Antibacterial Effects And Mechanism Of Plantaricin DLP1 On Pseudomonas Fluorescens

Pseudomonas fluorescens was a psychrotrophs spoilage microorganism that caused spoilage of aquatic products,dairy products and meat products.Bacteriocin is a secondary metabolite produced by lactic acid bacteria(LAB)that had antagonistic effects on other microorganisms.It had the advantages of safety,stability,high efficiency and broad spectrum,and was widely used in the field of food bio-preservation.In the study,bacteriocin-producing LAB with inhibitory activity to Pseudomonas were screened from natural ecological environment(traditional fermented food,the intestine of fish).In additional,the process of isolation,purification and identification of bacteriocin were analyzed.Antibacterial mechanism of bacteriocin was studied by three aspects of cell morphology,DNA transformation and protein expression.Finally,the feasibility of bacteriocin bio-preservative was explored.The main results were as followed:(1)146 strains LAB with inhibitory activity to Pseudomonas were screened from 639 strains LAB by Oxford cup agar well diffusion method.One bacteriocin-producing strain DLP1 was obtained from 146 LAB strains.Strain DLP1,isolated from turbot intestine,was identified as Lactobacillus plantarum by morphological,physiological and biochemical characteristics and 16S rDNA sequence analysis.Thus,this strain was designated as L.plantarum DLP1.(2)Plantaricin DLP1,formed by L.plantarum DLP1,was purified by ethyl acetate extraction followed by ultrafiltration,Sephadex G25 chromatography and high-performance liquid chromatography.The molecular mass of plantaricin DLP1 was determined as 1537 Da by Tricine-SDS-PAGE and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF-MS)analysis.The amino acid sequence of plantaricin DLP1 was DFGFDIPDEVEVR by N-termini sequencing analysis.Plantaricin DLP1 was identified as a new bacteriocin by BLAST comparison.It exhibited broad spectrum activity to food spoilage(Pseudomonas fluoreseens,Pseudomonas aeruginosa,Shewanella putrefaciens)and pathogenic bacteria(Listeria monocytogenes,Vibro harveyi,Staphylococcus aureus).In addition,plantaricin DLP1 was heat-stable(121? for 15 min)and kept active at the pH range of 2.0 to 6.0,but was sensitive to proteolytic enzymes.(3)Based on the aspects of cell damage and DNA transformation,antibacterial mechanism of plantaricin DLP1 to P.fluorescens was explored by scanning electron microscopy,transmission electron microscopy,fluorescence probe and flow cytometry.Firstly,plantaricin DLP1 passed through cell outer membrane by increasing the permeability of outer membrane.Secondly,plantaricin DLP1 dissipated the transmembrane potential and pH gradient of cell membrane,increased the permeability of cell membrane,destroyed the integrity of cell membrane with the pore formation,and resulted in the leakage of DNA and protein from intracellular.Additionly,plantaricin DLP1 inhibited the synthesis of DNA and increased the numbers of cell apoptosis.Finally,plantaricin DLP1 leaded to the cell death.(4)Antibacterial mechanism of plantaricin DLP1 was further studied from the level of protein expression.A total of 320 bacterial protein were significantly changed with plantaricin DLP1 treatment,among which 151 down-regulated proteins and 169 up-regulated proteins.Plantricin DLP1 passed through the outer membrane via the up-regulated porin OprO and was transferred across the cell membrane that is dependent on the up-regulated cell membrane peptide transporter TolQ.The up-regulated arabinose 5-phosphate isomerase,dTDP-4-dehydrorhamnose 3,5-epimerase and DegT/DnrJ/EryC1/StrS family aminotransferase involved the integrity of outer membrane indicated that plantaricin DLP1 destroyed the integrity of outer membrane.The up-regulated SstT and AtpD involved the permeability of cell membrane suggested that plantaricin DLP1 increased the permeability of cell membrane and resulted in the leakage of intracellular ionic material.The 30S ribosomal protein(S1,S3,S4,S5,S7),50S ribosomal protein(LI,L22),GltX,TrpS,AspS,ValS,PheT involved the synthesis of protein were down regulated,the result indicated plantaricin DLP1 inhibited the synthesis of protein.The down-regulated single-stranded DNA-binding protein,DnaJ,DnaK involved the DNA replication and NusG,RpoA,trigger factor involved the DNA transcription suggeated that plantaricin DLP1 inhibited the process of DNA replication and transcription.(5)Antibacterial mechanism of plantaricin DLP1 to P.fluorescens was predicted from the three aspects of cell damage,DNA transformation and protein synthesis.Firstly,plantaricin DLP1 passed through the outer membrane via the porin OprO and was transferred across the cell membrane that is dependent on the cell membrane peptide transporter TolQ,and then inhibited the synthesis of protein,DNA and RNA.Finally,plantaricin DLP1 destroyed the integrity of outer membrane and cell membrane,resulted in the leakage of intracellular components and the cell apoptosis and cell death.(6)Effects of plantaricin DLP1 act as bio-preservative for Paralichthys olivaceus fillets were evaluated by determination the index of sensory scores,microbiology,TVB-N,pH,texture properties.The result indicated that plantaricin DLP1 extended the shelf of Paralichthys olivaceus fillets from 6 days to 8-10 days and maintained good texture and sensory score by inhibiting the numbers of total viable counts and Pseudomonas fluorescens,delaying the increase of TVB-N and pH.Therefore,plantaricin DLP1 had promising potential as bio-preservative for the control of Pseudomonas in aquatic products.