Abstract: It was to optimize the fermentation conditions of neomycin producing by Streptomyces fradiae FIM-S38. The single factor experiments and orthogonal experiments were adopted to optimize the fermentation medium and conditions of FIM-S38 in shake flasks. The constituents of medium, the filling volume of shaking flask, the rotation speed and the temperature were the major factors that affected the yield of neomycin. The composition of glucose 6.0%, soluble starch 3.5%, soybean meal 2.0%, MgSO4 0.1% and CaCO3 0.3% was selected to be the medium formula, and the optimal fermentation parameters were as follows ：the filling volume 50 mL for a 500 mL shake flask, initial pH at 7.0, shaker’s rotating speed 250 r/min, temperature 35℃, fermentation time 6 d, and the titer of neomycin was 50% higher than that in control.

Key words: Streptomyces fradiae, Neomycin, Orthogonal experiment, Fermentation, Optimization