Optimization Of Fermentation Conditions And Purification Of Antimicrobial Lipopeptides Produced By Paenibacillus Polymyxa SC2

Paenibacillus polymyxa is a kind of plant-growth-promoting rhizobacteria（PGPR）which has obvious promotion effect on plant growth.It has been widely used in agricultural production.Paenibacillus polymyxa SC2 is isolated from the pepper rhizosphere and has the ability to produce lipopeptide antibacterial substances.And the yield of active substances produced by SC2 varies greatly due to different fermentation formulas.The fermentation conditions and medium formulation of the antibacterial active substances were optimized in this article and the antibacterial active substances were isolated and purified.The main contents are as follows:In this study,shaking flask culture was used to study the effects of environmental conditions and nutrition on the production of antimicrobial substances.Ashby medium is used as the basicculture medium,and by single factor experiments,the optimal sources of carbon,nitrogen,cultivation temperature,effects of adding inorganic salts and amino acids on fermentation were determined.Orthogonal design experiments were used to determine the ratio of carbon,nitrogen,and calcium carbonate.Then Response Surface Methodology was used to determine the ideal medium fermentation conditions with the highest inhibition rate.The results showed that Mn²⁺and Zn²⁺all had inhibitory effects on the production of extracellular antimicrobial substances of Paenibacillus polymyxa SC2,and the greater the concentration,the more obvious the inhibitory effect.Mg²⁺can shorten the fermentation cycle.At the same time,the study found that the addition of MgSO₄ can affect the production of antibacterial substances.Finally,the optimal medium for SC2 was determined to be sucrose43.60g/L、（NH₄）₂SO₄ 6.66g/L、CaCO₃ 6.26 g/L、MgSO₄ 0.2 g/L,KH₂PO₄ 0.2 g/L、NaCl0.2g/L.The optimal temperature and incubation time were 37°C and 60 h,respectively.After optimization,the yield of antibacterial substances increased from 295.0492μg/mL to 1470.86μg/m L,and raised about 4 times.In order to obtain relatively pure antibacterial substances and identify them,fermentation liquid was separated and purified.The main methods used in the study included extraction,Thin Layer Chromatography（TLC）,and High Performance Liquid Chromatography（HPLC）.The results showed:after the acidified heat treatment of the fermentation broth,the supernatant can be extracted by n-Butanol under alkaline conditions.Under acidic conditions,large amounts of impurities can be removed after anti-extraction,retaining its antibacterial activity.A brown crude extract is obtained after the anti-extraction water phase is concentrated by rotary evaporation and freeze-drying,and then high performance liquid chromatography（HPLC）analysis was performed.By analytical HPLC analysis,a total of 7 active peaks were found,and retention times were:5.5 min,7.5 min,9 min,12.5 min,16 min,17.5 min,23.5 min,respectively.The 7active peaks were collected and analyzed by mass spectrometry,and found that the molecular mass was mainly distributed between 1000-1200.Analysis by MS/MS,antibacterial substances produced by Paenibacillus polymyxa SC2 contained polymyxin P1,P2 and five other new polymyxin P substances.By preparative HPLC analysis,a total of 5 active peaks were found,and retention times were:11.1min,25.1min,27.8min,33.8min,34.9min.Each active peak was collected and concentrated and then subjected to secondary HPLC preparation.The elution conditions of each peak were optimized,and the antibacterial active substance was separated.Each active peak was collected and concentrated,prepared by secondary HPLC.The elution conditions were optimized,and the antibacterial active substances were separated.The content,molecular mass and structure were analyzed by mass spectrometry.The results showed:The active peak with a retention time at 11.1 min obtained two high activity peaks which molecular mass were 1190 and 1176,respectively,consistent with the molecular weight of polymyxin P2 and polymyxin P1,and structures are the same as polymyxin P2 and polymyxin P1,respectively.The active peak with a retention time at 34.9min obtained two high activity peaks which molecular mass were 1230 and 1258,respectively,and structure is not yet clear.