eparate extractions were performed on each of the
four samples per cultivar per storage time. Extractions were performed
under reduced light conditions. For each extraction, approximately 10
g of frozen berries was weighed and counted and allowed to thaw
partially at -20 °C. Extracts for all assays were prepared using acidified
methanol (0.1% HCl). This solvent maximizes extraction of anthocyanins (9), and studies in our lab demonstrated that acidified methanol
was superior to methanol/formic acid/water (10) for the recovery of
anthocyanins (data not shown). Preliminary studies (data not shown)
also demonstrated no significant difference in total phenolic content
between extracts prepared in acidified methanol and those prepared in
80% ethanol (11). Following 1:1 (w/v) addition of ice-cold acidified
methanol, the fruit was homogenized with a Polytron (Kinematica,
Luzern, Switzerland) homogenizer for 2 min. The homogenizer probe
was rinsed with an additional identical volume of acidified methanol,
and the rinsate was added to the homogenate. The homogenate was
filtered by gravity through 11 µm filter paper (P5, Fisher Scientific),
and the residue was mixed with a third volume of acidified methanol.
This was refiltered, and the filtrates were combined and standardized
to a final volume of 30 mL with acidified methanol. An 8 mL aliquot
was stored at -80 °C until the assays for antioxidant activity, total
phenolic content, and anthocyanin content were performed. Each extract
was tested in duplicate for each of the assays.