Study On The Standardization Detection Methods Of Antimicrobial Activity For Antimicrobial Peptides

As an ideal substitute for antibiotics,antimicrobial peptides have attracted much attention.However,there is no uniform standard or method for the detection of antimicrobial peptides,which not only leads the antimicrobial performance detection methods difficult to unify,but also makes the antimicrobial peptide products difficult to distinguish.Based on this,this paper mainly established the detection methods for the antibacterial ability of antimicrobial peptides,and developed a standard for activity determination.The researches were as follows:The antibacterial properties of antimicrobial peptides were tested,8 antimicrobial peptides and 3 commonly used indicator strains were collected,and the Oxford cup-agar diffusion method was used.The size of Oxford cup was 6.0mm±0.1mm in inner diameter,10.0mm±0.1mm in height and 7.8mm±0.1mm in outer diameter.Disposable Petri dish(90mm in diameter)was used and detection medium components were as follows: peptone10mg/L,beef paste 3mg/L,Na Cl 5mg/L,p H 7.2-7.4.The optimal conditions of Oxford cup-agar diffusion method were found as follows: the amount of culture medium was 15 m L,agar concentration was 1.5%,p H value was 6.0-7.0(except nisin),the concentration of indicator bacteria was 106 cfu/m L,the amount of antimicrobial peptide was 100μL,and the pre-diffusion time was 6-10 h.A standard curve was set up by measuring the diameter of bacteriostatic circle and the concentration of antibacterial peptide by Oxford cup-agar diffusion method.The titer was calculated according to the standard curve: the titer of Bacitracin against S.aureus ATCC 25923 was 489.42 U/mg.The titer of Bacitracin and Nisin against M.luteus ATCC 10240 was 134214.21 U/mg and 80141.53 U/mg,respectively.Polymyxin B had a titer of 33383.38 U/mg against S.typemurphy ATCC14028.The antifungal activity of antimicrobial peptides was tested,and 8 kinds of antimicrobial peptides and 2 kinds of commonly used indicator strains were collected.Compared with spore germination inhibition method and mycelial growth inhibition method,the more suitable method was mycelial growth inhibition method.The optimum conditions were as follows: culture medium was Czochralski medium,indicator strain was cultured for 48 h,the addition amount of plate medium was 20 m L.The suitable test conditions for polymyxin B against A.niger ATCC 16404 were as follows: culture time was 48 h,the diameter of inoculated cake was 8.00±0.02 mm,under these conditions,the EC50 of Polymyxin B was 0.68 mg/m L.The suitable test conditions for Polymyxin B against P.chrysogenum ATCC 10106 were as follows: culture time 72 h,inoculum diameter 5.80±0.02 mm,under these conditions,the EC50 of Polymyxin B was 0.45mg/m L.The antibacterial peptide was produced by fermentation of B.laterosporus S62-9,which was preserved in our laboratory,and further purified by preparative high performance liquid chromatography.The antibacterial peptide standard of brevilacterin 3was obtained,and the purity was over 98%.The titer of brevilacterin 3 against S.auraeus ATCC 25923 was 1743.21±8.21 U/mg.The storage stability of brevilacterin 3 was good,and the biological activity was kept above 97% after being stored for 6 months at room temperature.Through the above research,finally established the antibacterial peptide on antibacterial ability test method of standardized research,to achieve the preliminary study and formulation of antibacterial peptide products related standards.Through the above researches,the standardized methods for antibacterial ability test of antibacterial peptide were established,and the preliminary product standard of antibacterial peptide products was established.