2.4. Determination of the Polyphenol Oxidase (PPO) Activity

The determination of the PPO activity of the apple samples was done according to Kolodziejczyk et al. [31] and González et al. [32], with an adaption to a miniaturized procedure. About 10 g of the frozen sample was weighed and crushed in a mortar. Subsequently, 25 mL of a phosphate buffer (0.05 M, pH 7.0) were added and then mixed. The sample was incubated for 120 min. at 4 °C in the dark, centrifuged (15 min., 4 °C, 3225 g), and the supernatant used to determine PPO activity. First, 30 µL of the sample extract were given in a 96-well microtiter plate and either 270 µL of the phosphate buffer (0.2 M, pH 5.5) as blank value or 270 µL of a catechol solution (0.1 M in 0,2 M phosphate buffer, pH 5.5) were added. The enzyme activity was immediately determined at 25 °C by measuring the change in absorption over 10 min. at a wavelength of λ = 420 nm with a BioTek Synergy HT microplatereader (BioTek Instruments Inc., Winooski, VT, USA), whereby the change in absorption was recorded every 60 s. The enzyme activity was given as activity units per 100 g of fresh weight (f.w.) of a fruit sample. One unit is defined as the change of 0.01 in the absorbance value per minute [31,32].