Coppola, Francesca (2023) Wine oxidation and aging: a study on the key role of polyphenols and acetaldehyde. [Tesi di dottorato]

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Abstract

The present dissertation reports the results obtained from the activities conducted within the framework of the PhD focused on the project “Wine oxidation and aging: a study on the key role of polyphenols and acetaldehyde”. Aging is essential for improving wine quality, especially for red wine. It is no coincidence that high quality wines are traditionally produced by barrel aging, long and expensive methods that significantly affect wineries' cost factors and production capacities. Young, rough, and tart wine after fermentation with a cloudy, dim, and unstable structure requires adequate time for the reactions of oxidation-reduction, esterification, condensation, polymerization to impart a completely different sensory character. During this period, to guarantee chemical and biochemical changes within the wine and avoid alteration phenomena, it is fundamental to manage properly the main actor involved in wine aging process: the oxygen. The traditional aging method, called natural aging, is oak barrel aging. It involves storing wine in oak barrels for several months and has proven to be an effective method for producing many types of quality wines. The influence of this method on wine quality lies in the special permeability of wood to oxygen. Oak barrels have good permeability. Traces of oxygen can enter the vascular bundle of the wood and the cracks between the boards slowly and steadily, causing slow and continuous oxidation of the wine and allowing it to improve color, flavor and astringency character. In more recent years, the use of artificial methods to simulate the aging process of wine to shorten the aging time and improve its quality in a short period of time has become an area of research of great interest. Micro-oxygenation represents the most interesting technique of wine oxidation and aging. This method consists of adding a controlled amount of trace oxygen to wine to meet the oxygen demand required by the physical and chemical reactions of the different varieties that occur during the aging process, thus simulating the micro-oxygenated environment of the oak barrel aging process, promoting the maturation of the wine and improving its quality with particular reference to taste and structure to color stability, aromas as well as astringency. Micro-oxygenation-based aging technology overcomes the problems of time-consuming traditional aging, providing a new idea and a new direction for the development of the wine industry. Although the artificial aging technology, in particular the micro-oxygen technology, has made great progress, the dynamics and changes that occur in the polyphenols as well as the implication of neoformation complexes in the definition of the sensory characteristics of the wine they are clear enough. Sometimes the results of researchers from different teams are inconsistent and the detailed description of some mechanisms underlying the definition of sensory characteristics, such as astringency, is missing. It is known that acetaldehyde and polyphenols represent two components of extraordinary importance in defining the qualitative characteristics of wine. Understanding their role as single molecules, their evolution as well as their possible interaction among themselves and with other wine components would be of undoubted interest. Furthermore, it is clear that these compounds have a key role also in all issues related to health quality of wines and in the strategies useful for sustainable winemaking approaches. The study of relationship between sustainable techniques and polyphenols as well as the healthy effect of red wine inevitably involves the role of oxidation and acetaldehyde. Then it was the participation of acetaldehyde in the interaction between the different polyphenols and in the formation of new complexes molecules that was investigated. Furthermore, the effect of the each single polyphenol considered as a model and of the newly formed complex molecules involving the ethanal bridge was investigated with computational methods. This dissertation assesses the observations, and the main significant results were reported in five chapters. Chapter 1., based on the awareness that quality is not only linked to the alcohol composition of the wine, aims to be an overview of phenolic compounds and their recognized role. More specifically, the chapter distinguishes and preliminarily describes the different classes of phenolic compounds, then reports the main factors that underlie their presence, concentration and action in wine. Chapter 2 relying on polyphenols, aimed to estimate the position and role of wine in the Mediterranean diet as well as its relationship with health. In detail, the first part aims to provide a bibliometric analysis of the literature on the association between the Mediterranean diet, wine, and health. The results obtained from the data analysis show a strong relationship between the Mediterranean diet and red wine as well as their interconnection with health protection. Of interest in this regard is the link between wine included in the Mediterranean diet and topics such as 'risk reduction' 'cardiac risk' 'hypertension'. A certain focus on the relationship between red wine polyphenols and sirtuins, a specific family of proteins that mediate a wide variety of pathophysiological events, emerged from the analysis. However, it should be emphasized that there are really few studies related to the specific topic under research and bibliographic analysis. Based on these considerations, in the second half of chapter, the relationship between the phenolic content and the bioactivity of red wines was investigated. the phenolic profile and bioactivity of Italian red wines Gaglioppo, Magliocco, and Nerello Mascalese were characterized. NMR, HPLC/UV-Vis and spectrophotometric characterization showed that Magliocco was the richest wine in monomeric anthocyanins (two-fold), catechins, and low molecular weight phenolics (LMWP). In vitro evidence on the endothelial cell models of insulin resistance and hyperglycemia showed the ability of Magliocco to reduce reactive oxygen species (ROS) and cytokine release and to upregulate SIRT1 and SIRT6. Overall, the results indicated that the quantitative and qualitative phenolic profiles of red wines influence their in vitro beneficial effects on oxidative and proinflammatory milieu in endothelial cells, showing a positive modulation of SIRT1 and SIRT6, both implied in vascular aging. Chapter 3 focuses on wine oxidation and ageing, considering the concepts of sustainability and winemaking. The first part of the chapter provides an overview of the non-enzymatic oxidation of wine and the use of conventional