Study On The Isolation And Purification Of Spinosad

Spinosad, which is a novel microbial broad-spectrum insecticide, is secondary metabolites from the aerobic fermentation of a naturally occurring actinomycetes bacterium Saccharopolyspora spinosa. Spinosad is effective on a wide variety of crops. Attributes of spionsad included: high levels of activity against economical important pests, a short half-life, degradation to natural building blocks, and large margins of safety for mammals, birds, fish and even most beneficial insects. In this dissertation, process of isolating and purifying spinosad from fermentation broth by macro-porous adsorbent resin and solvent extraction were determined.At frist, the method of detection and analysis to spinosad was established, including thin layer chromatography and analysis high-performance liquid chromatography. HPLC was fast and accurate, which was very suitable for quantitative analysis of spinosad in fermentation broth and during extracting process. The chromatographic conditions were as following: chromatography column: RP-C18; mobile phase: Methanol : Acetonitrile 0.05% ammonium acetic = 49 : 49 : 2; detect wave length: 246nm; flow rate: 1.0ml/min; injection volume: 10ul; column temperature: room temperature.Process conditions of isolating and purifying spinosad from fermentation broth by macro-porous absorbent resin were determined. XAD-4 nonpolar resin was screened as the best adsorbent, and adsorbent conditions were optimized: the pH was maintained at 11, flow rate of adsorption was l/6(l/min), 2.0% NaCl was feasible, the quantity of adsorption spinosad was 1.09xl04 μg/ml wet resin. The desorption conditions were optimized: 0~70% Acetone was used for removing some impurity and pigment. 70% ~ 95% Acetone was used for eluting spinosad.Process conditions of isolating and purifying spinosad by solvent extraction were also determined: adjust the broth to pH 9.0, add an equal volume of acetone to the broth and filter, remove acetone by vacuum evaporation and add equal volume ethyl acetate, then recover the ethyl acetate extract by decanting off the immiscible aqueous portion; concentrate the ethyl acetate to 1/3 volume by vacuum; extract the concentrated ethyl acetate solution with equal volume aqueous 0.2M tartaric acid and separate the phases; adjust the concentrated aqueous solution to pH 9.0 with sodium hydroxide; separate the precipitate by filtration; dry under vacuum to get spinosad.As a result, compare with the method of macro-porous adsorbent resin and solvent extraction, the recycle rate were 64.86% and 68.27% respectively, however the purity were 95.0% and 91.0%.Compare with the standard table by dint of mass spectrum, both are same material.In addition, the results showed that spinosad had a good insecticidial activity to Lepidoptera, Diptera Thysanoptera Coleoptera and Homoptera pests by indoor experiments.