【摘要】 为了探究猪源枯草芽孢杆菌(Bacillus subtilis,B.subtilis) QXKL-3菌株对猪链球菌2型(Streptococcus suis type 2,SS2)的抑制效果，试验以培养B.subtilis QXKL-3获得的无细胞上清液(cell free supernatant,CFS)作为抑制剂，采用平板菌落计数法和打孔法评估QXKL-3 CFS对SS2的体外抑菌效果及其在不同条件下的理化特性(对温度的耐受性和在不同储存条件下的稳定性)，采用硫酸铵沉淀法提取抗菌物质，测定粗提物最小抑菌浓度(MIC)，利用液相色谱串联质谱(LC-MS/MS)联用技术进一步分析鉴定B.subtilis QXKL-3 CFS中的抑菌活性物质，并对抑菌活性物质进行KEGG通路分析。结果表明:B.subtilis QXKL-3 CFS可在10 min内完全杀灭SS2，其抑菌能力显著强于青霉素(1 mg/m L);当硫酸铵浓度为60%时抑菌圈直径最大，显著高于其他浓度(P<0.05)，通过60%硫酸铵沉淀得到的抗菌粗提物MIC为2.344 mg/m L; B.subtilis QXKL-3 CFS在121℃高压下处理30 min,-20℃、4℃及室温(25℃)条件下储存120 d仍具有较好的抗菌活性;经LC-MS/MS筛选鉴定出多种抑菌活性物质，包括表面活性素、杆菌烯、原儿茶酸等抑菌相关物质;涉及12个代谢通路，包括α-亚麻酸代谢、铁载体基团非核糖体肽的生物合成、次级代谢产物的生物合成等代谢通路。说明B.subtilis QXKL-3 CFS对猪链球菌具有较强的抑制作用，且具有多种抑菌活性物质，是一种极具前景的抗生素替代品。更多还原

【Abstract】 In order to explore the inhibitory effect of porcine-derived Bacillus subtilis(B.subtilis) QXKL-3 strain on Streptococcus suis type 2(SS2),the cell-free supernatant(CFS) obtained from the cultivation of B.subtilis QXKL-3 was used as an inhibitor in the experiment.The plate colony counting method and the punching method were adopted to evaluate the in vitro antibacterial effect of B.subtilis QXKL-3 CFS on SS2 and its physicochemical properties under different conditions(temperature tolerance and stability under different storage conditions).The antibacterial substances were extracted by ammonium sulfate precipitation method,and the minimum inhibitory concentration(MIC) of the crude extract was determined.Liquid chromatography-tandem mass spectrometry(LC-MS/MS) hyphenated technology was used to further analyze and identify the antibacterial active substances in B.subtilis QXKL-3 CFS,and KEGG pathway analysis was carried out on its antibacterial active substances.The results showed that B.subtilis QXKL-3 CFS could completely kill SS2 within 10 min,and its antibacterial ability was significantly stronger than that of penicillin(1 mg/m L).When the ammonium sulfate concentration was 60%,the diameter of the inhibition zone was the largest,which was significantly higher than that of the other concentrations(P < 0.05).The MIC of the crude antibacterial extract obtained by 60% ammonium sulfate precipitation was 2.344 mg/m L.The B.subtilis QXKL-3 CFS still had good antibacterial activity after being sterilized high-pressure treatment at 121 ℃ for 30 min and stored at-20 ℃,4 ℃,and room temperature(25 ℃) for 120 days.A variety of antibacterial active substances were screened and identified by LC-MS/MS,including surfactin,bacillaene,protocatechuic acid and other antibacterial-related substances.It was involved in 12 metabolic pathways,including such as α-linolenic acid metabolism,nonribosomal peptide biosynthesis of siderophore groups,and biosynthesis of secondary metabolites.It indicated that B.subtilis QXKL-3 CFS had strong antibacterial activity against Streptococcus suis,and had a variety of antibacterial active substances,which could be a promising substitute for antibiotics.更多还原