乳酸菌噬菌体的分离及生物学特性鉴定

Isolation and Biological Characterization of Lactobacillus Phage

为了解乳酸菌噬菌体的生物学特性,并为制定乳酸菌发酵生产过程中噬菌体污染的防控措施提供试验数据和参考,本研究通过斑点试验和双层琼脂平板法,以干酪乳杆菌(L.casei)ATCC 393、戊糖乳杆菌(L.pentosus)KLDS 1.0413、短小乳杆菌(L.brevis)ATCC 367为指示菌从乳制品、泡菜样品中分离乳酸菌噬菌体,通过透射电镜观察噬菌体形态,噬菌体基因组限制性内切酶作用分析其包装机制,并进行宿主范围和一步生长曲线的测定,SDS-PAGE分析噬菌体结构蛋白,对获得的噬菌体进行生物学特性鉴定。结果显示,分离获得3株烈性乳酸菌噬菌体,依次命名为Lc、Lpen和Lbre。电镜结果显示,噬菌体Lc、Lpen均由多面体头部和非收缩性尾部组成,而噬菌体Lbre由多面体头部和收缩性尾部组成。根据形态学分析,Lc和Lpen属于长尾噬菌体科(Siphoviridae),B1类,Lbre属于肌尾噬菌体科(Myoviridae),A1类。噬菌体基因组经限制性内切酶作用后,核酸电泳结果显示,噬菌体Lc、Lpen基因组均为异质平末端,其包装机制属满头包装,即pac-型,而Lbre含有黏性末端,其包装机制属于cos-型。宿主范围测定结果显示,噬菌体Lc、Lbre对宿主专一,而Lpen宿主谱较广。一步生长曲线显示,噬菌体Lc、Lpen和Lbre潜伏期分别为60、45和150 min,裂解期分别为45、90和105 min,裂解量分别为47、24和30 PFU/cell。SDS-PAGE分析显示,噬菌体Lc结构蛋白有7个,主要结构蛋白分子质量约为50 ku;噬菌体Lpen和Lbre结构蛋白均有5个,主要结构蛋白分子质量分别约为55和50 ku。综上,本研究分离获得3株乳酸菌烈性噬菌体,并对其主要生物学特性进行鉴定,对了解乳酸菌噬菌体及后续乳酸菌噬菌体污染的防治提供了试验数据和理论依据。

In order to understand the biological characteristics of Lactobacillus phage,and provide experimental data and reference for the prevention and control of phage pollution during fermentation production,in this study,Lactobacillus phages were isolated from dairy products and kimchi samples by spot test and double-layer agar plate method,L.casei ATCC 393,L.pentosus KLDS 1.0413 and L.brevis ATCC 367 were used as indicator bacteria.The morphology of the phage was observed by transmission electron microscopy,and the packaging mechanism of the phage was analyzed by the restriction enzyme activity of the phage genome,the host range and one-step growth curve were measured,the structural proteins of the phage were analyzed by SDS-PAGE to analyse the biological characteristics of the obtained phage.The results showed that three virulent Lactobacillus phages were isolated and named as Lc,Lpen and Lbre,respectively.The results of electron microscopy showed that phage Lc and Lpen were consisted of polyhedral heads and non-contractive tails,while phage Lbre was consisted of polyhedral heads and contractile tails.According to morphology analysis,Lc and Lpen belonged to the Siphoviridae family,B1 category,and Lbre belonged to the Myoviridae family,A1 category.The genomes of phages were extracted and subjected to restriction endonuclease.The results of nucleic acid electrophoresis showed that the genomes of phage Lc and Lpen both had heterogeneous blunt ends,and the packaging mechanism was full-head packaging,namely pac-type,while Lbre contained stickiness end,the packaging mechanism belonged to cos-type.The phage Lc and Lbre were host-specific,and the host spectrum of phage Lpen was broader.The one-step growth curve showed that the latent period of Lc,Lpen and Lbre were 60,45 and 150 min respectively,the burst period were 45,90 and 105 min respectively,and the burst size were 47,24 and 30 PFU/cell respectively.SDS-PAGE testing indicated that phage Lc had 7 structural proteins,while phage Lpen,Lbre had 5 structural proteins,their protein molecular weight were about 50,55 and 50 ku,respectively.In conclusion,three virulent Lactobacillus phages were isolated and their biological characteristics were indentified,which provided important experimental data and theoretical basis for understanding the prevention and control of virulent Lactobacillus phages contamination.