Screening Of Pseudomonas Bacteriophage And Its Application In Chilled Mutton

In recent years,the Mutton Industry in China has developed rapidly,but the chill ed mutton products are prone to corruption and deterioration in the process of transpor tation and storage,causing great economic losses.Pseudomonas is the dominant spoila ge bacteria of chilled mutton in the condition of aerobic cold storage.As a green,safe and effective antibacterial agent,phage can inhibit the growth and propagation of Pseu domonas and effectively delay the spoilage of chilled mutton.In this study,the biologi cal characteristics,genomics and preliminary application in chilled mutton of Pseudom onas were studied,which laid a foundation for the research and application of phage i n food field.The results are as follows:Isolate and screen Pseudomonas from the chilled mutton by plate coating method.Through colony morphology observation,physiological and biochemical test and 16S r DNA sequence determination,a total of 24 Pseudomonas were identified.6 phages(PSJ-1,PSY-1,vB PS-1,vB PS-2,vB PS-3,vB PS-4)were isolated from 24 isolates of Pseudomonas and two standard strains Pseudomonas aeruginosa PAO1 and Pseudo monas aeruginosa PAK.Two phages with the strongest cleavage ability and the widest cleavage spectrum were selected to study their biological characteristics.Transmission electron microscop y showed that phage PSY-1 belongs to Caudovirales and Myoviridae.Phage PSJ-1 bel ongs to Caudovirales and Siphoviridae.The optimal MOI of phage PSY-1 is 0.01and the optimal MOI of phage PSJ-1 is 0.1.The one-step growth curve of phage PSY-1 shows that its latent is about 10 min utes,growing period of 60 minutes,and the outbreak amount is 213 PFU/cell.The o ne-step growth curve of phage PSY-1 showed that the latent period is about 20 minut es,growing period of 30 minutes,and the outbreak amount is 32 PFU/cell.The bact eriophage PSY-1 can maintain its lytic activity under the conditions of 90? 1h and pH 2?10.The bacteriophage PSJ-1 can maintain its cleavage activity under the con ditions of 70? 1h and pH range of 2?11.The results of phage genome sequencing showed that the phage PSY-1 genome w as a linear double-stranded DNA,with a size of 92663 bp and GC content of 49%.14 tRNA-related genes were detected.Gene annotation using GeneMark and softberry showed that PSY-1 genome contains 169 Open Reading Frame(ORF),of which 38 co ding sequences predicted its function,and the remaining 131 were unknown coding se quences.Using RNA ligase nucleotide sequence to map the phylogenetic tree,it was f ound that the genome of phage PSY-1 was closely related to Pseudomonas phage vB_PaM_EPA1.The bacteriophage PSJ-1 can crack three(P5,P12,P18)different Pseudomonas st rains,has good acid-base resistance,and can survive in a wide range of temperatures.Therefore,choose the bacteriophage PSJ-1 for Bacteriostatic experiment of chilled mut ton.The results of bacteriostasis experiments showed that Pseudomonas bacteriophage PSJ-1 can effectively reduce the number of host bacteria and prolong the storage perio d of chilled mutton.