2.5.4. Lipoxygenase Activity

Kernels enzymatic extracts were obtained as described by Christopoulos and Tsantili [23]. An extraction solution was prepared by dissolving β-ME (5.0 mM), PVPP (1:100 *w*/*v*) and Triton X-114 (0.05:100 *w*/*v*) in 50 mM phosphate buffer (pH 6.6). Kernels (2.0 g) and the extraction solution (10 mL) were homogenized (6000 rpm, 40 s), filtered using glass wool and centrifuged (8000 rpm, 15 min, 4 ◦C). Supernatants were collected for determination of lipoxygenase (LOX) activity according to the procedure reported by Li et al. [24]. Solutions were made using 0.2 M borate buffer (pH 9.0). Linoleic acid dissolved in EtOH and 0.2 M borate buffer (1:1:1000 *v*/*v*/*v*) was employed as substrate stock solution to measure LOX activity. The stock solution (5 mL) was diluted completely in 20 mL of 0.2 M borate buffer and 5 mL of distilled water. The diluted solution (2 mL) and 0.2 M borate buffer (950 μL) were pipetted in a cell quartz and mixed by inversion. Next, enzymatic extracts (50 μL) were added and mixed by inversion. Absorbance was measured at 234 nm and registered every 10 s until 3 min of reaction in a Cecil CE 1010 UV-VIS spectrophotometer (Cecil Instruments Ltd., Cambridge, UK). LOX activity was calculated employing the molar extinction coefficient (ε) of hydroperoxides (26,800/M·cm) to express it as μmol of hydroperoxide produced per L of LOX per s (μmol/L·s) [25].
