Grape seed was separated from pomace of grape variety Prokupac, immediately after vinification process. Grape pomace was collected in winery (“Wine house Milinčić”), located in famous Serbian wine region Župa. Separated grape pomace seed was immediately grinded in a coffee grinder (Bosch MKM 6003 UC, BSH Hausgeräte GmbH, Munich, Germany) and PCs were extracted (1:10 w/v) by using 80% methanol (80/20 water:methanol), as described by Milinčić et al. [6]. After that, the grape pomace seed extract was evaporated (Heidolph, Laborota 4000, Schwabach, Germany) to dryness and reconstituted in Milli-Q water (SE) for further analysis and preparation of functional products.

Qualitative and quantitative analysis of PCs present in grape pomace seed extract were performed using an Accela Ultra high-performance liquid chromatography (UHPLC) system coupled to a linear ion trap-orbitrap mass spectrometer (LTQ OrbiTrap MS) equipped with a heated electrospray ionisation probe (HESI-II, ThermoFisher Scientific, Bremen, Germany) and Xcalibur software (version 2.1), as previously reported [6,8]. Quantification of all identified phenolic compounds was performed using available standards. The detection of phenolic compounds for which the standards were not been available was conducted based on their exact molecular masses and specific MS4 fragmentation. These compounds have been quantified and expressed as mg gallic acid; caffeic acid; catechin; quercetin–3-O-glucoside; or malvidin–3-O-glucoside per kg FW of seed, depending on the phenolic classes to which they belong.

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