Post-harvest withering plays a central role in determining the compositional and sensory characteristics of Valpolicella red wines [
1,
3]. From a quantitative point of view, the main physiological change associated with this traditional practice is water loss, that is carried out up to an average of 30% weight loss depending on wine style. This has major implication for grape composition, most notably increased concentration of metabolites such as sugars, phenolics, and certain aroma compounds, directly influencing composition of the resulting wine. Additional important consequences of increased sugar levels are related to changes in yeast metabolism, which can further impact wine composition. However, it has been recently shown that post-harvest withering is not simply a dehydration process, with many complex metabolic transformations beyond simple concentration taking place inside the berry, inducing important modifications in the pool of grape secondary metabolites, including volatile compounds [
17]. In consideration of this complex scenario, one of the purposes of the present study was to investigate how and to what extent withering of the grapes affects the volatile composition of the resulting wine. Second, and most important, this study had the objective to assess the potential of an alternative withering approach to modulate Corvina wine volatile composition. Although post-harvest withering is traditionally carried out in warehouses (locally called ”fruttaio”), there is an ongoing interest towards the exploration of alternative strategies that can be applied to obtain a suitable degree of over-ripening or withering, also with the aim of producing alternative wine types and styles [
2]. Among these, cane-cut on-vine has been shown to positively influence wine aroma and phenolic composition [
6,
7,
8,
9,
10,
11,
12]. In the present study, an alternative approach to on-vine withering, still based on blocking xylem flow but not involving cane cutting, was investigated in comparison with conventional fruttaio withering. As sugar levels at grape crush were similar for both withering modalities, any difference is expected to result from differences in grape composition in terms of secondary metabolites or interaction with yeast.
3.1. Influence of Grape Withering on Volatile Composition of Corvina Wines
Analysis of free and glycosidically-bound volatile compounds of the wines at bottling showed that withering of the grapes significantly affected wine aroma compounds, influencing the concentrations of various classes of volatiles. Free compounds can have a direct influence on wine aroma while the bound compounds can act as an aroma reservoir that is released during aging. At a general level, fermentation-derived volatiles such as esters, higher alcohols, and acids, as well as grape-related compounds such as certain norisoprenoids, were mostly associated with non-withered grapes, whereas withering resulted in higher wine content in terpenes and benzenoids (
Figure 1 and
Table 1). Among compounds known to impact red wine aroma, acetate esters (i.e., isoamyl acetate) and ethyl fatty acid esters (i.e., ethyl hexanoate and octanoate) were strongly influenced by withering, which resulted in a significant decrease in the concentration of nearly all the analyzed esters. Esters are related to red wine’s fruity character [
23] and are formed during alcoholic fermentation involving amino acid metabolism in the case of acetates, and fatty acid metabolism for ethyl esters [
24]. The production of esters by yeast is influenced by several factors, and different studies have reported an influence of grape maturity [
25] and levels of withering [
4,
9,
26] on wine ester content, suggesting and influence of must sugar content on ester production. In agreement with these reports, wines from withered Corvina grapes, which at harvest displayed additional 2 Brix compared to control grapes, showed lower ester content. In particular, acetate esters were more impacted, in spite of the fact that higher alcohol content, a precursor of acetates, was not so different across treatments and in some cases was even greater in withered samples. Ester/alcohol ratios were calculated to establish esterification rates of the different esters, and in the case of acetates it appeared clear that acetylation was much higher in fermentation of non-withered grapes (
Figure 3). Likewise, a higher acetylation rate was also observed for the control wine in the case of the ethyl ester of the branched chain fatty acid 3-methylbutanoic acid, also derived from amino acid metabolism. Conversely, although concertation of ethyl esters was higher in control samples, esterification of the corresponding fatty acid was similar in all treatments, so that it can be inferred that wine ester levels were determined by concentration of the corresponding fatty acid. It can be therefore assumed that, under our experimental conditions, withering impact on esters was due on one hand to reduced acetylation and on the other hand to reduced production of fatty acids, which would be in agreement with the observations of Saerens et al. (2008) [
27]. In the case of ethyl fatty acid esters, the reduced availability of short chain fatty acid precursors could be due to the greater availability of unsaturated fatty acids in musts from withered grapes [
28], which would result in reduced medium chain fatty acids biosynthesis [
29]. An increase in available lipids can also reduce the expression of the
ATF1 gene and therefore lower acetyl transferase activity catalyzing the acetylation reaction [
30]. Interestingly, ethyl acetate showed a completely different trend, its concentration increasing significantly in wines from withered grapes. Although acetyl transferase activities are expected to play a role in ethyl acetate formation by
S. cerevisiae, recent observations indicated that acetyl transferases other than Atf1 and Atf2 contribute significantly to production of this ester [
31], which could explain its different response to withering.
