2.4. Inoculum preparation and halophilic protease production in shake-flask culture

For inoculum preparation, a full loop of cells was transferred from a slant culture into a 250 mL Erlenmeyer flask containing 50 mL of SGC seed culture medium [26]. The seed culture was grown at 30 °C in a shaker incubator (Innova 400, New Brunswick Scientific Co., NJ, USA) at 200 rpm for 3 days with illumination.

For halophilic protease production, an inoculum (5%, v/v) was added into 250 mL Erlenmeyer flask containing 80 mL of M73 fermentation medium [25] with the following composition (g/L): gelatin 10, yeast extract 1.0, MgSO₄·7H₂O 10.0, KCl 5.0, CaCl₂·2H₂O 0.2 and NaCl 250 (pH 8.0). The flasks were incubated at 30 °C in a shaker incubator at 200 rpm for 6 days. After incubation, the culture broth was centrifuged at 8,000 rpm for 15 min at 4 °C using a Sorvall^® RC-5C plus superspeed refrigerated centrifuge (Kendro Laboratory Products, Newtown, CT, USA). The cell-free supernatant was used for determination of halophilic protease activity.