protective and antioxidant agents during vinification. From this examination, also emerges that numerous factors, such as increasing consumer attention to health concerns and restrictive legislation on preservatives, together with the quest for environmentally friendly production, have driven the interest of the scientific community and manufacturers in alternative methods to traditional protective and antioxidant agents. Based on these considerations, two case studies are presented in this chapter. The first case study is based on the need for information on the oxidative evolution of red wines by means of accelerated ageing tests. As already exposed in the previous chapters, phenols in wines vary in quality and quantity. It follows that it is of fundamental importance to assess how different types of red wines react to oxidation. This chapter reports the results of the study that evaluate the effect of three different oxygen addition systems on different red wines (Aglianico, Barbera, Magliocco, Gaglioppo and Nerello) with the aim of developing a tool for predicting the evolution of phenolic compounds in a short time. The results evidenced that oxygen saturation cycles and hydrogen peroxide addition tests have the same impacts on most of the wine chromatic properties and phenolic composition of the treated wines. Thus, both can be used as suitable methods to simulate red wine oxidation and to allow a discrimination among wines based on their possible evolution under oxygen exposure. The second case study aimed to investigate a sustainable tool, such as the use of chitosan, on the evolution of acetaldehyde in red wine before and after exposure to an oxidative stress designed to simulate wine ageing and, on tannins by assessing their reactivity towards salivary proteins, one of the mechanisms underlying the sensation of astringency in red wines. Chapter 4 turns its attention exclusively to acetaldehyde and the interactions it establishes with the main polyphenols in wine. After an initial description of the reasons for acetaldehyde accumulation in wine and its changes during ageing, the chapter reports the results of the case study: How acetaldehyde reacts with low molecular weight phenolics in white and red wines. The results highlighted that the chemical behaviour of acetaldehyde turned out to be different in white and red wines. Specifically, a dramatic loss of monomeric anthocyanins and a simultaneous massive formation of polymeric compounds was detected in red wine. The results regarding red wine highlighted that the anthocyanin moiety is not preferentially attacked by the protonated acetaldehyde, which seems to react first and preferentially with flavanols due to their better nucleophilicity when compared to anthocyanins. Subsequently, after the loss of a water molecule, the acetaldehyde-flavanol adduct attacks either another flavanol or an anthocyanin unit. In our studies conducted on real wines (pH =3.69), we only detected polymers containing just one anthocyanin unit, even if molecules of unreacted malvidin-3-O-glucoside were still present in the analyzed samples. Instead, in white wines the formation of vinyl-flavan-3-ol derivatives was mainly detected. Infact, the comparison of the total ion chromatograms (TIC) of the untreated and treated white wine brought to light a significant decrease of the relative abundance of some chromatographic peaks along with a concomitant increase of others. Based on the results, it can be assumed that flavanols in white wine are helpful to quench acetaldehyde by affording more stable compounds. In addition, in red wines the acetaldehyde led to the formation of ethylidene bridged red pigments. These latter positively enhanced the color properties of red wines. Chapter 5 addresses the description of the relationship between wine ageing and the perception of astringency. As reported in the previous sections, important relationships between anthocyanins and flavan-3-ols are established leading to the formation of new, more stable complexes involving the ethanal bridge. These newly formed adducts appear to be involved in the modulation of astringency. However, the prediction of astringency of a specific group of compounds must consider the molecular origin of astringency that is not yet fully understood, and the connection or contribution of the various proposed mechanisms in scientific literature is still unknown. The most accepted mechanism is based on the precipitation of salivary proteins by polyphenols. To date, the relationship with prolin rich protein PRPs, an important group of proteins subdivided into acidic, basic, and glycosylated PRPs, has been most investigated. PRPs associated with astringency are mostly basic and acidic. In addition to PRPs, other relevant salivary protein families that have been linked to astringency include α-amylase. Although α-amylase is the most abundant enzyme in saliva, the interaction of novel formation complexes and single molecules with α-amylase appears to be less investigated than with other proteins. On these bases, the final phase of the doctoral activity was directed toward the study of computational tools capable of investigating the interactions of anthocyanins and their polymeric pigments against salivary alpha-amylase; thus, an in silico molecular docking study was performed that could predict the preferred orientation of a receptor-bound ligand to form a stable complex. This computational investigation provides several insights into the mechanisms of interaction of salivary α-amylase binding with anthocyanins and their polymeric pigments that may be useful in enriching knowledge regarding the mechanisms involved in salivary protein precipitation and thus initiate the development of new wine aging strategies. Docking results indicated that both the two monomers as well as polymeric pigment showed excellent abilities to actively interact with the 1SMD protein by influencing its conformation. However, the binding energy between the three ligands and the enzyme is significantly different exerting different influence on astringency. The results confirm that catechin has a greater effect on astringency than malvidin. In addition, the astringency produced by the new formed pigment is greater than malvidin but lower than catechin. The results showed that docking tools can offer useful results regarding the interactions between polyphenolic compounds and salivary proteins. The kinetics and evolution of polyphenolic compounds during wine aging combined with computational study can offer extraordinary information for the development of appropriate aging strategies. This last activity was conducted in collaboration with the Randall Centre for Cell & Molecular Biophysics, within the Faculty of Life Sciences & Medicine at King’s College London.

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