Biological Characteristics And Inactivation Methods Of Limosilactobacillus Fermentum Phage

Lysogenic phages are widely distributed in Lactobacillus strains,which are hidden and difficult to detect,and once they are induced to become virulent phages,they may lead to fermentation failures and economic losses.Therefore,in this study,Limosilactobacillus fermentum IMAU32579 was used to obtain Limosilactobacillus fermentum phage through mitomycin C induction,and its tolerance,adsorption characteristics,inactivation conditions and whole genome sequence were studied to provide data and theoretical supports for understanding the biological characteristics of Lactobacillus phage and constructing phage prevention and control measures.The results showed that:（1）A Limosilactobacillus fermentum phage was successfully obtained by inducing Limosilactobacillus fermentum IMAU32579 with 1.9μg/m L mitomycin C,and was named LFP-VB1.The electron microscopy showed that this phage belonged to family Siphoviridae,with an isometric capsid of 62.86±2.01 nm,and a non contractile tail（approximately 259.26±2.00 nm long and 12.52±2.01 nm wide）.The optimal multiplicity of infection of this phage was 0.01.Its latent period was 30 min,the burst time was 60 min,burst size was 148.03±2.65 PFU/m L.（2）Temperature and p H had a significant effect on the survival rate of phage LFP-VB1.When the temperature was increased to 50℃,the survival rate decreased to78.36%after 30 min treatment;When the p H value is 2,it can be completely inactivated within 30 min.（3）Temperature,p H value,Ca²⁺or Mg²⁺cations and chloramphenicol had little effect on the adsorption of phage LFP-VB1.Under all temperature conditions（0,10,20,30,37,42,50℃）,the adsorption rate of phage could reach 90%;in the p H range of 4～11,the adsorption rate of phage could maintain 95%.The adsorption capacity was not significantly affected by the addition of Ca²⁺or Mg²⁺or chloramphenicol.（4）The phage could not be completely inactivated by the treatment with ethanol or sodium hypochlorite for 60 min.Treatment with 75%ethanol and 800 mg/L sodium hypochlorite for 60 min resulted in a 4.71 and 6.29 logarithmic reduction,respectively.Phage LFP-VB1 was extremely sensitive to high temperature.4 min of treatment at 63℃could completely inactivate it.（5）The genome of phage LFP-VB1 is a double-stranded DNA with a length of 43789bp and a G+C content of 46.06%,with 53 protein-coding regions.In comparison with the three published phage of Limosilactobacillus fermentum（LF1,phi PYB5 and JNU＿P5）in NCBI database,synteny analysis showed that phage LFP-VB1 had chromosomal deficiency,insertion,and inversion.