Study On The Isolation And Purification Of Josamycin

Josamycinwhich is a kind of antibiotic of Macrolides.It is a wide-spectrum antibiotic, Like as Erythromycin.With the little side effect and drugfastness.In this dissertation, process of isolating and purifying spinosad from fermentation broth by macro-porous adsorbent resin and solvent extraction were determined.First,the method of detection and analysis to josamycin 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 josamycin infermentation broth and during extracting process,The chromatographic conditions were asfollowing:chromatography column:RP-C₁₈;mobile phase:Methanol:phosphoric acid buffer =80:20;detect wave length:232nm;flow rate:1.Oml/min;injection volume:lOul; column temperature:room temperature.Process conditions of isolating and purifying spinosad from fermentation broth by macro-porous absorbent resin were determined.XAD-16 nonpolar resin was screened as the best adsorbent,and adsorbent conditions were optimized:the flow rate of adsorption was 0.5ml/min,the pH was maintained at10,1.0%NaCI was feasible.The desorption conditions were optimized:30%ethanol was used for removing some impurity and pigment.100%ethanol was used for eluting josamycin.Process conditions of isolating and purifying josamycin by solvent extraction were also determined:adjust the broth to pH9.0,then offcenter the broth remove the supernatant, and add equal weight calcium carbonate,vacuum desiccation the mycelial for 6 hour at 50℃.Use ethanol leaching the mycelial.by means of orthogonal experiment we get the optomized condition as follow:20ml ethanol for every 10g mycelial;leaching time is 36 hours;leaching temperture is room temperture;number of leaching times is three.In the Section of refining,select the silica gel like a 200 to 300 mesh,proportion of chloroform:methanol=2:3,as the eluent.Concentrate the eluent at the depressurization state(the vacuum degree is 0.095Mpa,the temperture is 50℃).Evaporated the solution and get the yellow powder of josamycin,to solute it with acidified water of pH3.5.then to change the pH of solution at pH9.0,park in 20-30 miniute,the primrose yellow of josamycin is separated out.In the process of determination of the crystal's activity,we find that the crystal of josamycin has the same bacteriostatic activity as the standard crystal which we are purchased.