2.1. Impact of the Vessel on the Wine Acidity and Elemental Composition
As shown in
Table 1, the wines fermented and aged in OVO/CNCR vessels showed the lowest titratable acidity and the highest pH among all treatments. However, no significant differences in the content of organic acids, potassium content, or conductivity were observed regardless of the type of vessel employed (
Table 1). These seemingly diverging results may have to do with the higher contents of other cations observed in the OVO/CNCR wines, which may disrupt the equilibrium between tartaric acid and hydrogen tartrate forms in dissolution, leading to wines with a higher pH and lower titratable acidity (
Table 1).
It should be noted that the wines were not subjected to a cold stabilization treatment, and all of them were unstable at the time of the analyses (according to the mini-contact test, a wine is considered unstable when the loss of conductivity is higher than 5%). However, wines elaborated in OVO/CNCR vessels showed the smallest conductivity loss, suggesting that the tartrate salts of OVO/CNCR wines were somewhat more stable when compared with the wines elaborated in the other vessels (
Table 1). This result could be related to the greater concentration of magnesium observed for OVO/CNCR wines (with an increase of approximately 60–80% when compared with the other vessels) since it has been described that the presence of magnesium cations (Mg
2+) increases the stability of tartrate salts [
20,
21,
22].
The OVO/CNCR vessels used for this study were not coated with a chemically inert lining such as epoxy resins, thus the wines were in direct contact with the concrete material, which, as previously documented [
23,
24,
25,
26], may have released inorganic elements such as silicon, sodium, magnesium, iron, and manganese into the OVO/CNCR wines (
Table 1). From these data, it seems that the enrichment of wines with these elements and the formation of inorganic salts could have altered the ionic strength and the equilibrium between tartaric acid and hydrogen tartrate forms in dissolution, leading to the changes observed in the pH and TA [
25,
27].
Considering that OVO/CNCR wines contain higher amounts of several of the elements studied in this trial, the lack of differences among the conductivities of the wines may be surprising (
Table 1). However, as mentioned before, the wines were not cold stabilized, and as a consequence, they were supersaturated with potassium ions (K
+), which could explain the lack of differences in conductivity [
28]. A noteworthy increase in the content of iron in the wines elaborated in OVO/CNCR when compared with the wines made in other vessels (with an increase of about 400%) was observed. This concentration is far from posing a metal instability risk but could have contributed to flavor changes via catalytic oxidative reactions [
29,
30]. This type of iron release from non-coated concrete vessels has been previously described [
26].
The release of inorganic compounds into the wines could also be expected for JAR/CLAY tanks [
31]; however, among the analyzed elements, JAR/CLAY wines only show higher amounts of copper when compared with the other wines. This is in agreement with a study conducted in Qvevri wines, suggesting that processing on clay amphoras could result in a metal composition equivalent to that of conventional wines [
14]. The copper concentration found does not imply a metal instability risk, but the function of copper as a catalyst for oxidative reactions may be a matter of concern [
29]. In any case, there is a great diversity of mineralogical compositions for concrete and clay [
32] and if a release of inorganic compounds from vessels to wine could take place, the specific chemical composition of the concrete or clay of which the vessels are made could impact the extent of the extraction phenomenon.
In addition, the results may also be influenced by the vessel shape and size as well as its surface-to-volume ratio, which determines the extent of the release of new elements into wine and the extent of their dissolution as inorganic salts. In this case, the vessel surface to wine volume ratios for OVO/CNCR and JAR/CLAY are quite similar: OVO/CNCR vessels have 79.1 cm2 per liter of wine (approximately 126 L of wine per m2 of concrete surface), and JAR/CLAY vessels have 82.0 cm2 per liter of wine (approximately 122 L of wine per m2 of clay surface). Thus, the differences in the release of inorganic compounds between the OVO/CNCR and JAR/CLAY vessels do not seem to be related to differences in the contact surface in this case.
