2.3. Extraction of Anthocyanin Fraction

Once collected, the whole blueberries (200 g FW) were immediately frozen at −80 °C for at least 24 h and subsequently freeze-dried. The resultant dry samples were independently mixed with 200 mL of acidified methanol (0.5% acetic acid) using a homogeniser [20,21,38], before being centrifuged at 3452 g for 10 min at 20 °C, and the supernatant fraction then being collected. Re-extraction was performed until the pellet was colourless. The supernatant of each variety was filtered, concentrated under vacuum at 35 °C to remove methanol, and then diluted 1:1 with water. The purification of the anthocyanins fraction of each variety was performed as previously described in the literature [6,20,21,23,63], using an Amberlite XAD-7 column (30 × 1.5 cm) previously activated with methanol, and then 300 mL of water. Samples were loaded onto the column and cleaned with 450 mL of water, in order to remove free sugars, pectin, and organic acid, among other polar compounds. The anthocyanin fraction was eluted with methanol/acetic acid solution (19:1, v/v) at 1 drop/s flow, concentrated under vacuum, frozen at −80 °C and freeze-dried to obtain the anthocyanin extract (Table 1).

Ratio of anthocyanin extract obtained from each blueberry variety.