Analysis Of Dominant Bacteria In Chilled Pork And Evaluation Of Bacteriophage Antiseptic Effect

Chilled fresh meat has become the main direction of the development of meat industry and the mainstream of raw meat consumption.However,its supply chain,including breeding,slaughtering,segmentation,processing,storage and transportation,is vulnerable to microbial contamination,leading to food spoilage and food safety problems.The microorganisms that cause the quality of chilled meat and food safety problems mainly include spoilage bacteria and food borne pathogenic bacteria,and phage is an ideal choice for disinfection and antiseptic biological agents because of its specific,non-toxic and harmless action mode to the target bacteria.In this study chilled fresh pork had been selected as the research object to clarify the evolution rule of spoilage bacteria and pathogenic bacteria under cold chain conditions.The dominant spoilage bacteria and their lytic phages were isolated and purified,and the bacteriostatic and antiseptic tests were carried out by optimized phages,which provided a theoretical basis for the application of phages in food preservation.In this study,four brands of chilled pork samples from Shandong Province were stored at 4?and 8?for 0,1,3 and 5 days respectively.The microorganism of pork samples with different cold storage time were sequenced by high-throughput sequencing technology of metagenomics.The diversity and structural composition of the microbial community were analyzed,and the evolution law of pork microbial community at low temperature was studied.At the same time,the dominant spoilage bacteria and pathogenic bacteria were obtained by traditional methods.For pathogenic bacteria,a total of 175 samples of slaughtering tools,slaughterers,water samples,body surface and meat samples were collected from a food factory,epidemiological investigation was carried out in slaughtering chain to analyze the epidemic characteristics and pollution status.Listeria monocytogenes were identified by PCR identification and PCR product sequencing analysis,serotypes and toxin genotypes of L.monocytogenes were detected to clarify their lineages,homology differences and virulence genes;for dominant spoilage bacteria,the corresponding phages were screened and isolated by double-layer plate method and co-culture method,and the bacteriostatic and antiseptic experiments of cold fresh meat were carried out based on this model.In terms of pathogenic bacteria,high-throughput sequencing analysis showed that the diversity of bacteria increased with the extension of cold storage time,and Listeria was the dominant cold chain growth pathogen with potential pathogenic risk.76 strains of Listeria were isolated from the slaughtering chain,in which 13 strains of Listeria monocytogenes.The contamination rate was 7.4%.Lineage II(1/2a,1/2C,3a and 3C)was the main serotype,accounting for 76.92%,and each strain of L.monocytogenes contained at least one virulence gene.The analysis of protophages in L.monocytogenes genome provides a new research idea for the research and development of phage products for the prevention and control of L.monocytogenes.The research of L.monocytogenes lays a foundation for exploring the relationship between pathogenicity,toxicity and drug resistance and eliminating potential safety hazards.In terms of spoilage bacteria,the results of high-throughput sequencing showed that there was no significant difference in the dominant bacterial species of different brands of chilled pork samples,but there was a big difference in the bacterial structure.The distribution of dominant bacteria in the same sample at different time points was similar,and the growth of dominant bacteria in different low temperature conditions was different.In general,Firmicutes had more growth advantages,Aeromonas and aroma bacteria were the dominant spoilage bacteria.The most isolated Aeromonas was selected(separation rate was 12%)as the host bacteria to isolate lytic phages.The results showed that the cleavage rates of two phages were high,with the titers higher than 10~8pfu/m L,the optimal infection complex number 1 and 0.1,the optimal growth temperature 37?,the optimal p H value wide,and the good thermal stability.The results showed that the time of spoilage was prolonged by 1-2days,the total number of colonies was reduced,and the trend of p H value from decrease to increase was slowed down in the chilled fresh pork treated by the phage PAs6.It indicated that the phage had a certain potential in Chilled Pork preservative,which provided a new theoretical basis and technical means for the development of a truly safe,green and pollution-free natural preservative.