Optimization Of Fermentation Conditions And Purification Of Aureobasidium Pullulans PA-2 Lipopeptide Substance

Microbial pesticides have the characteristics of being easily degraded and not easily polluting the environment,and they have attracted the attention of scientific researchers.Since 2013,our laboratory has continuously screened microorganisms in nature,and finally isolated a strain with good inhibitory effect on pathogenic microorganisms from the leaves of susceptible poplars in Pingan County,Qinghai Province.After the identification of the strain,it was Aureobacidium pullulans,and named the strain PA-2.On this basis,this experiment studied the antibacterial secondary metabolites of Aureobasidium pullulans PA-2.In order to clarify the type of bacteriostatic substance of A.pullulans PA-2,this study used in situ acid hydrolysis-ninhydrin color method to qualitatively identify the bacteriostatic substance,and determined its antibacterial activity by agar punch diffusion method.Inorder to increase the yield of A.pullulans PA-2 bacteriostatic substance,this experiment used a central composite design（CCD）to construct a response surface from five aspects:rotation speed,temperature,liquid volume,inoculation volume,and initial p H of the medium.Method to optimize the culture conditions of A.pullulans PA-2 in liquid fermentation.In order to further understand the stability of antibacterial active substances,this experiment measured the stability of antibacterial active substances from six aspects:temperature,p H,ultraviolet irradiation,metal ions,organic solvents,and proteases,and applied high performance liquid chromatography to The crude material is further separated and purified.The specific test results are as follows:1.The bacteriostatic substance of A.pullulans PA-2 was determined to be a cyclic lipopeptide substance after preliminary identification:The substance has a significant inhibitory effect on S.aureus,S.cerevisiae,E.coli,M.cerasella and P.teres.The diameters of the inhibition zone were 3.65 cm,1.95 cm,2.15 cm,1.35 cm,and 2.18 cm,respectively.2.The optimized liquid fermentation conditions of A.pullulans PA-2 are as follows:inoculation amount 6.85%,rotation speed 216.24 r·min^-1,temperature25.80℃,liquid volume 124.87 m L,p H 7.1.Under these conditions,the predicted yield of lipopeptides by the model is 0.94 g·L^-1,which is actually 0.92 g·L^-1,which is51%higher than the yield before optimization(0.61 g·L^-1).3.Results of stability determination of A.pullulans PA-2 lipopeptides:The ambient temperature of-80～100℃will not affect the antibacterial activity of the crude lipopeptide extract.The high temperature of about 121℃can obviously reduce the antibacterial activity of the crude extract,and the inactivation rate is 16%.When the solvent pH is 3,the antibacterial activity of the crude lip opeptide extract is the strongest,the neutral environment will weaken the antib acterial activity of the crude lipopeptide extract,and the alkaline environment will completely disappear the antibacterial activity of the crude lipopeptide extr act.Ultraviolet irradiation within 8 hours will not affect the antibacterial activity of the crude extract.The four metalions of Na⁺,K⁺,Ca²⁺and Fe³⁺have no effect on the antibacterial activity of the crude lipopeptide extract.The crude extract is completely soluble in methanol,slightly soluble in ethanol,and inso luble in acetonitrile,acetone and chloroform.Trypsin,proteinase K and Pepsin will not have a significant impact on the antibacterial activity of crude lipopeptide extracts.4.Using a C18 reverse analysis column（4.6mm×250mm）,methanol and water as mobile phases,the best separation and purification conditions were obtained for the first time:methanol:water=75:25,flow rate 1 m Lmin,injection vol ume 20μL,detection Wavelength 220 nm;The crude peptide extract was prep ared witha C18 reverse preparation column（30mm×250mm）to obtain 5 g component 1 and 1 g component 2.Component 1 has inhibitory effect on E.coli and S.aureus.Both E.coil and S.aureus have no inhibitory effect;Using a C18 reverse analysis column（4.6mm×250mm）,using acetonitrile and water as mobile phases,the best separation and purification conditions for component 1are:nitrile:water=10:90,flow rate 1m L·min^-1,injection Volume 20μL,detectio n wavelength 220 nm;Use a C18 reverse preparation column（30mm×250mm）to prepare component 3 and obtain three active components.After concentrati on and drying,3 g component 3,1 g component 4 and 0.5 gcomponent 5 are obtained.E.coli,S.aureus as the target bacteria was tested for antibacterial activity,and it was found that the substances collected in components 3,4,and5 had inhibitory effects on the above two kinds of bacteria.Among them,the substances corresponding to component 3 were effective against E.coli and S.au reus has the largest and clearest bacteriostatic zone diameter,and the strongest bacteriostatic activity;The minimum inhibitory concentration of crude lipopeptide extract against E.coli was 1.0 mg·m L^-1,and the minimum inhibitory concentr ation against S.aureus was 0.5 mg·m L^-1.