3.5.4. Determination of Polyphenol Oxidase Activity

PPO activity was spectrophotometrically measured in the enzymatic extracts. The enzyme activity was detected according to the method described by Zocca et al. (2011) with some modifications [77]. The absorbance was recorded at 400 nm and 25 °C using a spectrophotometer (Varian Carry 50 Bio UV/Vis, Agilent Technologies, Santa Clara, CA, USA). The reaction sample included 1 mL of 10 mM catechol in sodium citrate buffer (0.1 M, pH = 6) and 10 μL of PPO extract. The blank sample contained 10 μL of PPO extract and 1 mL sodium citrate buffer (0.1 M, pH = 6) without the substrate. The absorbance was monitored continuously for 180 s considering the linear part of the PPO kinetic curve. For calculating the specific activity of PPO, one unit of enzyme activity was defined as the increase of 0.001 in absorbance per min, and the obtained value was divided by the protein content of PPO extracts, which was measured using the method described by Bradford (1976) [78].