| Salmonella is an important food-borne gram-negative intestinal pathogen that mainly threatens young birds under 3 weeks of age.The high mortality rate after infection has affected the development of chicken farming and public health safety seriously.Chinese chicken industry has caused huge economic losses by Salmonellosis.Although the chemical environment bactericide has a good bactericidal effect,its longterm residue will cause damage to human health.Phages are bacterial viruses and natural enemies of bacteria.The bacteriophage has high specificity of host and kills bacteria without harming the human body.The bacteriophage is widely found in the ocean and land,and its existence is found in various environmental conditions.Its environmental adaptability and characteristics make the bacteriophage have the potential for development as an environmental bactericidal biological agent.The phage cocktail preparation is a mixture of various bacteriophages with different functions.It is artificially synthesized according to the purpose of broadening the bacteriophage’s lysis spectrum and enhancing the bacteriophage’s bactericidal ability.In this study,mitomycin C(MMC)was used to induce and purify the salmonella phage from the salmonella prephage gene successfully,and preliminary analysis of its biological characteristics,the biological characteristics of the bacteriophage determine its ability to adapt to the living environment,invade the host bacteria ability and so on.Due to the disadvantage of a single bacteriophage with a narrow bacteriophage spectrum,its application is limited in many ways.In this research,the newly mixed bacteriophage was mixed with the laboratory-environmentally isolated lytic bacteriophage pSQC73,which was applied to the development of salmonella bacteriophage preparations in vitro.Related research is as follows:1.The induction and purification of salmonella phageIn this research,mitomycin C(MMC)reagent method was used to induce salmonella of different serotypes,and 16 salmonella phages were induced successfully.These 16 strains of salmonella phages were purified three times by the double-layer plate method,respectively,and 15 strains of phage capable of forming a stable plaque morphology were obtained,with an induction rate of 31.91%.After the determination,the titers were all above 108pfu/ml,and the titers were higher.Observation of the morphology of phage by transmission electron microscope revealed that the induced phages included long-tailed phages and short-tailed phages.The successful induction and purification of salmonella bacteriophages provided important biomaterials for the later development of environmental bactericidal preparations of salmonella bacteriophages.2.Analysis of biological characteristics of salmonella bacteriophageThe purpose of this research was to investigate the biological characteristics of salmonella phages under different conditions.First,using 46 strains of salmonella stored in the laboratory to determine the phage profile of 16(and a total of 16 unpurified phage)strains of salmonella phages,it was found that pP14 had a broader lysis profile.Subsequently,pP14 was subjected to thermal stability test and acid-base stability test,and found that its high temperature resistance and acid-base resistance are strong:the mixed salmonella bacteriophage preparation can be completely destroyed when heated at 80℃ for 1 h and when the pH is less than 3 or higher than 12.Ethanol inactivation experiments showed that the phage pP14 was more sensitive to ethanol,and 50%ethanol could completely inactivate it.At the same time,the titers of long-tailed pP14 and short-tailed pG8 were measured for six months,and the potency of the lytic bacteriophage pSQC73 was measured two years later.It was found that the titers of the three phages remained stable on the order of magnitude.The storage effect is good at four degrees,and it is not easy to decay.In addition,according to the bacteriostatic experiment,it can be seen that the bacteriostatic time of the phages pP14 and pSQC73 can reach about 6 hours.From the above characteristic analysis,it can be inferred that the properties of the two phages are relatively stable,the environment has less influence on them,and they have the potential to be the environmental bactericides.3.In vitro application and effect evaluation of Salmonella phage cocktail preparationThe purpose of this research was to investigate the in vitro bactericidal effect of salmonella phage cocktail preparations and to make up for the limitations of a single phage host spectrum.This experiment shows that the mixed preparation of salmonella phage pP14 and pSQC73 can kill salmonella typhimurium and salmonella pullorum in a certain period of time.The cocktail preparation can kill more than 90%of bacteria within 1 h,and the high-efficiency sterilization effect is sustainable 3 h,continuous sterilization can reach 6 h,experiments show that the lower the bacterial concentration,the better the bacteriostatic effect.The results show that the mixed preparation has a good killing effect on the salmonella serotypes common in livestock and poultry breeding environments,and has a long maintenance time.It is easy to remove,and has the characteristics of continuous bacteriostasis.It has the potential to be developed into an environmental disinfectant that can be used in production. |
