Characterization of bioactive compounds of dealcoholized wine: valuation of distillation process

Red wine is an important source of dietary intake of phenolic compounds with antioxidant activity and that are related to the prevention of cardiovascular diseases and cancer. These compounds are a major by-product of the wine distillation process used to produce the spirits required for production of Port Wine. This thesis aims to add value to the phenolic compounds resultant from wine distilleries by analyzing their composition, studying their interactions with the wine polymeric components, evaluating their stability upon storage, and assaying potential in vitro biological effects. This will allow to define applications as food ingredients for these compounds. Two red wines (RW1 and RW2) were used as source of phenolic compounds. In order to study these compounds, each wine was heatevaporated at atmospheric pressure, allowing to obtain the respective dealcoholized wine (DW1 and DW2). The polysaccharides and phenolic compounds present in the DW were fractionated by solid phase extraction (SPE) using C-18 sep-pak cartridges, allowing to obtain the hydrophilic and hydrophobic fractions. The latter were further separated in phenolic acids-rich, procyanidins-rich and anthocyanins-rich fractions that were used to evaluate their contribution to the total antioxidant activity of DW and for the detailed phenolic characterization of DW by HPLC. Comparable amounts of total phenolic compounds (1.3 g/L for RW1 and DW1, and 3.1 for RW2 and DW2), tannins (1.2 g/L for RW1 and DW1 and 1.6 for RW2 and DW2), and anthocyanins (0.24 g/L for RW1 and DW1 and 0.41 for RW2 and DW2) were obtained. Also, the antioxidant activity of RW and DW, assayed by DPPH and ABTS, resulted in similar values, allowing to infer that the distillation process carried out did not promote relevant loss of phenolic compounds. The anthocyanins-rich fraction achieved the highest contribution to the total wine antioxidant activity. Both DW were dialyzed to obtain the wines polymeric material (WPM1 and WPM2). The WPM1 and WPM2 accounted for 1.1 and 1.3 g/L of solid material. They were composed of polysaccharides (31 and 36%) and proteins (10 and 12%) associated with phenolic compounds (32 and 43%). Sugar analysis showed that mannoproteins and arabinogalactans were the main polysaccharides present. From the extraction of WPM with methanol resulted a methanol insoluble (PMi) and a methanol soluble fraction, which still contained both carbohydrate and phenolic compounds, showing a strong interaction between these compounds. To determine the activation energy of phenolic release from the wine polymeric fractions, dialysis of DW, WPM and PMi, were performed using four different concentrations, and five temperatures (5-40 oC) until a steady state was reached. The Ea was 25 and 61 kJ/mol for WPM1 and PMi fraction, showing that wine phenolic compounds can be differently associated with the polymeric matrix and a fraction can be even strongly associated into this matrix. In order to evaluate if a selective interaction exists between the phenolic compounds, the wine polymeric material was fractionated, allowing to obtain a mannoprotein-rich (MP) fraction using concanavalin A affinity chromatography and 3 arabinogalactan-rich fractions (AG0, AG1 and AG2) by anion-exchange chromatography. Using these compounds, the diffusion of nine monomeric anthocyanins through a dialysis membrane in presence of the WPM, MP and AG fractions was evaluated. In addition, the dialysis of free wine phenolic compounds was performed as a blank experiment. All polymeric fractions showed capacity for retaining the different anthocyanins, although in different extents. The higher retention capacity was observed for the acylglucosylated when compared with the glucosylated anthocyanins. The AG fraction had a higher contribution than MP fraction for retention capacity of wine polymeric material, especially when the anthocyanins were acetylated. The phenolic compounds extract (PCE) as a powder, was stored in different conditions of light and atmosphere, at room temperature for 1 year in order to study forms to preserve the antioxidant properties of these phenolic compounds in long-term storage conditions. It was observed that the PCE stored in the dark, inside a desiccator under a nitrogen atmosphere, preserved 95% of the initial antioxidant activity. It was also evaluated the best conditions to preserve the anthocyanins when in solution, stored at two temperatures (5 and 30oC) during 3 months. The presence of 0.5 g/L of a polysaccharide-rich fraction in a wine solution stored at 30 oC promoted the protection to the anthocyanin content, especially of coumaroylated anthocyanins. The potential biological effects of phenolic compounds were evaluated in different in vitro cellular systems using the fractions: WPM, WPS (wine polysaccharides), WPC (wine phenolic compounds), PA-E (phenolic acids fraction), PR-E (procyanidins-rich fraction), and APP-E (anthocyanins and polymeric procyanidins-rich fraction). A higher cell survival rate of HT-29 colon carcinoma cells was observed when they were exposed to two oxidative agents (UV radiation and H2O2) in presence of PR-E and APP-E extracts. Also, the WPS, WPC, PR-E and APP-E extracts showed anti-inflammatory properties using LPS-induced NO production in RAW 264.7 macrophage cells, where the APP-E extract (0.19 mg/mL) exhibited the highest capacity. In order to further elucidate the antioxidant properties of wine extracts in human cells, red blood cells (RBC) were selected as a metabolically simplified model system. The wine extracts (WPM, WPS, WPC, PR-E, and APP-E) showed anti-hemolytic effect of hydrogen peroxide (H2O2) and 2,2 ́-Azo-bis (2-amidinopropane) dihydrochloride (AAPH) induced RBC hemolysis. The results obtained allowed to conclude that the dealcoholization process of wines at ambient pressure preserved the main antioxidant characteristics of phenolic compounds. These compounds may contribute for cell defenses against oxidative agents, namely having potential antiinflammatory and anti-hemolytic activities, promoting cell viability. The interaction of wine phenolic compounds with the polymeric material allows to predict a continuous and gradual dosage of red wine anthocyanins upon ingestion, contributing for a longer period of their exposure and, as a consequence, of their potential health benefits.