2.2. Impact of the Vessel on the Wine Color and Phenolic Composition
As shown in
Table 1, no significant differences were observed for the color parameters of wines, with the only exception of the red-green CIELab coordinate (a*), which was slightly greater for the OVO/PE and OVO/CNCR wines when compared with JAR/CLAY wines. In fact, differences in color among the wines are almost nonexistent and could not be observed by the naked eye [
33]. The lack of differences in the color intensity and lightness (L*) among vessels may indicate similar rates of wine oxidation, in line with the expected increase in absorbance at 420 nm observed during the oxidation process of white wines [
34]. Moreover, regarding the total phenols by a spectrophotometric approach (I
280), JAR/CLAY wines showed the highest total phenolic content, followed by OVO/PE wines. However, when the phenolic composition was analyzed by RP-HPLC-DAD after SPE extraction from the wine matrix, only small differences were observed. Thus, the differences observed among the total phenolic contents are related to the method used, which corresponds to the optical density of diluted wine at 280 nm [
35], and as a result of the low content of phenolic compounds, it is possible that proteins, glycoproteins, and inorganic compounds greatly contribute to wine absorbance at 280 nm [
36].
Among all of the phenolic families described in white wines [
37], only hydroxycinnamic acids were detected and quantified by SPE followed by RP-HPLC-DAD analysis, while neither flavonols nor stilbenes detected at quantifiable amounts. The quantification results of the hydroxycinnamic acids are shown in
Table 2. According to these results, no differences in the total hydroxycinnamic acid content were found among wines elaborated in the different kinds of vessels. Slight differences could be observed regarding the esterification pattern of hydroxycinnamates. OVO/CNCR wines showed the highest ethyl ester content and the lowest tartaric acid ester content. Thus, regarding the color and phenolic composition of the wines, it seems that the vessels used during winemaking did not have a significant impact on the phenolic content. This is consistent with previous results in which stabilized wines were analyzed immediately after alcoholic fermentation [
19].
2.3. Impact of the Vessel on the Wine Turbidity and Polysaccharide Content
Although not scientifically proven, one of the benefits attributed to oval-shaped vessels for wine production is that such a shape would favor the formation of convection currents inside the liquid, thus preventing suspended solids from settling at the bottom of the vessel and favoring the release of polymeric carbohydrate substances from suspended solids into the wine. Like so, it has been described that the shape of the bottom of the vessels influences the contact surface between wine and the settled solids [
3]. The turbidity results shown in
Table 1 agree with this idea, since the cylindrical vessels (CYL/INOX, flat-bottomed) showed the lowest turbidity of all, whilst the wines elaborated in JAR/CLAY, having an inverted oval shape, showed the highest turbidity results. Therefore, the results seem to agree with the hypothesis that oval-shaped vessels increase the suspended solids of wines regardless of the vertical orientation of the ovoid.
At this point, it should be noted that in this trial, aging on lees was performed without racking off the gross lees, mainly for two different reasons: On the one hand, winemakers are increasingly worried about the sustainability of wine production and the use of environmentally friendly practices in wineries. In that sense, racking the wines implies the use of greater amounts of water due to vessel cleanup requirements. On the other hand, it has been described that the use of aging on lees without racking enriched the mannoproteins of chardonnay wine while contributing to the solubilization of grape polysaccharides [
17].
The soluble polysaccharides of wines were analyzed by High Resolution Size Exclusion Chromatography coupled to a Refraction Index Detector (HRSEC-RID) after their precipitation with cold acidified ethanol. Regarding the soluble polysaccharide profiles, some differences could be observed among the wines fermented and aged in the different kinds of vessels (
Table 3,
Figure 1). Briefly, the JAR/CLAY wines showed a lower average molecular mass (M
n) for all of the studied fractions, while the CYL/INOX wines showed a higher average molecular mass (M
n) for all of the studied fractions. OVO/PE and OVO/CNCR wines showed intermediate molecular weights, but statistical differences were mainly observed between CYL/INOX and JAR/CLAY wines. It has been previously reported that aging on lees resulted in the degradation of grape berry polysaccharides such as arabinogalactans and arabinans [
18]. The results seem to indicate that the type of vessel used during wine aging could impact the rate of hydrolysis of wine polysaccharides, maybe due to a greater enzymatic activity from yeast lees.
