Exoprotease activity
Exoprotease activity was assessed using the azocasein degradation assay, following a previously reported method (Husain et al., 2017). P. aeruginosa was cultured with and without chitosan extract supplementation (10 and 100 μg/mL). The cell-free supernatant was obtained by centrifugation. One hundred microliters of the supernatant were mixed with 0.3% azocasein (1000 μl) in a 0.05 M Tris-HCl solution containing 0.5 mM CaCl2 at pH 7.5. The reaction mixture was incubated at 37 °C for 15 minutes, followed by the addition of 500 μl of trichloroacetic acid (10% w/v) to stop the reaction. After centrifugation at 12000 rpm for 10 minutes, the optical density of the supernatant was measured at 400 nm.