Effect-Directed Extraction of Grape Pomace: Optimizing Antioxidant and Antibrowning Efficacy

Abstract

The increasing interest in valorizing agricultural by-products has positioned grape pomace as a rich source of bioactive compounds. This study developed an effect-directed extraction (EDE) approach guided by bioactivity quantification on thin layer chroma-tography (TLC). Twelve grape pomaces were screened based on antioxidant and tyro-sinase inhibitory properties. Using hydroalcoholic solvent (ethanol:water, 1:1), the two most promising sources (Malbec from San Rafael) were subjected to response surface methodology (RSM) to optimize extraction of anti-browning and antioxidant com-pounds visualized as TLC spots. Temperature and time were optimized, and samples were analyzed using TLC coupled with DPPH and laccase inhibition bioautography. Antioxidant compounds showed retention factor values on TLC plates of 0.37 and 0.75 (DPPH/ABTS-active), while laccase inhibition occurred at Rf 0.35, coinciding with the primary tyrosinase inhibition zone. However, subsequent bioassay-guided HPLC frac-tionation and LC-HRMS/MS analysis revealed that tyrosinase and laccase inhibitions are mediated by distinct co-migrating compounds within this bioactive zone, highlighting a synergistic multi-target effect in the optimized extract that is retained throughout the process. The primary tyrosinase inhibitor at Rf ~0.35 was structurally elucidated as an acylated anthocyanin, consistent with malvidin-3-O-(p-coumaroyl)glucoside (m/z 639.1735). Optimized extracts were evaluated on Pink Lady apple slices at different timepoints. Browning index was reduced 25% versus control at 15h, confirmed by sig-nificantly lower ΔE values (p<0.05). The process requires only food-grade solvents and conventional equipment, facilitating scale-up for grape pomace generated worldwide. Validating the EDE philosophy, this TLC-guided approach successfully tracked and preserved the primary anti-tyrosinase activity from the crude waste matrix down to the unequivocally identified molecule, contributing to circular economy objectives in the wine industry.