Effects of different oenological technologies on the evolution of anthocyanin derivatives determined by LC-MS during red wine fermentations. Encapsulation of phenolic grape pomace extracts for cosmetic applications

Resumen

This PhD thesis is focused on to two topics related to wine-making: (i) the study of the influence of novel oenological techniques on the phenolic composition of wine during its elaboration, and (ii) the valorization of grape pomace waste resulting from wine elaboration.Phenolic compounds occur naturally in plants and fruits. Red grapes are one of the fruits with the highest content of phenolics, which account for their color, flavor and antioxidant and anti-inflammatory properties. Wine is a popular and appreciated beverage elaborated through a fermentation process from grape must and grapes. In Spain, red wine is of great importance, especially in the area of La Rioja, where red wine has a Protected Designation of Origin (PDO). Red wine is rich in phenolic compounds, mainly anthocyanins and anthocyanin derivatives. Pedoclimatic conditions of gravevine areas affect the chemical composition of grape berries, and thus of the wine produced from these grapes. Wine elaboration techniques also influence the composition of wine and its evolution during the wine-making process and its maturation in the long term.In this work, different oenological technologies were evaluated to study their influence on the phenolic composition of wine during its elaboration process. For this purpose, wine elaborations were performed in 2018 and 2019 in Bodegas Faustino, under controlled conditions at pilot scale, employing the main grape cultivar under the PDO "Rioja" named Tempranillo. The oenological technologies studied were: (i) microoxygenation during malolactic fermentation (MLF); (ii) ionic exchange technologies to lower the pH of the grape must at the beginning of alcoholic fermentation (AF); (iii) tannin addition at the beginning of AF; and (iv) the use of a mixture of must from different grape cultivars. An experimental design including all the variables of the oenological technologies was carried out, and the wine elaborations were monitored and sampled from the start of AF until the end of MLF.In the must and wine samples, 68 phenolic compounds (13 anthocyanins, 27 anthocyanin derivatives and 28 tannins) were determined during the wine-making process by HPLC-DAD and HPLC-DAD-ESI(+)-MS/MS. The concentration phenolic profiles of 8 anthocyanins and anthocyanin derivatives, selected because of their relevance in wine characteristics, were treated mathematically in order to study their evolution kinetics, i.e. extraction from grape, formation and degradation. The kinetic constants were statistically compared (i) between different compounds under the same oenological conditions, in order to study their different stability and evolution kinetics; and (ii) between different oenological conditions employed during wine elaboration, to observe the influence of the oenological techniques on the evolution kinetics.Results of the concentration measurements and the consequent kinetic study showed that the main anthocyanin derivatives present in must and wine are pyranoanthocyanins (i.e. Vitisin A, Vitisin B and derivatives with vinylphenol), followed by anthocyanin-flavan-3-ol condensation products, both direct (F- A+ or A+-F) or indirect, linked by an ethyl bridge (A+-ethyl-F). Vitisin B reaches higher concentration levels than the other compounds during wine elaboration; however, its stability is lower in the long term, leading to a higher degradation of the compound and a lower concentration in wine at the end of fermentations. In contrast, Vitisin A shows a higher stability during fermentations, leading to a more important contribution of this compound to wine color at the end of the elaboration. Malvidin-3-O-glucoside-vinylphenol showed a similar stability as Vitisin A, and thus, it also contributes to wine color. Among the anthocyanin-flavan-3-ol condensation derivatives, the order of stability found is F-A+ > A+-ethyl-F > A+-F; catechin-malvidin-3-O-glucoside (F-A+) being the most important regarding wine color contribution at the end of fermentations.Related to the studied oenological variables, the use of a secondary grape cultivar mixed with the main grape cultivar Tempranillo compared to the use of the single main grape cultivar, led to an increase in the formation constants of pyranoanthocyanins and a decrease in those of anthocyanin-flavan-3-ol condensation products. The application of ionic exchange resulted in a decrease in the concentrations of pyranoanthocyanins and an increase in the concentrations of the anthocyanin-flavan-3-ol condensation products at the end of the fermentations. The addition of tannins and the use of microoxygenation during MLF did not produce significant changes in the concentration of the studied compounds or their kinetic constants.Grape pomace residues from the wine elaborations were employed in order to obtain extracts rich in phenolic compounds with potential cosmetic applications. These extracts were analyzed by HPLC-DAD and HPLC-DAD-ESI(+)-QToF/MS: 13 anthocyanins were identified and quantified, and 24 minor phenolics (flavan-3-ols, flavonols, hydroxycinnamic and hydroxybenzoic acids) were identified. The extracts were formulated in liposomal vesicles, i.e. liposomes and transfersomes, with the aim of enhancing their health beneficial properties and making them suitable for application on skin. The nanoformulations prepared were characterized in terms of size, shape and stability by transmission electron microscopy (TEM), dynamic light scattering (DLS) and zeta potential, and their antioxidant activities and total phenolic contents were assayed by means of the DPPH and FRAP and Folin-Ciocalteu assays respectively. Furthermore, the cytocompatibility of the nanoformulations containing the extracts, as well as their inhibition capacity of intracellular Reactive Oxygen Species (ROS) generation were evaluated. Nanoformulation of the extracts obtained from the grape pomace residues of wine-making process yielded stable, safe, effective and usable formulations for the treatment of skin under oxidative stress conditions. The in vitro studies highlighted the ability of the vesicle formulations to counteract ROS overproduction, enhancing the effect of the extracts in the cells.