一株短乳杆菌烈性噬菌体的分离鉴定与生化特性研究

Isolation,identification and characterization of a virulent phage of Lactobacillus brevis

短乳杆菌是应用于食品发酵中的重要菌株,噬菌体污染是导致发酵失败的主要因素之一,对食品发酵生产有着巨大的危害。从发酵泡菜水中分离纯化短乳杆菌烈性噬菌体,研究其生物学性质。利用双层平板法分离和纯化噬菌体,通过负染色电镜形态和核酸类型鉴定。采用核酸内切酶处理后电泳鉴定核酸类型、基因组大小和包装机制。通过双层平板法测定噬菌体效价,实验测定最佳感染复数、一步生长曲线、温度和pH等物理化学因素及常见消毒剂对其存活的影响。分离纯化到1株烈性噬菌体,命名为φB23。其由多面体结构的头部和弯曲的长尾构成,φB23基因组为双链DNA,大小约为52.4 kb,包装机制为黏末端连接。最佳感染复数为0.001,潜伏期为30 min,裂解期为90 min,裂解量为(103.2±9.0)pfu/cell。常用的巴氏灭菌温度为63℃和72℃,分别处理10 min和5 min能较好地灭活;在pH 3.0~8.0保持稳定,75%乙醇处理10 min能使其丧失活性,500 mg/L次氯酸钠处理5 min能完全灭活。φB23鉴定为尾噬菌体目(Caudovirales)长尾噬菌体科(Siphoviridae)的噬菌体,表征并分析了φB23的生化特性。实验结果为进一步阐明短乳杆菌噬菌体侵染机制和制定有效的噬菌体防治方法提供了基础。

Lactobacillus brevis is an important strain used in food fermentation.Bacteriophage contamination is one of the main factors resulting in the failure of fermentation,which has caused great losses to food fermentation production.A virulent bacteriophage of L.brevis was isolated and characterized from fermented pickle water.Double-layer plate method was used to isolate and purify phage.The isolated phage was identified via the characteristics in negative staining observed with transmission electron microscopy(TEM),and its nucleic acid type.The phage genome was digested with various nuclease,and analyzed with electrophoresis subsequently to determine the type of nucleic acid,genome size and packaging mechanism.The phage titer was determined by double plate method.Characterization of the phage was investigated by determination of the optimal multiplicity of infection,one-step growth curve,and the effect of temperature,pH,other physicochemical factors and common disinfectants on their survival.A virulent phage namedφB23 was isolated and purified.It consisted of a head with a polyhedral structure and a long-curved tail.The genome was double-stranded DNA,about 52.4 kb in size,and packaged with sticky terminal ligation in mechanism.The optimal multiplicity of infection was 0.001.One-step growth curve showed that the latent time and burst time were 30 min and 90 min,respectively,and the burst size was(103.2±9.0)pfu/cell.φB23 could be effectively inactivated by pasteurization under temperature of 63℃and 72℃,treated for 10 min and 5 min,respectively.It was stable in the range of pH 3.0-8.0.In terms of disinfection effect of biocidal,φB23 was inactivated by 75%ethanol treatment for 10 min,and completely inactivated by 500 mg/L sodium hypochlorite treatment for 5 min.φB23 was identified as a phage belongs to the family Siphoviridae in the order Caudovirales,and its biochemical properties were characterized and analyzed.The results provide a basis for further elucidating the phage infection mechanism against L.brevis and developing effective phage control methods.