The Isolation,Identification And Purification Of Phage JS25 And Its Bacteriostasis In Milk

Milk is a kind of nutritive food,its quality is related to human health. The raw milk is often contaminanted by pathogenic bacteria because of the prevalence of bovine mastitis.Staphylococcus aureus is a kind of important pathogenic bacterium of bovine mastitis.Antibiotics application caused a remarkable increase in antibiotic residuce and the multiple drug resistance.It is necessary to find a new solution to control the pathogenic bacteria in milk. As a potential microbicides in food, phage has become a research hotspot.In this study,a lytic bacteriophage named vB_SauM_JS25(JS25) was isolated from a dairy farm in Jiangsu.We characterized its biological properity,tested the bactericidal effect in virro,and developed the purification techniques,and studied the application effect in milk. The details were listed listed below:1.The isolation and identification of phage JS25A new bacteriophage named vB_SauM_JS25(JS25) was isolation with S. aureus ATCC6538 as host strain.The phage was morphologically classified as Myoviridae and its genetic material was dsDNA.Its latent period,rise period,and average burst size were approximately 10min,60min and 21PFU/cell,respectively.The adsorption time and adsorption rate were 5min and 92%, respectively. Phage JS25 was able to remain active in a temperature range from 37℃ to 50℃ and in a pH range between 6 and 9.1t had about fifteen structural proteins and its genome size was about 128kb.lt was proved to have broad host ranges.2.The optimal enrichment conditions of phage JS25The optimal enrichment conditions of phage JS25 were investigated with respect to phage titer by culture temperature,culture time,incubate time and MOI.When cultivating temperature of 37℃, training time is 6h, incubation time for 5 min, MOI for 100,the largest number of progeny phages were got after making phage enrichment. 3.The optimal purication conditions of phage JS25The optimal purication conditions of phage JS25 were investigated with respect to the yield of phage, removal rate of protein and enterotoxin by PEG,ultracentrifugation and filtration.The conclusion is that:PEG6000 had more advantages than PEG8000 in phage purification,but the ultracentrifugation was the best of all, in other words, afer filter,the bacteriophage mixture will achieve the best results by ultracentrifugation in speed of 35000×g, time for 1.5 h.4.The bacteriostatic action of phage JS25 in the milkStudied on the relationship of the four factors on bacteriostatic rate of phage in the milk:phage titer,storage temperature, milk with different concentrations of nutrients and fat. Sterile water was served as a control group. The results show that phage JS25 can restrain bacteria growth in the milk by high titer of phage or cryogenic storage.