## 3 Results  
To understand the impact of the timing and level of the acidification on red wine evolution, four samples treated with two levels (low and high) of acid as musts (LmM and HmM) or wines (LmW and HmW) were analyzed just after the end of the alcoholic fermentation (EAF) and after 12 and 24 months of aging. The evolution of acetaldehyde is shown in Figure 2. In control wine (pH 3.2), it increased over two years of aging. Conversely, after the first year of aging, a stabilization (LmM, LmW, HmM) or even a decrease (HmW) was observed. Acetaldehyde is a highly reactive molecule, and its variation trend detected in wines LmW, HmM and HmW could be ascribed to its quick consumption in reactions with flavans [18] that were more abundant in wines obtained through a maceration at higher pH values (Table S1) [12]. Data on the evolution of the vanillin index supported this hypothesis (Figure 3). The decrease in vanillin index in LmW, HmM and HmW was significant after one year of aging, while in 3.2 and LmM wines, a significant difference was observed only after two years of aging. Vanillin index can be considered an indirect, inverse measure of the oxidative polymerization of flavanols via methyl methine bridge, since acetaldehyde [19], like vanillin [20], reacts with the C8 and C6 positions of the flavanol A ring. As reported, part of these reactions could involve anthocyanins [10]. The content of total monomeric anthocyanins (Figure 4), determined by HPLC, corroborated the importance of choosing the acidification timing appropriately. Differences among samples were not significant at the end of the alcoholic fermentation but, after one year of aging, samples that had undergone late acidification showed lower contents of monomeric anthocyanins. This could be ascribed to the involvement of these native pigments in reactions of polymerization favored in late-acidified wines due to the higher concentration of flavanols [12]. Figure 3: Evolution of vanillin index during aging of experimental wines. Codes assigned to wine samples are as in Figure 2. EAF: end of alcoholic fermentation. Wine type at the same moment (A,B) and each wine through time (a–c) sharing the same letters are not significantly different. Figure 2: Evolution of acetaldehyde during aging of experimental wines: 3.2, control wine at pH 3.2; LmM, wine originally at pH 3.5 acidified during AF (alcoholic fermentation) to have pH 3.2; LmW, wine originally at pH 3.5 acidified seven days after AF to have pH 3.2; HmM, wine originally at pH 3.7 acidified during AF to have pH 3.2; HmM, wine originally at pH 3.2; EAF, end of alcoholic fermentation. Wine type at the same moment (A–C) and each wine through time (a–c) sharing the same letters are not significantly different. Data on large polymeric pigments (LPPs) (Figure 5) showed that the formation of polymeric structures was enhanced in wines obtained through the maceration of grapes at higher pH and that this effect is already significant after the end of alcoholic fermentation. These pigments are of great relevance as they contribute to the stability of the color intensity of wines over time. Experimental data showed that the later the acidification was performed, the higher the production of these long pigments occurred over time. BSA-reactive tannins (Figure 6), compounds with high molecular weight that also can be formed during wine production, oxidation and aging [21], are more abundant in wines obtained at higher pH values and then acidified. Figure 4: Evolution of total monomeric anthocyanins during aging of experimental wines. Codes assigned to wine samples are as in Figure 2. EAF: end of alcoholic fermentation. Wine type at the same moment (A,B) and each wine through time (a–c) sharing the same letters are not significantly different. Figure 5: Evolution of large polymeric pigments during aging of experimental wines. Codes assigned to wine samples are as in Figure 2. EAF: end of alcoholic fermentation.