The increasing global demand for food has intensified agro-industrial production, leading to the accumulation of large amounts of waste materials with significant environmental impact. Among these, grape pomace (GP), the main solid by-product of the winemaking industry, represents a valuable source of bioactive polyphenols and therefore a promising matrix for sustainable valorization strategies. In this work, Deep Eutectic Solvents (DESs), and particularly Natural Deep Eutectic Solvents (NADESs), were employed as green extraction media for the recovery of bioactive compounds from Apulian grape pomace. A preliminary screening was performed on Petit Verdot and Cabernet Sauvignon grape pomaces using seven DESs and hydroalcoholic mixtures as reference solvents. Solid–liquid extractions were followed by the development and optimization of an HPLC-DAD analytical method for the qualitative and quantitative determination of selected phenolic compounds. A 20 μL volume of each diluted and filtered sample was injected into the C18 column at 25 °C. The mobile phase, delivered at a flow rate of 0.7 mL/min, consisted of a binary gradient of acidified water containing 5% acetic acid solution and methanol. Standard solutions of phenolic compounds were prepared in methanol, and detection was simultaneously performed at five wavelengths: 260, 280, 325, 360, and 560 nm. Antioxidant and antibacterial assays were also carried out to support the screening of the extraction media. Choline chloride/ascorbic acid (ChCl/AA), choline chloride/urea (ChCl/U), and betaine/lactic acid (Bet/LA) emerged as the most promising systems. Subsequently, seventeen red and white Apulian GP varieties were extracted using the selected NADESs and further characterized by HPLC-DAD analysis employing eleven phenolic standards. Organic Merlot and conventional Sangiovese were among the richest cultivars, while Bet/LA and ChCl/AA proved to be the most effective extraction media. To obtain a more comprehensive characterization of the extracts, LC-ESI-QTOF-MS semi-quantitative metabolomic profiling was performed, allowing the identification of up to sixty-two bioactive metabolites and highlighting higher recoveries for Bet/LA-based extracts compared to ChCl/AA ones. Chromatographic analysis was performed on a C18 column using a binary mobile phase composed of a gradient of water and acetonitrile, both containing 0.1% formic acid. Diluted samples (5 μL) were injected at a flow rate of 0.4 mL/min. Mass spectrometric analyses were carried out using a QTOF-MS system equipped with a Dual Jet Stream electrospray ionization source operating in both positive and negative modes. Metabolites were tentatively identified through accurate mass measurements and database matching based on a custom library of phenolic compounds commonly identified in grape pomace by mass spectrometry. Results, expressed as mg GAE/kg DW using a gallic acid calibration curve, provided semi-quantitative estimates useful for comparing the extraction solvents and bioactive compounds. Additional bioactivity assays revealed promising antioxidant and antimicrobial properties of the extracts, although the intrinsic antioxidant contribution of ascorbic acid significantly influenced ChCl/AA-based samples. In vitro digestion experiments combined with metabolomic analyses also highlighted the biostability of Bet/LA extracts and suggested possible interactions between grape pomace polyphenols and probiotic microorganisms, supporting a potential prebiotic effect and opening future perspectives for the development of symbiotic formulations. Overall, this work demonstrates how green NADES-based extraction combined with advanced HPLC-DAD and LC-MS analytical characterization can support the sustainable valorization of grape pomace into ready-to-use bioactive extracts with potential nutraceutical and feed supplement applications.
From winery waste to ready-to-use extracts: sustainable NADES-based extraction and advantage analytical profiling
Roselli Vincenzo
2026-01-01
Abstract
The increasing global demand for food has intensified agro-industrial production, leading to the accumulation of large amounts of waste materials with significant environmental impact. Among these, grape pomace (GP), the main solid by-product of the winemaking industry, represents a valuable source of bioactive polyphenols and therefore a promising matrix for sustainable valorization strategies. In this work, Deep Eutectic Solvents (DESs), and particularly Natural Deep Eutectic Solvents (NADESs), were employed as green extraction media for the recovery of bioactive compounds from Apulian grape pomace. A preliminary screening was performed on Petit Verdot and Cabernet Sauvignon grape pomaces using seven DESs and hydroalcoholic mixtures as reference solvents. Solid–liquid extractions were followed by the development and optimization of an HPLC-DAD analytical method for the qualitative and quantitative determination of selected phenolic compounds. A 20 μL volume of each diluted and filtered sample was injected into the C18 column at 25 °C. The mobile phase, delivered at a flow rate of 0.7 mL/min, consisted of a binary gradient of acidified water containing 5% acetic acid solution and methanol. Standard solutions of phenolic compounds were prepared in methanol, and detection was simultaneously performed at five wavelengths: 260, 280, 325, 360, and 560 nm. Antioxidant and antibacterial assays were also carried out to support the screening of the extraction media. Choline chloride/ascorbic acid (ChCl/AA), choline chloride/urea (ChCl/U), and betaine/lactic acid (Bet/LA) emerged as the most promising systems. Subsequently, seventeen red and white Apulian GP varieties were extracted using the selected NADESs and further characterized by HPLC-DAD analysis employing eleven phenolic standards. Organic Merlot and conventional Sangiovese were among the richest cultivars, while Bet/LA and ChCl/AA proved to be the most effective extraction media. To obtain a more comprehensive characterization of the extracts, LC-ESI-QTOF-MS semi-quantitative metabolomic profiling was performed, allowing the identification of up to sixty-two bioactive metabolites and highlighting higher recoveries for Bet/LA-based extracts compared to ChCl/AA ones. Chromatographic analysis was performed on a C18 column using a binary mobile phase composed of a gradient of water and acetonitrile, both containing 0.1% formic acid. Diluted samples (5 μL) were injected at a flow rate of 0.4 mL/min. Mass spectrometric analyses were carried out using a QTOF-MS system equipped with a Dual Jet Stream electrospray ionization source operating in both positive and negative modes. Metabolites were tentatively identified through accurate mass measurements and database matching based on a custom library of phenolic compounds commonly identified in grape pomace by mass spectrometry. Results, expressed as mg GAE/kg DW using a gallic acid calibration curve, provided semi-quantitative estimates useful for comparing the extraction solvents and bioactive compounds. Additional bioactivity assays revealed promising antioxidant and antimicrobial properties of the extracts, although the intrinsic antioxidant contribution of ascorbic acid significantly influenced ChCl/AA-based samples. In vitro digestion experiments combined with metabolomic analyses also highlighted the biostability of Bet/LA extracts and suggested possible interactions between grape pomace polyphenols and probiotic microorganisms, supporting a potential prebiotic effect and opening future perspectives for the development of symbiotic formulations. Overall, this work demonstrates how green NADES-based extraction combined with advanced HPLC-DAD and LC-MS analytical characterization can support the sustainable valorization of grape pomace into ready-to-use bioactive extracts with potential nutraceutical and feed supplement applications.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
