.380 AU/m L,respectively.The OD_600nm and drop range of p H of B.subtilis was also maximal which were 0.55±0.001and 4.95±0.048respectively.Thus,the co-culture of the two strains was carried out by selecting glucose and yeast extract as the best carbon source and nitrogen source.Secondly,the model system of L.paracasei HD1.7 with B.subtilis was set up and to investigate the inductive mechanisms of B.subtilis,as well as ecological relationship between each other.As control,culture of L.paracasei HD1.7 added AI-2 and monocultures of both were also prepared.The main results are as follows:(1)Resultes indicated that a significant increase in bacteriocin production was observed in culture of L.paracasei HD1.7 added AI-2 and with B.subtilis(4.39%and4.54%increase at 36h)compared to monoculture.Similarly,a significant increase in the transcriptional level of quorum sensing genes(lux S,prc K and prc R)of L.paracasei HD1.7 was observed in co-culture system compared to monoculture.The transcriptional level of lux S increase 7.74 fold(24h,culture of L.paracasei HD1.7 added AI-2)and2.72 fold(36h,co-culture of L.paracasei HD1.7 with B.subtilis).The transcriptional level of prc K increase 1.60 fold(24h,culture of L.paracasei HD1.7 added AI-2)and2.51 fold(24h,co-culture of L.paracasei HD1.7 with B.subtilis).The transcriptional level of prc R increase 3.00 fold(24h,culture of L.paracasei HD1.7 added AI-2)and3.11 fold(24h,co-culture of L.paracasei HD1.7 with B.subtilis).B.subtilis could screted a sigal molecule which is similar to the function of AI-2.It could also increase the production of Paracin 1.7 and transcriptional level of quorum sensing genes of L.paracasei HD1.7.(2)The co-cluture and monoculture of B.subtilis with L.paracasei HD1.7 were investigated.Results indicated that the growth of B.subtilis was inhibited at 8h and was inhibited completely at 36h in co-culture.And no spore was detected in the process of fermentation.The sporulation genes were detected and analysized by quantitative reverse transcription PCR.The transcriptional level of spo0A,sig E,sig F and sig G of B.subtilis dropped in co-culture compared to monoculture(drop 52%-92%).(3)We used i TRAQ-LC-MS/MS/MS technology to establish the differences proteome expression system of L.paracasei HD1.7 in co-culture and monoculture.A tatol of 1914proteins were identified,and direct comparisons showed that 162 proteins incresed and167 proteins decreased in co-culture with B.subtilis.Bioinformatics analysis of differentially expressed proteins by GO and KEGG,indicated that the differentially expressed involved in biosynthesis of amino acids,two-component system,ABC transporters system and other pathways.In conclusion,the bacteriocin production and cell numbers of L.paracasei HD1.7 were increased significantly when L.paracasei HD1.7 co-cultured with B.subtilis compared to monoculture.Using i TRAQ-LC-MS/MS technology to identify differentially expressed proteins of L.paracasei HD1.7 in co-culture compared to monoculture.The differentially expressed proteins involved in biosynthesis of amino acids,two-component system,ABC transporters system and other pathways.And significant up regulation of histidine kinase may be relatived to two component system and synthesis of bacteriocin.And we could also observe that L.paracasei HD1.7 can play a significant role in preventing the growth and sporulation of B.subtilis.Therefore,L.paracasei HD1.7 is the amensalism for B.subtilis,but B.subtilis is the commensalism for L.paracasei HD1.7.