Quantification of Carob Flour Phenolics

The carob flour total phenolic content (TPC) was extracted in methanol:H₂O:acetate (50:49.5:0.5) for 24 h at 4 °C in the dark and quantified based on the modification of Singleton et al. [22 ], described previously by Kyriacou et al. [6 (link)]. Quantification was based on the absorbance at 765 nm determined on a Jasco V-550 spectrometer (Jasco Corp., Tokyo, Japan) against linear calibration (R² > 0.99) with gallic acid (50−500 mg L⁻¹) as an external standard and the results were denoted as g kg⁻¹ flour dw in gallic acid equivalents.
Carob flour content in condensed tannins (proanthocyanidins) was determined according to a modification of the vanillin methods of Sun et al. [23 (link)] and Sepperer et al. [24 (link)], described previously by Antoniou et al. [25 (link)]. Lyophilized flour 0.5 g was extracted in 10 mL methanol:H₂O:HCl (50:40:10) at 4 °C for 24 h in the dark. Quantification was performed at 500 nm against calibration with catechin external standards (0.025–0.5% w/v in methanol), and the results were expressed in mg catechin equivalents (CE) per g dw.