Inoculum preparation and fermentation

Pure cultures of eight identified potent keratinolytic bacteria were used as inoculant for shake flask fermentation of keratinase where each sample was run in triplicate. First, bacterial inoculum was prepared in nutrient broth culture by incubating overnight at 37 °C in a rotary shaking incubator. Submerged fermentation was carried out in an Erlenmeyer shake flask fermenter. Basal modified medium was used for keratinase production containing feather meal powder (10 g/l), NH₄Cl (1.0 g/l), NaCl (1.0 g/l), KH₂PO₄ (0.8 g/l), K₂HPO₄ (0.6 g/l), MgCl₂.6H₂O (0.5 g/l), yeast extract (0.2 g/l), and pH (7.5) with 5% bacterial inoculum [29]. The fermented organisms were cultivated in 250-ml cotton-plugged Erlenmeyer flasks after 72 h shaking at 150 rpm. After incubation, fermented broth was centrifuged at 8000 rpm for 15 min at 4 °C. Cell-free supernatant was collected and preserved for the estimation of keratinase activity as described previously [30].