Study On The Effect Of Antibacterial Peptides Lac-B23 From Lactobacillus On Pseudomonas Fluorescens Biofilm

In the food sector,especially in the dairy industry,biofilms may be the source of intractable contaminants,leading to food corruption,as well as public health problems.Pseudomonas fluorescens belongs to the food-borne thermophilic bacteria,it is difficult to inactivate at high temperature which can secrete protease and lipase and other metabolites,resulting in the production and processing equipment to form biofilm,resulting in continuous pollution.The antimicrobial peptide Lac-B23 is produced by the Lactobacillus in the metabolic process by ribosomal secretion,with broad-spectrum antibacterial activity,because of its natural non-toxic,no residue,high efficiency,good stability,no resistance to drugs such as the food industry has become a popular natural microbial source of food preservatives.In this study,the antimicrobial peptide Lac-B23 was used to reduce the biofilm of Pseudomonas fluorescens,the titer was determined,the titer of the antimicrobial peptide Lac-B23 was 2560 AU/m L,which proved that the antibacterial activity was high and the the minimum inhibitory concentration of Pseudomonas fluorescens was 640?g/m L,and the antibacterial effect was obvious.The biofilm model was successfully constructed by crystal violet dyeing method,and the best biofilm formation time was 24 h.Scanning electron microscopy showed that the three-dimensional network structure of the biofilm was destroyed,indicating that the biofilm decomposition;we used laser confocal microscopy to observe the extracellular polysaccharide and protein,found that when the antimicrobial peptide Lac-B23 treated with biofilm,green and red fluorescence weakened,indicating that the extracellular polysaccharide and protein content decreased,which indicated that the antimicrobial peptide Lac-B23 damage the biofilm,the skeleton structure of biofilm was decomposed;phenol sulfuric acid method for quantitative analysis of extracellular polysaccharide,when the antimicrobial peptide Lac-B23 treated with biofilm and the content of extracellular polysaccharide was decreased by 30%.The content of eDNA was determined by agarose gel electrophoresis when the antimicrobial peptide Lac-B23 treated with biofilm,and the eDNA content was increased to about 7 times of the control group,indicating that the antimicrobial peptide Lac-B23 may kill the bacteria or cause bacteria autolysis,resulting in the eDNA content increased.Transmission electron microscopy revealed that the integrity of the membrane was destroyed,indicating that the antimicrobial peptide Lac-B23 increased membrane permeability,leading to bacterial content outflow and the bacteria death.When the antimicrobial peptide Lac-B23 treated with the biofilm,the bacterial activity was detected by the Bacterial Activity Kit.The results of laser confocal microscopy showed that the red fluorescence enhanced,indicating that most of the bacteria in the biofilm appeared to die,and the results were same as the fluorescence microscopy.Flow cytometry was used to determine the apoptotic status of the biofilm,it was found that when the antibacterial peptide Lac-B23 treated the biofilm,the apoptosis of the membrane was increased and the cell integrity was decreased,the results showed that the antibacterial peptide Lac-B23 could damage the bacteria in the biofilm.We used RT-PCR to detect the changes of related genes,and the related genes were flagellar gene,2,4-DAPG biosynthesis enzyme gene,regulation lapA adhesion hormone secretion gene,periplasmic cysteine protease gene,signal transduction protein gene,and soluble pyrimidine nucleoside transhydrogenase gene,the results showed that these genes were down-regulated by 0.66,0.90,0.39,0.52 and 0.72 times,respectively.Because Pseudomonas fluorescens has flagellar structure,we have detected the motility of Pseudomonas fluorescens and found that the motility of Pseudomonas fluorescens was changed obviously under the action of antimicrobial peptide Lac-B23.