Study On The Biofilm Formation Characteristics Of Contaminated Bacteria And Genes Expression Of Pseudomonas In Chilled Pork

Chilled pork is nutritious and susceptible to microbial contamination during production and distribution.The surfaces of moist and nutrient-rich utensils in processing chain provide good environment for microbial reproduction and biofilm formation.After biofilm formation,bacteria become more adaptable to the environment,which causes meat spoilage,cross contamination,foodborne diseases prevalence and food poisoning.Pseudomonas is one of the dominant spoilage bacteria that causes the deterioration of chilled pork.Pseudomonas fluorescens and Pseudomonas putida can rapidly grow and proliferate at low temperaturs,while having strong capacities of biofilm formation,which are potential threat to chilled pork industry.Study used the culture method combined with test tubes and slides,and the quantitative biofilm method of crystal violet staining to investigate the biofilm formation laws of mixed flora on the surfaces of glass and stainless steel in chilled pork at 4℃,15℃ and 26℃.While analyzing the diversity of membrane microorganisms with metagenomic sequencing technology,isolating and identifying biofilm-forming microbes with physiological and biochemical identification and 16S rRNA sequencing technology,screening bacteria with strong biofilm-forming ability by 96-well plate method,and detecting correlations between genes expression and biofilm formation of Pseudomonas with real-time fluorescene quantitative polymerase chain reaction.It is significant to provide theoretical basis and guidance for preventing and controlling bacterial biofilm contamination in the production of chilled pork,and to ensure the quality and safety of chilled pork.The results are as follows:(1)When bacterial concentration in the eluent of chilled pork was 10~5CFU/mL,at 4℃,15℃and 26℃,the mature stage of bacterial biofilm was reached on 7th d,4th d and 0.5th d,respectively.At different tempretures,the quantity of biofilm formed on the surfaces of stainless steel was greater than that of glass,and there were significant differences in biofilm formation(p<0.05).(2)By separation and purification of plates,from the biofilm mixed flora in chilled pork,22 strains with strong biofilm-forming abilities,including Escherichia coli,Hafnia,Citrobacter,Pseudomonas and so on were isolated and identified,among which,the dominant bacteria were Hafnia and Escherichia coli.The microorganism diversity analysis of macrogenomic technology detected 61 species,among them,Halomonas,Pseudomonas and Acinetobacter were dominant bacteria,the three species were up to 90%.The abundance of Pseudomonas rapidly increased by 53.6%during the formation of biofilm from 12 h to 24 h at 15℃,which accounted for a significant advantage.At the same time,three Pseudomonas isolates with strong biofilm-forming ability were selected.(3)The Pseudomonas fluorescens with strong biofilm-forming ability was selected for membrane formation.At 4℃,15℃and 26℃,the amount of its biofilm gradually decreased with increasing temperature.The biofilm on surfaces of stainless steel peaked at 8th d,3th d and 0.5th d at different temperatures.On the surfaces of glass,biofilm reached maturity at different temperatures at 10th d,4th d and 0.5th d respectively.(4)Real-time quantitative PCR(RT-qPCR)was used to detect related genes expression of Pseudomonas in free and biofilm state.Free and biofilm cells were collected for RT-qPCR to detect biofilm related genes rpoZ(RNA polymerase gene),algR(alginate gene)and lapA(adhesin gene),quorum-sensing gene ppuR and internal reference gene atpC at 26℃,12 h.The expression of lapA,algR,rpoZ and ppuR genes in Pseudomonas mature biofilms increased significantly,and different biofilm-forming surfaces had significant influences on the expression of related genes.(5)RT-qPCR was used to detect related genes expression of Pseudomonas and Proteus in mixed biofilm-forming state.The mixed cells were collected for RT-qPCR and the rpoZ,algR,lapA and ppuR genes(internal reference gene atpC)were detected.Compared with single Pseudomonas,the expression of lapA,algR and rpoZ of Pseudomonas in mixed bacteria at free and biofilm states were significantly decreased,but the ppuR gene had different expression results.