一株虹鳟源枯草芽孢杆菌的分离鉴定及生物学特性研究  

Isolation,Identification and Biological Characterization of a Bacillus subtilis Strain from Rainbow Trout(Oncorhynchus mykiss)

作　　者：樊丹 邓福容 李绍戊[1,2] 卢彤岩 刘红柏[1,2] 王荻 Fan Dan;Deng Furong;Li Shaowu;Lu Tongyan;Liu Hongbai;Wang Di(Heilongjiang River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Harbin 150070, China;Key Laboratory of Aquatic Animal Diseases and Immunology of Heilongjiang Province, Harbin 150070, China;National Demonstration Center for Experimental Fisheries Science Education, Shanghai Ocean University, Shanghai 201306, China;National Pathogen Collection Center for Aquatic Animals, Shanghai Ocean University, Shanghai 201306, China)

机构地区：[1]中国水产科学研究院黑龙江水产研究所,黑龙江哈尔滨150070 [2]黑龙江省水生动物病害与免疫重点实验室,黑龙江哈尔滨150070 [3]上海海洋大学水产科学国家级实验教学示范中心,上海201306 [4]上海海洋大学国家水生动物病原库,上海201306

出　　处：《中国海洋大学学报（自然科学版）》2022年第2期41-49,共9页Periodical of Ocean University of China

基　　金：中央级公益性科研院所基本科研业务费专项(HSY202007M);现代农业产业技术体系项目(CARS-46);冷水性鱼类病害防控创新团队项目(2020TD43)资助。

摘　　要：为筛选对虹鳟(Oncorhynchus mykiss)有潜在益生效果的菌株,本研究从健康虹鳟幼鱼肠道中分离到1株潜在益生菌株RT-BS07,采用形态学观察和分子特征分析对该菌株进行了鉴定,并评价了该菌株的抑菌性、耐受性、黏附性和安全性。形态学观察显示,菌株RT-BS07为革兰氏阳性杆菌,有圆形或椭圆形芽孢。16S rRNA比对结果显示,该菌株与枯草芽孢杆菌(Bacillus subtilis)NCBI3610株的同源性为99.86%。结合16S rRNA基因序列分析和形态学观察结果,确定菌株RT-BS07为枯草芽孢杆菌。菌株RT-BS07具备产蛋白酶和纤维素酶能力,其酶活力分别为(0.039±0.002)和(0.393±0.002)U/mg;该菌株可有效抑制温和气单胞菌(Aeromonas sobria)、杀鲑气单胞菌(A.salmonicida)和鲁氏耶尔森氏菌(Yersinia ruckeri)等致病菌。菌株RT-BS07在pH为2~10 LB液体培养基中均可存活;在盐度为2%~8%条件下存活率为5.70%~93.28%,其存活率随盐度升高而降低。体外黏附试验结果表明,菌株RT-BS07可大量黏附在虹鳟前肠黏膜,不破坏肠道组织完整性,对左氟沙星、环丙沙星、氧氟沙星和链霉素等18种抗菌药物敏感。本研究分离筛选得到的枯草芽孢杆菌RT-BS07株具有产消化酶性能,其抗逆性能强,对虹鳟无毒害作用,可作为虹鳟健康养殖及益生菌制剂研发源益生菌候选株。To obtain the potential probiotics,one bacterial strain was isolated by comparing the enzyme production characteristics of the isolates from the intestines of healthy rainbow trout(Oncorhynchus mykiss)juvenile and named RT-BS07.The results of morphological observation showed that the strain with round or oval spores was Gram-positive bacillus,and its colony with fold and hump on the edge was milky white,rough and opaque.According to 16S rRNA gene sequencing analysis,the gene of RT-BS07 was 99.86%similar to that Bacillus subtilis(NCBI3610).Combined with 16S rRNA gene sequence analysis,RT-BS07 strain was identified as B.subtilis.The strain had the enzyme production capacity of protease and cellulase,which was(0.039±0.002)and(0.393±0.002)U/mg,respectively.It can effectively inhibit Aeromonas sobria,A.salmonicida and Yersinia ruckeri,and the antibacterial zone area was 5.34,3.27 and 2.40 cm2,respectively.RT-BS07 strain can survive LB liquid medium at pH 2~10.The survival rate ranged from 5.70%to 93.28%when the salinity varied between 2%and 8%,decreasing when the salinity increased.Adhesion test in vitro showed that RT-BS07 strain can adhere to the anterior intestinal mucosa without destroying the integrity.Antimicrobial susceptibility test indicated that RT-BS07 strain was sensitive to 18 antimicrobial agents such as levofloxacin,ciprofloxacin,ofloxacin,streptomycin among others.These results indicated that the isolated B.subtilis stain RT-BS07 can be used as a candidate strain,providing strain resources for healthy aquaculture of rainbow trout and the development of probiotic preparation.

关 键 词：虹鳟 枯草芽孢杆菌 产酶性能 抑菌特性 黏附特性 

分 类 号：S942.1[农业科学—水产养殖]