渤海湾仿刺参养殖圈内贝莱斯芽胞杆菌B-VE的分离鉴定及其产抗菌脂肽的研究

为分离筛选具有广谱抑菌作用的细菌并研究其抗菌物质,实验利用琼脂扩散法和牛津杯法,以溶壁微球菌、金黄色葡萄球菌、铜绿假单胞杆菌、大肠埃希菌为指示菌,从渤海湾大连海域仿刺参养殖圈中分离筛选出9株活性菌,2株具有广谱抑菌效果,其中1株抑菌效果最强。通过形态学和分子生物学检测分析,鉴定该菌株为贝莱斯芽胞杆菌(Bacillus velezensis),命名为B-VE。通过酸沉法分离发酵液中的抑菌物质,利用飞行时间质谱对其分子量进行检测,初步确定其隶属于抗菌脂肽类物质,抑菌物质包括的三种物质分别为伊枯草菌素(Iturin)、芬荠素(Fengycin A)和杆菌霉素(Bacillomycin D)。实验发现该抗菌脂肽类粗提物具有较强的pH和温度耐受性,可以耐受多种蛋白的酶解作用,且在多种有机溶剂中可以保持较强的抑菌效果。利用扫描电镜检测发现其主要通过破坏细菌的细胞膜对溶壁微球菌产生抑菌作用。结果表明,贝莱斯芽胞杆菌B-VE广谱抑菌,所产抗菌脂肽对细菌具有较强的破膜作用,且理化性质稳定,具有较好的开发利用价值。

Isolation and Identification of Bacillus velezensis Strain B-VE from Sea Cucumber Breeding Environment in Bohai Bay and Study on Its Antimicrobial Lipopeptides

In order to isolate and screen out broad-spectrum antimicrobial strains and excavate their antimicrobial substance, experiments isolated and screened out 9 active microbial strains from a sea cucumber breeding circle in Dalian Bohai Bay, adopted agar diffusion method and Oxford cup method, and take Micrococcus lysodeikticus, Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli as the indicator bacteria. Two of them had broad-spectrum antimicrobial effects, and one strain of both having the strongest antimicrobial effect. Based on morphological and molecular bio-testing analysis, the strain was identified as Bacillus velezensis and named as B-VE. After isolated by acid precipitation, the antimicrobial substances in the B-VE fermentation broth were identified by MALDL-TOF MS. Analyses showed that the substances belonged to the antimicrobial lipopeptides, and three of them are Iturin, Fengyc A, and Bacillomycin D. Experiments had found that crude lipopeptide extracts could damage the bacterial cell membrane and kill the indicator bacteria M. lysodeikticus using scanning electron microscopy analysis. Further study confirmed that the crude antimicrobial lipopeptide extracts of the strain B-VE had a strong pH/temperature tolerance range. It also can tolerate enzymatic hydrolysis of various proteins and can maintain relative strong inhibition in organic solvents. The above results showed that strain B-VE are a broad-spectrum antimicrobial strain that produces antimicrobial lipopeptides. It has a strong membrane-breaking effect on bacteria and stable physical and chemical properties, and it has good development and application value.