| Lactobacillus paracasei HD1.7 was isolated from Chinese sauerkraut fermentation broth,which secreted a kind of bacteriocin Paracin1.7.The Paracin1.7 could efficiently inhibit the growth of Pathogenic microorganism and food spoilage bacteria.Earlier research has proved that the expression of Paracin1.7 secreted by L.paracasei HD1.7can be enhanced during the co-culture of L.paracasei HD1.7 and B.subtilis.The work included the investigation of flora relationship between L.paracasei HD1.7 and B.subtilis that is helpful to pull the theoretical researchment of Quorum Sensing(QS)of L.paracasei HD1.7 to a new level;the mechanism illumination of interspecific relationship between L.paracasei HD1.7 and B.subtilis by separation and purification of addition released by B.subtilis and researchment of the effect of the addition on L.paracasei HD1.7 by transcriptomics technology;the exploration of competition and evolution strategies between L.paracasei HD1.7 and some strains of Bacillus.First of all,the L.paracasei HD1.7 was co-cultured with cells-free fermentation broth,viable cells and deadly cells of B.subtilis.The results showed that antibacterial effects of Paracin1.7 were 113.78% and 129.53% higher and the up-regulated expressions of prc K and prc R that encoding gene of Paracin1.7 were 1.32 and 1.58 times more than that of the control in the case of the co-culture of L.paracasei HD1.7with cells-free fermentation broth and viable cells of Bacillus subtilis.Furthermore,the up-regulated expressions of Lux S that is the QS relative gene were 3.31 and 7.23 times more than that of the control in the same case.These datas could be explained by the addition released by B.subtilis that leads to variations of L.paracasei HD1.7.Study on B.subtitls impact L.paracasei HD1.7 transcriptome discovery,significant upregulation of 51 genes,significantly regulated genes 76;enrichment of the most significant metabolic pathways were histidine metabolism,alanine,aspartate and glutamate metabolism,valine,leucine and isoleucine degradation,fructose and mannose metabolism,pyrimidine metabolism and phosphotransferase system.Secondly,to primary confirm the ecological relationship between L.paracasei HD1.7 and some strains of Bacillus,the L.paracasei HD1.7 was co-cultured with the no-cells fermentation of B.megatherium,B.pumilus,B.licheniformis and B.laterosporus.The results showed that antibacterial effects of Paracin1.7 were 108.12%?112.74%?107.89% and 105.73% higher than that of the control.The q RT-PCR results demonstrated that the cells precipitates of the strains could efficiently up-regulate the Lux S gene expressions,which were 1.23,3.24,2.51 and 2.03 times more than that of the control respectively.B.subtilis had the best effect.At last,the addition was separated and purified by three step purification method.Step one,the ammonium sulfate precipitation was seclected to extract crude enzyme with the concentration of 60% of ammonium sulfate determined by the test of optimal concentration detection.Step two,the addition was further purified by CM Sepharose Fast Flow.Step three,the addition was finally purified by Superdex 75.The finally purified addition was then co-cultured with L.paracasei HD1.7.The results illuminated that the antibacterial activities of the experiment was higher that the control strain.The titer increase by 118.68%,respectively.The expression level of Lux S gene was higher than the control,which was 1.85 times more than the control.The pure samples by SDS-PAGE electrophoresis to test its degree of purity,the molecular weight was about30 k Da,LC-MS analyzed showed that the stimulating factor might be a new peptide.The B.subtilis stimulating factor was separated and purificated.This stimulating factor can promote L.paracasei HD1.7 bacteriocin generate gene prc K,prc R genes and the QS relative gene Lux S up-regulated,it might be a novel peptide.This research could provide foundation for the bacteriocin production and the QS mechanism,it laid a foundation for the study of flora relations. |
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