C
6 alcohols were also found to discriminate, with a high level of significance, control wines from the two withering modalities. C
6 alcohols contribute to the “leafy” and “herbaceous” odors of wines [
32]. In control samples
cis-3-hexenol showed and odor active value (OAV, calculated as ration between concentration and odor threshold) higher than one therefore potentially contributed to wine aroma (OAV = 1.3). Instead, in withered samples, the C
6 alcohols had OAV values lower than one. C
6 alcohols are formed during berry crushing by enzymatic oxidation of grape unsaturated fatty acids, initiated by grape lipoxygenase enzymes [
33]. Zenoni et al. (2016) [
17] reported a decrease in the expression of lipoxygenase genes during withering of Corvina, which could explain the decrease in C
6 alcohols observed here. However, other studies indicated an opposite trend [
8,
16], an increase in C
6 aldehydes and alcohols during postharvest grape dehydration of Malvasia grape was reported [
8], suggesting that more complex patterns could occur.
Various terpenes were affected by withering, although trends varied depending on the specific molecule. The importance of monoterpene alcohols and cyclic terpenes to Corvina wines aroma was recently described, in particular for linalool [
34,
35]. In the present study, linalool was the main monoterpene alcohol detected and its concentration was significantly decreased by withering, in agreement with previous findings [
17,
36]. Considering that in control wines linalool had an OAV = 1.1, a possible contribution to wine aroma characteristics can be expected, whereas in wines from withered grapes this was not the case (OAV = 0.59 and 0.66 for fruttaio and on-vine respectively). Terpenes are produced in grapes through both the 1-deoxy-
d-xylulose-5-phosphate/methylerythritol phosphate (DOXP/MEP) pathway and the mevalonic acid (MVA) pathway. In Corvina the influence of withering on these pathways is complex, with upper steps of the pathway being downregulated but late biosynthetic steps upregulated [
16]. In addition to free forms of terpenes, grapes also contain non-volatile glycosylated forms of these compounds, which in Corvina can contribute significantly to terpenes level in finished through enzymatic and chemical hydrolysis during vinification [
35,
37]. Although in the present study differences in glycosidically-bound terpenes in the finished wines were relatively small, we observed generally higher concentrations of bound terpenes in withered wines (
Table 2). Contrary to linalool, citronellol, the second most abundant monoterpene alcohols detected, increased with withering. In non-aromatic grapes such as Corvina, formation of citronellol is connected to the ability of yeast to reduce available geraniol including the portion derived from hydrolysis of geraniol glyosidic precursors [
37,
38]. Bound geraniol in finished wines increased in one of the withering modalities, supporting a possible contribution of bound geraniol to free citronellol levels. In wines from withered Pinot noir, Moreno et al. (2008) [
9] also observed increased wine citronellol content. Among other terpenes, withering was consistently associated with increased contents of linalool oxides, limonene and 1,4-cineole. Small increases in the content of terpinen-4-ol in wine were also observed with withering, in agreement with the observations of Zenoni et al. (2016) [
17]. The contribution of linalool oxides, limonene, 1,4-cineole and terpinen-4-ol to wine aroma seemed to be limited because their concentrations were found to be lower than the respective odor thresholds.
Comparted to terpenes, norisoprenoids, were affected to a smaller extent by withering, with concentration of the potent odorant β-damascenone decreasing. The formation of this compound during winemaking is associated with multiple pathways involving acid- or yeast-mediated hydrolysis of different precursors [
39,
40]. The negative influence of withering on damascenone wine content could be due to complex factors and requires further investigation, also considering that other norisoprenoids such as 3-oxo-α-ionol had an opposite behavior and were found in higher concentrations in withered samples.