The total polysaccharide content was greater for wines fermented and aged in clay jar vessels, mainly due to the higher content of FI (the polysaccharides with the greatest molecular mass) and FIV (the oligosaccharide fraction), given that no significant differences were observed among the wines for FII (the polysaccharides with a medium molecular mass) or FIII (the polysaccharides with low molecular mass). It has been previously reported that during aging on lees, wines were enriched with large polysaccharides (100–120 kDa) [
38], and no differences on large polysaccharides were reported at the end of alcoholic fermentation [
19]. Thus, the differences observed among the FI of wines fermented and aged in the different kinds of vessels were probably related to the aging on lees period. Although the wines fermented and aged in CYL/INOX vessels showed the lowest turbidity, they contained more large polysaccharides (FI) than OVO/CNCR wines; thus, the correlation between the suspended solids during aging and wine polysaccharide content does not seem as clear as winemakers expected. To clarify this issue, a statistical correlation analysis (using Pearson’s approach) among polysaccharide fractions and wine turbidity was performed (
Table 4). The correlation between the wine turbidity and soluble polysaccharide content was very weak (
p > 0.05) for the FI, FII, FIII, and total polysaccharide contents, while the correlation between the wine turbidity and FIV showed a Pearson’s correlation coefficient of 0.82 (
p < 0.05). Thus, results suggest that higher turbidity during aging on lees could enhance the content of oligosaccharides in the resulting wines. A correlation between yeast macromolecule release during alcoholic fermentation and the turbidity of fermentation media has been previously described [
39]. However, no differences in the wine turbidity or polysaccharide content were observed under the conditions of a previous study, in which the wines were analyzed immediately after alcoholic fermentation [
19]. Therefore, the differences in turbidity and polysaccharide content observed in this study should be mainly attributed to the changes generated during aging over the lees period. Guilloux-Benatier and colleagues stated that the production of yeast macromolecules depends on the initial content of colloids prior to alcoholic fermentation, while they did not find evidence of higher enrichment with polysaccharides when the turbidity was higher during yeast autolysis [
39].
2.4. Impact of the Vessel on the Volatile Composition of Wine
The wines fermented and aged in the different kinds of vessels were subjected to SPME-GC-MS analysis to determine their volatile composition profiles (
Table 5). Forty-four volatile compounds were identified and quantified, including 23 esters, eight alcohols, three acids, one aldehyde, eight terpenes (six monoterpenes and two sesquiterpenes), and one nor-isoprenoid. In general terms, the wines fermented and aged in JAR/CLAY vessels showed lower contents of several of the quantified volatile compounds. Regarding the volatile compound contents grouped by chemical family, significant differences were only found for acetates, other esters (all esters other than ethyl esters and acetates), and carboxylic acids, while no differences were observed for ethyl esters, alcohols, or terpenes. Considering acetates, other esters, and carboxylic acids, the CYL/INOX and OVO/CNCR wines showed the highest contents, while the JAR/CLAY wines showed the lowest. In fact, JAR/CLAY wines showed a statistically lower content of total volatile compounds (
Figure 2) than CYL/INOX wines.
Although a sensory analysis of samples was not performed, the higher content of volatile compounds could indicate a higher potential aromatic intensity for CYL/INOX wines. However, it is well-known that some of the volatile compounds with high impact on wine sensory perception were found in very small concentrations in the wine matrix, and the correlation between the analytical determination of volatile compounds and wine sensory perception is much more complex than could be expected [
40].
