Isolation And Identification Of E.coil O157:H7 Phages And Preparation And Application Of Its Microcapsule

O157:H7 is an enterohemorrhagic Escherichia coli（EHEC）that can cause hemorrhagic diarrhea,hemorrhagic colitis（HC）,hemolytic uremic syndrome（HUS）and thrombocytopenic purpura（TTP）,which poses a threat to global public health and food safety.Antibiotics are usually the first choice for the treatment of bacterial diseases.However,due to excessive and inappropriate use of antibiotics in recent years,the problem of antibiotic resistance has become more and more serious.Therefore,it is urgent to find an effective alternative to antibiotics.Phage is a kind of bacterial virus,which has the advantages of strong specificity,self replication and proliferation,safety,no residue and abundant source,and is one of the ideal substitutes for antibiotics.Phage has been effective in the prevention and treatment of bacterial diseases in animals,but the current problem is the lack of suitable dosage forms.Especially in the treatment of animal intestinal diseases,the activity of oral phage is easily destroyed by gastric acid,digestive enzymes and bile salts,resulting in the loss of antibacterial activity.Microcapsulation technology uses polymer materials to wrap the core（solid,liquid or gas）to form a dense barrier around the core to reduce its reaction with the external environment.By microencapsulation,the physical and chemical properties of active substances can be improved,including improvement of stability,prevention or reduction of the inactivation,and extension of the storage time.Recently,there have been many reports on microencapsulated phage,proving the effectiveness and feasibility of phage microcapsulation.The main purpose of this experiment is to isolate phage which has strong lysis effect on E.coli O157:H7,determine its biological characteristics and prepare microencapsulated phage,so as to solve the problem that phage is easy to be destroyed by gastric fluid and enzyme after oral administration,improve the storage stability of phage,and provide reference for phage therapy,especially for the treatment of gastrointestinal diseases.1 Isolation and identification of phage and study of its biological characteristicsE.coli O157:H7 preserved in the laboratory was selected as the host bacteria,and five phages were isolated and purified from 200 fecal samples collected from a chicken farm in Nanjing.Phage PNJ1901 had a strong lytic effect on E.coli O157:H7.The biological characteristics such as morphology,optimal multiple infection（MOI）,thermal stability,p H stability and one-step growth curve of phage PNJ1901 was investigated.The results showed that phage PNJ1901 belonged to Myoviridae family.The edge of the plaque was transparent and neat,with the diameter of about 1 mm.MOI was 1.Phage PNJ1901 survived well when the temperature is below 60℃,however,the titer decreased significantly at 70℃,and the phage was inactivated after50 min treatment.Phage PNJ1901 was resistant to p H 4-11.The one step growth curve showed that the latent period of phage PNJ1901 was 8 min,and the burst size was 74.2 Preparation and application of microencapsulated phageIn order to improve the stability of phage in gastrointestinal tract,sodium alginate and Ca Cl₂were used as carriers to microencapsulate phage PNJ1901.The characteristics of microencapsulated phage in vitro were studied,and the therapeutic effect of microencapsulated phage in vivo was evaluated.The results showed that the encapsulation efficiency of microencapsulated phage was 77.767±2.636%,and the size was 1.759±0.013 mm.Compared with free phage,the resistance to simulated gastrointestinal conditions and stability of storage of microencapsulated phage was significantly improved:Free phage PNJ1901 was inactivated after incubation in SGF with p H 2 and 2.4 for 15 and 30 min,respectively,while microencapsulated phage PNJ1901 only decreased by 0.528±0.053 log₁₀PFU/g and 0.242±0.049 log₁₀PFU/g after incubation in SGF with p H 2 and 2.4 for 240 min.The titer of free phage decreased by 0.915±0.022 log₁₀PFU/m L and 1.787±0.029 log₁₀PFU/m L after incubation in 1%and 2%bile salt for 180 min,respectively,while that of microencapsulated phage only decreased by 0.012±0.012 log₁₀PFU/g and0.099±0.058 log₁₀PFU/g.The release rate of microencapsulated phage in SIF reached70.313%and 90.104%after 6 h and 24 h incubation.After 15 weeks of storage at 4℃,the titer of free phage decreased by 2.519±0.042 log₁₀PFU/m L,while the microencapsulated phage only decreased by 1.211±0.146 log₁₀PFU/g.In addition,the therapeutic effect of microencapsulated phage is better than that of free phage.Therefore,phage microencapsulation has a potential to be an effective form of phage therapy.