4.6. Measurement of Anthocyanin Biosynthesis-Related Enzymes Activity

The main enzymes (CHS, CHI, F3H, F3′H, F3′5′H, DFR, LAR, ANS, and ANR) activity was measured by corresponding plant ELISA kits (Shanghai Fusheng Industry Co., Ltd., Shanghai, China). Sample (0.5 g) was ground into a fine powder in liquid nitrogen, and 4.5 mL phosphate-buffered saline (PBS, pH 7.4, 0.01 mol/L) was added to the powder. The mixture centrifuged at 5000 rpm for 15 min at 4 °C. The supernatant was transferred into the tube for enzyme activity assays and then measured according to the manufacturer’s recommendations. Briefly, (a) all reagents were balanced at room temperature for 20 min before starting the assay. (b) 10 μL of testing sample and 40 μL of sample diluent were added into the same microplate well. (c) 100 μL of horseradish peroxidase (HRP)-conjugate reagent was added to each well. Then, the microplate was immediately sealed with an adhesive strip and incubated for 60 min at 37 °C. (d) The liquid in microplate wells was discarded, and the microplate was patted dry with absorbent papers. Then the microplate wells were washed with washing solution (400 μL). The microplate was let stand for 1 min and then the microplate was patted dry on an absorbent paper, and the washing process was repeated for five washes. (e) 50 μL of chromogen solution A and 50 μL chromogen solution B were added to each well on the microplate. Then, the microplate was gently mixed and incubated immediately at 37 °C in dark for 15 min. (f) 50 μL of stop solution was added to each well and the optical density value was recorded at 450 nm in a microplate reader within 15 min.