From a qualitative and quantitative point of view, esters are the major family of volatile compounds released during autolysis, beginning after approximately four months of aging [
40]. Usually, short-chain (C3–C5) and medium-chain (C6–C12) acyl esters increase at the beginning of this lytic process [
41]. Given that the raw grapes and wine production were the same for all the vessels, the differences in short-chain and medium-chain esters observed among wines could be attributed to the kind of vessel used during winemaking (
Figure 2).
Several compounds released from lees can interact with volatiles, modifying the perception of the wine aroma. Thus, the release of fatty acids from yeasts into wine triggers the synthesis reactions of esters and aldehydes [
42], or the increase in enzymes with esterase activity in the media may even alter the volatile profile of wines, mainly affecting fruity nuances [
43]. Moreover, yeast mannoproteins may interact with aroma compounds and decrease their volatility [
44], which leads to a reduction in the volatile compound concentration when determined by GC-MS [
45,
46]. A strong binding capability of volatile compounds by the insoluble fraction of yeast autolysates has been described [
45,
47,
48]. The reported results agree with this idea since the wines produced in JAR/CLAY wines showed the highest turbidity values (mostly due to insoluble yeast fractions) and lower contents of volatile compounds (
Table 5,
Figure 2). Moreover, a negative correlation between the ester fractions (both small chain and medium-chain esters) and the polysaccharide contents is observed, also suggesting the loss of aroma compounds due to the presence of insoluble solids (
Table 4). These data suggest that an adsorption phenomenon of volatile compounds by yeast (and/or grape) cell walls took place during winemaking.
Despite the difficulty of predicting wine sensory properties from its volatile compound profile, C6 compounds have been largely related to vegetal scents [
49]. As shown in
Figure 3, the JAR/CLAY wines showed the lowest amount of C6 compounds, while the OVO/CNCR wines showed the greatest. In addition, JAR/CLAY wines also showed the highest amount of ethyl heptanoate (
Table 5), which corresponds to a fruity ester compound with an odd carbon backbone that comes from grape precursors since they cannot be synthesized by yeasts during alcoholic fermentation [
50]. Regarding such differences in volatile profiles of wines from the different tanks, it seems that the use of different vessels could be a useful tool to modulate the wine aromatic profile, enhancing or masking its fruity or vegetal scents.
2.5. Multivariate Analysis
Given the high amount of collected chemical data, a multivariate statistical analysis was performed (principal component analysis, PCA) to understand whether variability among wines is explained by the kind of vessel used during alcoholic fermentation and aging over lees. As
Figure 3 shows, only 54% of the variability observed is explained by the two main principal components (PC 1 explains 34% of the variance and PC 2 explains the remaining 20%), but 116 variables were used to perform the multivariate analysis. Gray globes in
Figure 3 show sample grouping according to Euclidean cluster analysis by using the same 116 variables. According to the multivariate analyses, JAR/CLAY wines differ from the others since they were cluster-grouped in the negative region of PC 1. The main reason for that aggregation (the loadings for each variable resulting from PCA can be found in
Table 6) is their lower content of most of the analyzed volatile compounds in addition to their hydroxycinnamic acid and polysaccharide profiles. OVO/PE wines showed negative PC 1 scores, as happened with JAR/CLAY wines. However, OVO/PE wines are not distinguishable from CYL/INOX wines or OVO/CNCR wines by the PC 1 score distribution. Regarding PC 2, which explains 20% of the data variability, an interesting distribution of scores is observed when the vessel’s material is considered. JAR/CLAY and OVO/CNCR wines, corresponding to the noninert materials (they could be attacked by acidic solutions such as wine releasing some inorganic compounds consequently), showed negative PC 2 scores. In contrast, CYL/INOX and OVO/PE wines, corresponding to inert materials (they could not be attacked by acidic solutions such as wine), showed positive PC 2 scores. This distribution suggests that the material of which the vessel is made has an impact on the overall chemical composition of the resulting wine. Therefore, under the conditions of the trial, the fermenting and aging of lees wines in clay jars led to a more distinctive chemical composition than when the other kinds of vessels were used.