6 SUMMARY OF THE OBSERVATIONS
6.3 A MINO ACIDS AFTER MATURATION OVER LEES OF WHITE WINES IN DIFFERENT ANTIOXIDANT
The analysis of VOCs in wines under different antioxidant conditions, during fermentation and after fermentation, showed that varieties behaviour is different. A short maturation over lees prior to bottling is another oenological practice already being used in same cases[87]. This technique is frequently used on sparkling wines however, is being increasingly used to ensure wine stabilization[87]. The contact with lees by a short period can provide additional aromatic profile[33][87][88]. The application of different doses of SO2 on the alcoholic fermentation step of winemaking describing the simultaneous impact on the yeast nitrogen metabolism or by taking a role as a protective agent for yeast against stress conditions during fermentation, such as the in-creasing presence of ethanol levels was already reported[89][90]. Ageing and storage conditions affect the amino acids content of wines. Indeed, keeping wines over lees tends to increase nitroge-nous substances, since yeasts autolysis occurs and intracellular enzymes slowly hydrolysed yeasts cells, releasing amino acids to the wine medium[50][51]. Additionally, when wines undergo a cold stabilization, aiming to achieve tartaric stabilization, followed by filtration step before bottling, the amino acid content is reduced[26]. During storage, wine VOCs profile also changes, affecting wine aroma and quality[91]. SO2 has an important role to prevent microbiological spoilage and oxidation, both affecting negatively VOCs profile and hence the organoleptic characteristics of the final product[92].
For that matter, two musts of Arinto variety (variety that presented higher sensibility to fer-mentation conditions in the previous studies) from vineyards at Évora were fermented. This variety was fermented with two concentration ranges considered low (0, 15, 30 and 45 mg/L of SO2) and high (0, 50 and 100 mg/L of SO2). Wines were kept over lees for 3 months with a second antioxidant condition. For the lower range three more conditions were added (0, 30 and 60 mg/L of SO2) after fermentation and for the highest range only two additional conditions were considered (0 and 60 mg/L of SO2), Table 6-4 summarize the conditions applied.
Table 6-4 Antioxidant conditions applied for the amino acid content on Arinto wine variety.
SO2 addition Before fermentation (mg/L) After fermentation (mg/L)
Lower range 0; 15; 30; 45 0; 30; 60
Higher range 0; 50; 100 0; 60
The amino acids were analysed by HPLC-DAD after 3 months in contact with the lees.
Total amino acid content for lower range dose were higher for wines with a second addition of 60 mg/L of SO2, been the initial concentration of 15 mg/L of SO2 combined with 60 mg/L of SO2 the one with the higher increase. The increase can be related to the yeast autolysis after the decline of viable cells during maturation on lees[50][51][93]. Since the factors known to affect autolysis (pH, temperature, ethanol and the yeast strain) were kept constant, results suggest that SO2 doses may also play a role in amino acid released by yeasts autolysis. In all the conditions studied for this range, Figure 6-9, proline was the more representative one. accounting for more than 43% of total amino acids concentration. Proline and arginine are two of the major amino acids present in the must and proline is only metabolized in high nitrogen starvation media. The observed results may indicate that for higher concentrations of SO2, the medium is more microbiologically protected, with a lower ni-trogen requirement[94][95].
Figure 6-9 Graphical representation of total amino acid content observed on Arinto must fermented with lower range doses of SO2 with the error bar represented.
A PCA analysis was performed to evaluate if the SO2 applications could influence the amino acid content of wines. To construct the PCA illustrated in Figure 6-9, the concentration of each ami-no acid was used and ami-normalize by Z-ami-normalization, to mitigate a predominance of a variable. It was possible to observe that for lower range doses 90.78% of the system variance was explained by the first and second principal component, PC1 with 81.56% and PC2 with 9.22%, respectively. The pre-dominant factor responsible for sample distribution was the second SO application. Regarding the
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Figure 6-10 PCA illustrating the simultaneous projection of the Arinto wines fermented with lower range SO2 doses, in relation to the conditions of SO2 applications and the amino acid content. Applications before fermentation: blue – 0 mg/L of SO2, black – 15 mg/L of SO2, orange – 30 mg/L of SO2 and yellow – 45 mg/L of SO2. Before fermentation: triangle – 0 mg/L of SO2, lozenge – 30 mg/L of SO2 and square – 60 mg/L of SO2. Grey dots represent the amino acid.
