副溶血性弧菌拮抗菌的筛选鉴定及抑菌物质特性研究

Isolation and identification of antagonistic bacteria aganist Vibrio parahaemolyticus and its characteristics of antibacterial substances

为获取既可抑制食源性致病菌又可抑制水产致病菌的益生菌株,以人畜共患致病菌副溶血性弧菌(Vibrio parahaemolyticus ATCC17802)为指示菌,采用点种法初筛以及打孔扩散法复筛,得到1株来源于上海东海海水的H19菌株,其抑菌物抑菌圈直径达(17.16±0.28)mm。通过形态观察及16SrDNA序列分析可知,菌株H19为枯草芽孢杆菌(Bacillus subtilis H19,GenBank登录号:MG383451)。将菌株H19进行抑菌谱试验,探究菌株H19抑菌物的理化性质,并且抑菌物通过硫酸铵沉淀及不同孔径透析袋截留粗提,粗提物经蛋白酶处理及Tricine-SDS-PAGE蛋白质凝胶电泳可知,该抑菌物具有广谱抑菌活性,既可抑制革兰氏阳性菌又可抑制大部分革兰氏阴性菌,且具有较好的热、pH以及紫外线稳定性,该抑菌物可能为2种新型抗菌肽,分子量分别在6.5~9.5kDa以及27.0~35.0kDa。由此可见,枯草芽孢杆菌H19所产抗菌肽具有防治食源性疾病以及水产疾病的应用潜力。

In order to screen a probiotic strain against foodbornepathogens and aquatic pathogens, the main zoonotic pathogenic bac- teria of maricuhure Vibrio #arahaemolyticus ATCC17802 was used as indicator bacterium. The target prohioties was screened using the spot-on-lawn method and rescreened by the agar diffusion method, and the diameter of inhibition zone of antibacterial substances reached （17.16±0.28） ram. Through the combination of morphology and 16S rDNA sequence analysis, strain H19 was identified as Bacillus subti lis （ GenBank accession number： MG383451 ）. Antimierobial spectrum of B. subtilis H19 was tested, meanwhile the physical and chemical properties of antimierobial substances produced by H19 was studied. The supernatant of the H 19 fermentation broth was subjec- ted to ammonium sulfate precipitation and dialysis against several dif- ferent pore-size membranes, and two novel kinds of antibacterial peptides with molecular weight of 6.5--9.5 kDa and 27.0-35.0 kDa respectively were obtained based on Tricine-SDS-PAGE protein gel e- lectrophoresis and protease treatment. It had broad-spectrum antimi- crobial activity, which could inhibit both Gram positive bacteria and most of Gram-negative bacteria, and showed good heat, pH and UV stability. Consequently, the antibacterial peptide produced by B. sub tilis H19 had the potential to antagonize the pathogenic bacteria for the prevention and control of foodborne and aquatic diseases.