枯草芽孢杆菌体外抑菌试验及其抑菌蛋白的鉴定  

Antibacterial test in vitro of Bacillus subtilis and identification of its antibacterial proteins

作　　者：尹杨燕 彭昊[2] 李小宁 李军[2] 李常挺 白慧丽 马春霞[2] 钟舒红[2] 陶立[2] 兰美益[2] 韦天超[1] 廖玉英 YIN Yangyan;PENG Hao;LI Xiaoning;LI Jun;LI Changting;BAI Huili;MA Chunxia;ZHONG Shuhong;TAO Li;LAN Meiyi;WEI Tianchao;LIAO Yuying(Institute of Animal Science and Technology,Guangxi University,Nanning 530004,China;Guangxi Veterinary Research Institute/Guangxi Key Laboratory of Veterinary Biotechnology,Nanning 530001,China)

机构地区：[1]广西大学动物科学技术学院,广西南宁530004 [2]广西壮族自治区兽医研究所,广西南宁530001

出　　处：《畜牧与兽医》2022年第12期57-63,共7页Animal Husbandry & Veterinary Medicine

基　　金：广西重点研发计划(AB21238003,AB18221074);国家水禽产业技术体系建设专项(CARS-42)桂科专项(18-3,19-3,21-6);国家现代农业产业体系广西肉鸡产业创新团队建设专项(nycytxgxcxtd);南宁市重点研发计划(20202106,20212023);良庆区重点研发计划(202109),良庆区重大科技专项(202118);防城港市重点研发计划(AB21014016);武鸣区重点研发(20210102)。

摘　　要：旨在深入研究枯草芽孢杆菌发挥抑菌作用的相关抑菌蛋白,进一步了解枯草芽孢杆菌抑菌机制。本研究以枯草芽孢杆菌GX20200511-1株为研究对象,采用体外共培养及牛津杯法测定体外抑菌活性,低温离心和超滤法初步分离枯草芽孢杆菌的胞外蛋白,利用液相色谱串联质谱(LC-MS/MS)进一步分析鉴定枯草芽孢杆菌发酵产物中的抑菌成分。结果表明枯草芽孢杆菌GX20200511-1对沙门菌、金黄色葡萄球菌和大肠杆菌均具有良好的抑菌效果,显著降低共培养24 h后的指示菌数量。经LC-MS/MS鉴定分析,共检索出455种蛋白质,通过Uniprot蛋白数据库对所有蛋白质进行分析,检测出6种可信度较高且与抑菌作用相关的蛋白质,分别是壳聚糖酶(chitosanase)、聚酮生物合成3-羟基-3-甲基戊二酰ACP合成酶PksG(polyketide biosynthesis 3-hydroxy-3-methylglutaryl-ACP synthase PksG)、伊枯草菌素合成酶(iturin synthetase)、表面活性素合成酶(surfactin synthetase)、丰原素合成酶(fengycin synthetase)和tRNA核酸酶WapA(tRNA nuclease WapA),分子量分别为31.395、46.766、296.6、402.43、48.86和173.525 kDa。本研究枯草芽孢杆菌GX20200511-1可能通过代谢产物中的多种抑菌蛋白协同发挥抗菌作用。In order to further study the possible antibacterial proteins of Bacillus(B.) subtilis and further understand the antibacterial mechanism of this bacterium, B. subtilis GX20200511-1 strain was chosen to be the object of this study, and the Oxford cup method was used to determine the antimicrobial activity of the strain. Proteins were extracted from the B. subtilis GX20200511-1 stain by centrifugation at low temperature and by ultrafiltration, and the antibacterial activities of the proteins were measured. Antimicrobial components from B. subtilis metabolites were further analyzed by LC-MS/MS. The results showed that B. subtilis GX20200511-1 had a strong antibacterial effect on Salmonella, Staphylococcus aureus and Escherichia coli, and it significantly reduced the number of indicator bacteria after 24 hours of co-culture. The GC-MS/MS analysis showed that 455 proteins were retrieved. All the proteins were analyzed by means of the UniProt protein database. Six kinds of proteins were obtained which were not only corrected with bacteriostasis but also possessed higher credibility. The proteins were chitosanase, polyketide biosynthesis 3-hydroxy-3-methylglutaryl-ACP synthase PksG, iturin synthetase, fengycin synthetase,surfactin synthetase, and tRNA nuclease WapA. Their molecular weights were 31. 395, 46. 766, 296. 6, 402. 43, 48. 86, 173. 525 kDa, respectively. The identification of the antimicrobial proteins extracted fromB. subtilisGX20200511-1 in this study laid a foundation for further isolation and purification of the components of the proteins. It was speculated thatB. subtilisGX20200511-1 might play an antibacterial role by the coordination of the antibacterial proteins in metabolites.

关 键 词：枯草芽孢杆菌 体外抑菌 抑菌蛋白 超滤法 液相色谱串联质谱 

分 类 号：S858[农业科学—临床兽医学]