Optimization of Culture Conditions for Production of Protease by Two Bacterial Strains, Bacillus licheniformis BF2 and Bacillus subtilis BH4 Isolated from the Digestive Tract of Bata, Labeo bata (Hamilton)

Abstract

The culture conditions for extracellular protease production by two bacterial strains, Bacillus licheniformis BF2 and Bacillus subtilis BH4 isolated from the foregut and hindgut regions, respectively, of bata (Labeo bata, Cyprinidae) were optimized to obtain maximum level of protease. Both strains were cultured in TSA broth for 24 h at 37 ± 2 °C. This was used as the inoculum for the production medium. The fermentation medium was seeded with 1.0, 2.0, 3.0, 4.0, and 5.0 % inoculum (TSA) and incubated in static culture at 37 °C to standardize the inoculum size for fermentation. The effects of fermentation condition, moisture, pH, temperature, inoculum size, and nitrogen sources on protease production by the isolated bacterial strain were studied. The strain, BH4, produced more protease in comparison to the strain BF2 isolated from the same fish in solid-state fermentation. Enzyme production in both the isolates increased in an optimum pH range of 6.0–6.5. Maximum protease production by both the strains was obtained at 40 °C. To standardize the fermentation period for protease production, production rate was measured at 12-h intervals up to 120 h. Enzyme production increased for 72 h of fermentation in both strains, and decreased thereafter. The enzyme production increased with increased inoculum size up to 3.0 percentage points. Beef extract as the nitrogen source was most effective in optimizing enzyme production by both the isolated strains. The information generated from the present investigation will help to standardize the requirements for optimum production of protease by protease-producing fish gut bacteria.