The total amino acid concentration for wines fermented with the higher range dose presented a smaller variation (Figure 6-10), comparing with the ones fermented with lower doses (Figure 6-9).
Wines without a second addition of SO2 presented a total content of amino acids similar regarding the second addition. When a second addition of SO2 at 60 mg/L was applied to wines fermented without SO2 and with 50 mg/L of SO2 a higher increase of the amino acid content was observed. Re-garding the individual amino acid, proline was also present in higher concentration, as previously observed in wines fermented with lower range doses. In these conditions asparagine was the second higher residue, probability duo the metabolic preferential amino acid assimilation, similar to proline case[50].
Figure 6-11 Graphical representation of total amino acid content observed on Arinto must fermented with higher range doses of SO2 with the error bar represented.
Observing PCA of higher range doses, Figure 6-11, 93.18% of the system variance was ex-plained by the first and second principal component, PC1 with 77.97% and PC2 with 15.21%, respec-tively. The same Z-normalization was done. The predominant factor responsible for sample distribu-tion in this range was the second SO2 application. When no SO2 was added after fermentation sam-ples were more similar. These results suggest that SO2 contribute to yeast autolyses promoting dif-ferent increasing profiles on amino acid when in contact with lees[87].
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Since these results indicate that this variety are sensitive to the antioxidant environments on fermentation and maturation on lees, the same doses were applied in the presence and absence of bentonite. Bentonite is an additive applied on wine that as the ability to remove proteins in wine and reduce the protein instabilities of the final product[44][45]. However, bentonite present affinity nonspecific and can impair the quality of wine[85].
Arinto musts were fermented with 0, 50 and 100 mg/L of SO2, since was the conditions were Arinto must presented less differences and the fermentation occur in the presence and absence of bentonite for each condition. After fermentation 0 and 60 mg/L of SO2 were added, and the wines were kept on lees for 3 months.
A PCA analyse using the amino acid content of each amino acid identified (concentration of each amino acid was normalized by Z-normalization) is showed in Figure 6-12, where 81.35% of the system variance was explained by the first and second principal component, PC1 with 71.73% and PC2 with 9.62%, respectively. PC1 clearly separate wines by the second antioxidant condition (0 and 60 mg/L of SO2) and PC2 separate wine by presence or absence of bentonite. The results indicate that the second antioxidant addition promote an amino acid content alteration on wines. Wines fermented with 100 mg/L of SO2 were less sensitive to the conditions applied.
Figure 6-13 PCA illustrating the simultaneous projection of the Arinto wines in relation to the conditions of SO2 applications and the amino acid content fermented with and without bentonite. Applications before fermentation: blue – 0 mg/L of SO2, black – 50 mg/L of SO2 and orange – 100 mg/L of SO2. Before fermentation: triangle – 0 mg/L of SO2 and square – 60 mg/L of SO2. Grey dots represent the amino acid. Wines fermented with bentonite – inside fill with dots and wines fer-mented without bentonite – inside fill with lines.
Through the results obtained, it was possible to observe that different antioxidant conditions, either in fermentation or in lees maturation, modify the amino acid content of wine obtained by the same must. In samples fermented without SO2 (samples shown in blue, Figure 6-12) we see a greater spacing according to the presence or absence of bentonite. The samples without bentonite and without SO2 are very similar to the samples fermented with 50 mg/L of SO2 (samples represented in black, Figure 6-12). Except for those that fermented without bentonite and were kept on lees with 60 mg/L of SO2 in the second addition. Regarding the samples fermented with 100 mg/L of SO2 (rep-resented in orange, Figure 6-12), regardless of the other conditions, they present less dispersion between samples. Also, the presence of bentonite during fermentation changes the amino acid composition although it is not a determining factor.
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