| Lactic acid bacteria(LAB)have been widely used in fermentation industry,such as milk,meat,vegetables and feedstuffs.However,the infection of Lactobacillus phages would result in delaying of fermentation process or terminating completely,thereby making the product quality reduced,which caused fermentation failure and serious economic losses.In this study,the phages were isolated from dairy products and kimchi,its main biological characteristics and influence of different physical and chemical factors on the fermentation process were analyzed.And many experimental datas and theories were provided in order to control the phage pollution in the fermentation production,to protect the industrial strains and to formulate a series of effective preventive and control measures.Three virulent Lactobacillus phages were isolated from dairy products and kimchi by using spot test and double-layer agar plate method,one strong-lysis L.casei ATCC 393 strain named as Lc,one strong-lysis L.Pentosus KLDS 1.0413 phage named as Lpen and one strong-lysis L.brevis ATCC 367 phage named as Lbre and studied their major biological characteristics.By electron microscopy our study found that three phages were consisted of polyhedron head,but Lc and Lpen were composed of heterogeneous flat end,while Lbre consisted of contraction tail.Phage Lc and Lpen were classified as Siphoviridae Branch,B1 Class;Lbre was classified as Myoviridae Branch,A1 Class,which based on morphological analysis.The genomes of three phages were extracted and treated with restriction endonuclease action.Lc and Lpen genome contained heterogeneous flat ends,belonged to the pac-type,but Lbre contained cohesive ends,the packaging mechanism was the cos-type.So it One-step growth curve of phages showed the latency of Lc、Lpen、Lbre were 60 min、45 min and 150 min,the burst period of of Lc、Lpen、Lbre were 45 min、90 min and 105 min,the burst size of Lc、Lpen、Lbre were 47 PFU/cell、30 PFU/cell and 24 PFU/cell,respectively.Phage Lc and Lbre had a host-specific character,while Lpen host spectrum was broader.By analyzing the influence of physical and chemical factors on the phages in the external environment(-20℃,0℃,20 ℃,32 ℃,37 ℃,42℃),phages were less sensitive to external environmental temperature.As the temperature increased,the phages activity was slightly reduced.Different concentrations of ethanol and isopropanol showed different resistance to phages,less than a live phage could be detected when Lc、Lpen and Lbre treated with 25%、35%and 10%ethanol for 40 min、30 min and 10 min,respectively;less than a live phage could be detected when Lc、Lpen and Lbre treated with 25%isopropanol for 20 min、50 min and 20 min,respectively.Other disinfectant 0.05%bromogeramine,0.05%KMn04 and 0.1%formaldehyde were commonly used,phages were inactivated after treating 5 min.A lower tolerance to UV,the titer of phage decreased by 2-4 orders of magnitude when Lc、Lpen and Lbre treated with UV for 1h.By analyzing the influence of different physical and chemical factors on phage and host bacteria in fermentation medium,the phage was highly sensitive to high temperature,Lc、pen、Lbre can be inactivated when treated at 56℃ for 20 min、45 min、10 min,respectively.Phage has strong resistance to acid and alkali,the detection results of phage stability showed that Lc、Lpen and Lbre maintained their viability within the range of pH 3-13,but the host bacteria are eosinophilic,their activity was relatively stable in the range of pH 4-9.The results of the influence of Ca2+ or Mg2+ were showed that host bacteria can grow normally in MCM,MRS-Ca,MRS-Mg and MRS medium,but the phage titer decreased by 2 to 4 orders of magnitude when phage Lc,Lpen and Lbre were not added Ca2+ and/or Mg2+.Within the standard range of T-20 addition,the host bacteria grew normally,but phage Lc titer decreased by 3-4 orders of magnitude via treating 2%T-20;phage Lpen was inactivated when they were added 2%T-20;Lbre had a lower tolerance to T-20.Different concentrations of NaCl as a food additive,the host bacteria could grow normally during the fermentation process,phage Lc was treated with 5%NaCl,its potency decreased 2 orders of magnitude;and the phage Lpen and Lbre can be inactivated when treated with 5%NaCl.In this study,three virulent lactobacilli phages were isolated and their main biological characteristics、the influence of different physical and chemical factors on the fermentation process were studied.The study aims to provide a theoretical basis for protecting industrial strains,establishing effective control phage contamination measures,and for in-depth understanding interactions of phage-host. |
