一株枯草芽孢杆菌噬菌体的生物学特性分析及抗性菌株的诱变筛选

摘要

枯草芽孢杆菌工业生产发酵过程中经常出现噬菌体污染的问题,分离筛选以枯草芽孢杆菌为宿主菌的噬菌体,分析其生物学特性并采用自发突变方法筛选噬菌体抗性菌株.以枯草芽孢杆菌KC-260为宿主菌,从车间异常发酵液中分离纯化得到一株噬菌体,将其命名为P260,富集培养后测定效价,对其热稳定性、最佳感染复数、宿主范围、抑制剂和消毒剂耐受性等生物学特性进行了研究.为防治噬菌体P260的污染,利用自发突变的方法筛选对噬菌体P260有抗性的枯草芽孢杆菌突变菌.结果显示,分离纯化得到一株效价为3.45×1010 PFU/mL的噬菌体P260,该噬菌体具有一定的宿主专一性,对凝结芽孢杆菌和短小芽孢杆菌不敏感.噬菌体P260在高温条件下很容易失活,不耐热,70℃处理15 min存活率下降极为明显;25 mg/L的ClO2对P260有极强的杀灭作用;0.5％的草酸铵和柠檬酸铵对该噬菌体有一定的抑制作用.P260感染宿主菌的最佳感染复数为0.01,增殖的潜伏期为15 min,45 min进入裂解期.通过自发突变的方法获得了抗噬菌体菌株ZF-260,发酵过程中添加噬菌体后ZF-260的生长不受影响,说明ZF-260是一株可以抗噬菌体P260的优良菌株.研究了噬菌体P260的生物学特性,获得了遗传稳定且活菌数高的抗性菌株ZF-260.%Bacteriophage infection usually occurs in industrial fermentation,thus this work aims to isolate and screen bacteriophage with Bacillus subtilis as host,to analyze its biological characteristics,and to screen anti-phage strains via spontaneous mutation.With B.subtilis KC-260 as the host bacterium,a bacteriophage,designated as P260,was isolated and purified from the fermentation broth.Its titer was measured after enrichment culture,and then its biological characteristics such as thermal stability,optimal multiplicity of infection,host range,inhibitor,disinfectant-tolerance,etc.were investigated.In order to prevent the contamination from phage P260,anti-phage 260 strains were screened by spontaneous mutations.As results,titer of P260 reached 3.45 x 1010 PFU/mL,it had a certain host specificity,and not sensitive to Bacillus coagulans and Bacillus pumilus.P260 easily lost its activity at high temperature,i.e.,not heat-resistant,and its survival rate under 70℃ condition for 15 min decreased significantly.ClO2 in 25 mg/L had a strong exterminate effect on P260.The 0.5％ ammonium oxalate and ammonium citrate presented inhibition to P260.Optimal multiplicity of infection of phage P260 to the host was 0.01,one step growth curve showed that the incubation period was 15 min and the lytic stage started after 45 min.An anti-phage strain ZF-260 was obtained by spontaneous mutation.ZF-260 was not affected when adding phage P260,which indicated that ZF-260 was an excellent strain of anti-phage P260.In conclusion,the biological characteristics of phage P260 was studied,and ZF-260 with stable genetic and high viable cell count was obtained.