Study On Preparation Of Polyoxin B And Extraction Of Fermentation Broth

Polyoxin,which belongs to the type of pyrimidine nucleosides antibiotics,is characterized by high efficiency and low toxicity.Polyoxin has good efficacy on the controlling of plant diseases caused by a variety of pathogenic fungi.At the same time,polyoxin is safe to natural enemies and plants,and low resistance.Although the raw pesticide and formulation of polyoxin have been registered in China by many manufactures,the large-scale production was achieved in a few companies.Several key technical issues are need to be solved in the production of polyoxin.Because of no commercial reference substance,the content of polyoxin in technical and formulation was mainly determined by the evaluation of bioactivity.There is no refine procedure in the production of polyoxin technical,and which resulted in the content of active ingredients in the final product is low.To solve these problems,the key techniques such as preparation of active ingredients and refine of technical polyoxin were investigated in this paper.Polyoxin is a multi-component antibiotic,and there are more than 20 similar pyrimidine nucleoside compounds in the fermentation broth,all of which are water-solubility compounds that are difficult to be separated and purified.Polyoxin B is a relatively large component in fermentation broth,and also an effective component for quantitative detection in product quality standards of most enterprises.In this paper,ion exchange chromatography,activated carbon adsorption,gel chromatography,medium pressure ion pair chromatography and high pressure ion pair chromatography were used to establish the preparation process of polyoxin B in the fermentation broth,and the purity of the final product reached more than 97%,which could meet the requirements for the quality detection of polyoxin raw pesticide and quality control of formulations.HPLC method for the analysis of polyoxin technical was established by using trifluoroacetic acid as ion pair reagent,and the specific chromatographic conditions are as follows:Bomai Unisil C₁₈ column（4.6*250 mm,size 5μm）,mobile phase:methanol/0.1%trifluoroacetic acid=2/98,flow rate:0.8 m L.min^-1 column temperature of 35℃,detection wavelength of 260 nm.By comparing the adsorption and analytical behavior of polyoxin on different types of ion exchange resins,the purification process of polyoxin was preliminarily studied,and finally the 001×7 cation exchange resin was selected as the adsorption material.The optimal adsorption conditions were as follows:the fermentation broth p H value was 4.0,the flow rate was 2 BV/h,and the sample concentration was 1mg/m L.The optimal desorption conditions were as follows:the desorption agent was 0.4 mol/L ammonia water,the flow rate was 2 BV/h,the elution volume was 3 BV,and the desorption rate was up to85.6%.The results of this study have a good reference value for improving the industrialization level of polyoxins.