3.3. Extraction Procedure

The dried samples (40 mg) were processed according to diverse extracting conditions designed according to the different levels of the variables referred in Table 5 with 1.5 mL of solvent (ratio ~1:40, w/v).

Symbols and coded factor levels for the considered independent variables.

The ethanol used contained different proportions of hydrochloric acid to obtain a distinct pH that was hypothesized to influence the efficiency of the polyphenol extraction according to Periago et al. (2002) [54]. Extractions were performed in a water bath for 30 min using different temperatures. Then, the extracts were centrifuged at 5000 rpm for 15 min and at 4 °C (Sigma 2-16K, Osterode am Harz, Germany). The supernatants were collected into a volumetric flask of 5 mL. The solid residue was extracted two more times under matching conditions, and the extracts were pooled in the volumetric flask. The repeated extractions were performed to overwhelm possible saturations of the extracting solvent and thus, guarantee the attainment of all the polyphenolic burden of the grape pomace. The final volume was made up to 5 mL to reduce the variability between samples attributable to dilution issues. Finally, the extract was filtered through a 0.22-µm inorganic membrane filter (ANOTOP 10 plus, Whatman, Maidstone, UK) and preserved at 4 °C until analysis.