Diversity And Control Of Biofilm-forming Bacterial Flora In Food Processing System And The Detection Of G~+ Bacteria Using Nisin-EGFP Probe

Biofilms are aggregation of bacterial flora and their extracellular polymeric substances;comparing to their planktonic form,this adaptation made bacteria had more resistant abilities to the environment changes and the antimicrobial substances.The formation of biofilm may give rise to many negative effects,including increasing the risk of food-borne diseases.In food processing system,the formation of biofilms,which is favored by the accumulation of food residue,impropriate sanitation,wet circumstances,the erosion of the surface and interior of the food processing equipment would foster the contamination of food products and lead to a great threat to food safety.The present work focused on analyzing the bacterial community,biological control and detection of the biofilm in order to solve the above problem.To analyze the bacterial flora in food processing system,biofilm samples were collected from the surface and interior of the equipments in milk and meat industry,their DNA were extracted,qualified,broke into pieces,and then sequencing using the Illumina platform through bacterial 16S rRNA V4 regions.The sequencing clean data were analyzed by bioinformatic techniques.Results indicated that at plylum level,Proteobacteria,Firmicutes,and Bacteroides have the highest abundance in the biofilm community in milk industry,the other phyla were Acidobacteria,Actinobacteria,Cyanobacteria_Chloroplast,Deinococcus-Thermus,Firmicutes,Planctomycetes;Firmicutes and Proteobacteria have the highest abundance in the biofilm community in meat industry,the other phyla were Acidobacteria,Actinobacteria,Crenachaoeta,Fusobacteria,Planctomycetes,Syneristetes,Verrucomicrobia.The strains in each biofilm samples were isolated cultured-dependent,identified by molecular biological method and tested by crystal semi-quantified assay to define their biofilm formation abilities.Results revealed that Corynebacterium callunae,a strong biofilm formation ability bacteria,Acinetobacter baumannii,Acinetobacter junii,Enterococcus faecalis and Stenotrophomonas maltophilia,four moderate biofilm formation ability bacteria,were isolated in the biofilm sample of milk industry;Enterobacteriaceae bacterium and Enterococcus faecalis,strong biofilm formation ability bacteria,Enterococcus faecalis and Lactococcus lactis,moderated biofilm formation bacteria were isolated in the biofilm sample of meat industry.To analyze the relationship between the ycbR gene and the adherence of E.coli,ycbR gene was amplified using the DNA of E.coli O157:H7 as the template through the primers designed according to genebank,the amplified ycbR gene was then cloned to pET28a vector.Recombinant YCBR protein was expressed and purified.Purified protein was subcutaneously injected to rabbits to prepare antisera.The titer of the sera was 1:800,1:800,and 1:1600 under 0.5,2,and 5g/mL antigen consistency.The results of an adherence assay in the presence of anti-YCBR-His antibodies indicated that antibodies blocked the adherence of E.coli O157:H7 to HEp-2 cells.The reductions of the adherence were 15%,35%,and 75%at 1:50,1:20,and 1:10 dilution of the anti-YCBR.To investigate the biological control strategy of the biofilm adapted in the food processing equipment,the Biofilm formation inhibition of PA003 against Escherichia coli,Salmonella enterica serovar Typhimurium,Staphylococcus aureus,and Listeria monocytogeneson stainless steel,polyvinyl chloride and glass slideswere conducted in terms of exclusion,displacement and competition.The numbers of biofilm cells of these pathogens on stainless steel,polyvinyl chloride and glass slides coupons were effectively reduced at approximately 4 log CFU/coupon respectively.Considering the binding ability of nisin to G⁺bacteria and the stable fluorescent ability of EGFP protein,a fluorescent recombinant Nisin-EGFP protein probe was constructed using Nisin as the receptor and EGFP as the fluorophore,in order to detect gram-positive bacteria.The nisin and egfp gene was amplified separately according to the sequence published in Gene Bank using unique primers.pET42a-spaN-egfp without protein linker beween the nisin and EGFP and pET28b-spaN-egfp with a His-Met protein linker between the two proteins were constructed.The recombinant nisin-EGFP probe was expressed and then purified by Ni-NTA.The binding specificity of the recombinant protein was performed on Listeria.monocytogenes,Staphylococcus aureus,and Micrococcus luteus,and then detected under fluorescent microscopic.The fluorescent results evaluated that the Nisin-EGFP probe could examine the Gram-positive bacteria at a 10⁸ CFU mL^-1.The present work enlightened the understangding of the biofilm forming in food processing system through the investigation of the biofilm forming mechanism and the method of detection,and provided an evidence for controlling the biofilm in food processing system and the food safty.