2.3. Shake flask fermentation with selected medium
Alkaline protease was produced in an optimized production medium of pH 8.0 containing 1% galactose, 0.5% casein, 0.55% peptone, 0.2% KH2PO4, 1% Na2CO3 and 0.2% MgSO4•7H2O; 4% inoculum was added to the production medium and incubated with continuous shaking. Shaker fermentations were carried out at 37 ◦C for 72 h with controlled agitation at 150–200 rpm. At the end of the fermentation period, the whole culture broth was centrifuged at 5000 rpm for 30 min to remove debris, and the supernatant was collected and used for further experiments.