Pyocyanin extraction and quantification
Pyocyanin extraction in Pseudomonas Broth (PB) was carried out using a previously described method. PB medium consisted of MgCl2 (1.4 g/l), peptone (20 g/l), and K2SO4 (10 g/l) (26). P. aeruginosa was cultivated in PB medium with different concentrations of chitosan extract (10 and 100 μg/mL) for 18 hours. The supernatant (5 mL) from the overnight culture was extracted with chloroform (3 mL), and the resulting organic phase was re-extracted with 0.2 N HCl (1.2 mL). The absorbance of the pink aqueous phase was measured at 520 nm. To determine the pyocyanin concentration, the absorbance was multiplied by 17.072, as previously stated (Qais et al., 2019).