Antibacterial Activity And Mechanism Of Bovine Lactoferrin Peptides Expressed By Lactococcus Lactis

Bovine lactoferrin peptides are short peptides with various biological activities released from the N-terminus of bovine lactoferrin hydrolyzed by pepsin under acidic environment.Bovine lactoferrin peptides have various functions such as antibacterial,antiviral,antiparasitic,and immune regulation.Bovine lactoferrin peptides are used in food,safe and non-toxic side effects,no residual hidden dangers,and will not pose a potential threat to the environment,are widely used as food preservatives,nutritional additives,etc.The broad-spectrum antibacterial activity of bovine lactoferrin peptides and its killing effect on antibiotic-resistant strains make it have broad development prospects in the field of new antibacterial peptide drugs.However,the source of bovine lactoferrin peptides is limited and the production cost is high.In recent years,the foreign gene expression systems are used greatly to reduce the production cost of bovine lactoferrin peptides,which provides a basis for its application in production practice.As a food-grade microorganism,Lactococcus lactis has a natural probiotic effect and it will not be inhibited by bovine lactoferrin peptides,so it is an ideal host bacteria for expressing bovine lactoferrin peptides.Therefore,this study used bovine lactoferrin peptides expressed by Lactococcus lactis and clarified its antibacterial activity and mechanism to provide a theoretical basis for lactoferrin peptides expressed by the recombinant bacteria as a microecological preparation for production.This study was based on the Lactococcus lactis bacteria p AMJ399-LFcin BA/MG1363 expressing bovine lactoferrin peptides that had been constructed successfully in the laboratory.First of all,the monoclonal antibody of anti-LFampin that a type of bovine lactoferrin peptides was prepared,and bovine lactoferrin was used as the immunogen to immunize BALB/c mice intraperitoneally.The spleen cells of immunized mice and myeloma cells SP2/0 were prepared by cell fusion technology.The LFampin was used to screen and clone the fused cells.Finally,three hybridoma cells capable of stably secreting anti-LFampin monoclonal antibody were prepared and named as: 2C5,2C6,3E2.Then analyzed the biological characteristics of monoclonal antibodies such as stability,specificity,subtype,etc.The results proved that the three selected hybridoma cells could stably secrete monoclonal antibodies and could bind to LFampin specifically.It was determined that the subtypes of 2C5 and 2C6 antibodies were Ig G1 and 3E2 was Ig M.The monoclonal antibody 2C6 and the laboratory-protected anti-LFcin monoclonal antibody 4E10 were purified,and the purified monoclonal antibodies were labeled with TRITC and FITC respectively.The 2C6 and 4E10 antibodies were used to identify the recombinant Lactococcus lactis p AMJ399-LFcin BA/MG1363 expressing bovine lactoferrin peptides and the concentration of the bovine lactoferrin peptides expressed by supernatant of the recombinant bacteria was 24.39 ?g/m L and the concentration of bovine lactoferrin peptides in the bacteria was 33.51 ?g/mg.In order to explore the effect in vitro cytotoxic of the bovine lactoferrin peptides expressed by recombinant Lactococcus lactis,CCK-8 was used to detect its toxic effect on RAW 264.7 cells,and the hemolysis rate of red blood cells in mice was determined.The results showed that bovine lactoferrin peptides expressed by recombinant bacteria had no obvious cytotoxic effect on mouse red blood cells and RAW 264.7 cells.In order to investigate the antibacterial activity of bovine lactoferrin peptides expressed by recombinant Lactococcus lactis,the Oxford Cup method was used to determine the inhibitory effect of bovine lactoferrin peptides expressed by recombinant bacteria on 35 strains of bacteria.The broth microdilution method was used to determine the minimal inhibitory concentrations.The results showed that the lactoferrin peptides expressed by Lactococcus lactis had different degrees of inhibition on 25 strains of pathogenic bacteria used in the experiments.Its antibacterial concentration range was 16-128 ?g/m L,but it had no obvious inhibition on 10 kinds of probiotics and enterococci.It was proved that the bovine lactoferrin peptides expressed by the recombinant bacteria had a broad-spectrum antibacterial activity,but had no obvious antibacterial activity against specific probiotic bacteria.In order to investigate the antibacterial mechanism of bovine lactoferrin peptides expressed by recombinant Lactococcus lactis,the scanning electron microscopy,transmission electron microscopy and fluorescence microscopy were used to observe the effect of bovine lactoferrin peptides expressed by recombinant bacteria on cell structure.The gel retardation assay was used to investigate the effects of lactoferrin peptides expressed by Lactococcus lactis on bacterial DNA and the adhesion test was used to investigate the effect of Escherichia coli,Salmonella gallinarum,Staphylococcus aureus,Streptococcus pyogenes on Caco-2 and IPEC cells.The results showed that the bovine lactoferrin peptides expressed by the recombinant bacteria had different degrees of destruction on the integrity of Escherichia coli,Staphylococcus aureus,Pasteurella multocida,and Salmonella gallinarum.Its main target is the bacterial cell membrane,The results of gel retardation experiments showed that the bovine lactoferrin peptides expressed by the recombinant bacteria could inhibit the migration of bacterial DNA,which could bind to bacterial DNA to inhibit bacterial growth.The results of adhesion experiments showed that the bovine lactoferrin peptides expressed by recombinant bacteria could reduce the adhesion of bacteria to cells effectively.The above experimental results showed that the bovine lactoferrin peptides expressed by recombinant bacteria had a broad-spectrum antibacterial activity and had no obvious cytotoxic effect on eukaryotic cells,so it could be safely applied to the body.The bovine lactoferrin peptides expressed by recombinant bacteria acted on multiple targets simultaneously to exert antibacterial activity,reducing the risk of a single target being prone to drug resistance.This study provides a theoretical basis for the application of Lactococcus lactis expressing bovine lactoferrin peptides as a microecological preparation for production.