The benzenoids ethyl vanillate, and benzyl alcohol were found in higher concentrations in withered samples, unlike previously reports by Bellincontro et al. (2016) [
4], albeit with more intense withering.
Considering that wines made from withered grapes are generally destined to age, the volatile profile evolution of withered and non-withered samples was investigated by means of a model aging protocol [
34]. Data showed that after model aging, the three sample modalities showed differences for a smaller number of compounds compared to young wines. Variations in compound concentrations are reported in
Table 5. Esters remained one major factor discriminating wines from withered grapes after aging (
Table 3 and
Figure 2), with control wines exhibiting higher levels of ethyl fatty acids and acetate esters. Trends during aging were, however, different, with withered wines typically showing reduced losses of even increases in some cases compared to control. Esters can be formed or degraded according to wine pH and the ester/acid ratio. As a consequence, esters produced in a higher amount by yeast during fermentation, such as isoamyl acetate, tend to decrease during aging while branched-chain fatty acid esters increase [
41,
42,
43]. Considering that the pH of the different samples was similar, we can conclude that by reducing esters formation during fermentation, withering resulted in reduced ester losses. One exception to this was observed for ethyl hexanoate, for which the rate of hydrolysis was similar in all treatments.
Aging patterns of certain terpenes also showed differences that could be associated with withering. For example, linalool in control wines during aging decreased from being sensorially active (OAV = 1.1) to an OAV < 1. Instead, in withered wines, linalool concentration tended to increase with aging. Particularly in the Fruttaio samples, after aging linalool had an increase of 8.3 µg/L, 1.5 times higher than young Fruttaio wines. This increase could be due to a higher content of glycosylated precursors in withered samples. However, the analysis of bound compounds in young wines did not show significant differences between samples that could explain the observed differences. It may be that different precursors forms of linalool exist in our samples that were not quantified with the employed method.
cis-Linalool oxide increased more markedly in wines from withered grapes, and this could be attributed to acid hydrolysis of glycosidic precursors (diendiol) [
44], and 3,7-dimethyloct-1-ene-3,6,7-triol (triol) [
45]. This last pathway seemed more consistent in this sample set, as both
cis- and
trans-linalool oxide bound precursors did not decrease with aging. 1,8-Cineole also displayed substantially different behaviors during aging between wines from withered and non-withered grapes, with concentration increasing during aging only in withered wines. This could be due to the fact that young wines from withered grapes exhibited significantly higher content of tepinen-4-ol, which we have recently shown to be a precursor to 1,8-cineole in Corvina wines [
34].
β-Damascenone evolution with aging also highlighted a major difference associated with withering. Evolution of β-damascenone in Corvina wines during aging is characterized by a complex trend with an initial increase followed by a decline [
34], reflecting simultaneous release from precursors (until available) followed by degradation through various reactions [
46,
47]. In the present study, the concentration of β-damascenone after model aging remained stable in control wines, and increased in withered samples reaching, in the case of on-vine samples, the level of control (
Figure 4). Samples withered in fruttaio showed the most important increase of β-damascenone.
The (
E)-1-(2,3,6-trimethylphenyl)buta-1,3-diene (TPB) also showed interesting differences in aged wines. A significant important increase has been observed after aging only in control samples. A slight increase occurred in samples withered in fruttaio while on-vine samples did not show any changes with aging. TPB has a tobacco aroma at low concentrations and geranium like odor at higher concentrations [
48]. Its concentration in Corvina wine has been correlated with wine ageing [
34]. It has been suggested that in red wine rich in tannins, TPB could react with polyphenols, resulting in a lower concentration like in Shiraz and Cabernet Sauvignon (Janusz 2003). It is reported that withered wines have a higher polyphenol content [
10,
12] while Corvina is known to be poor in polyphenols, so a TPB–tannin reaction may explain the lower content found in aged withered samples.
TDN variation was higher in fruttaio samples; however, it should be noticed that at the end of model aging samples of all the three modalities, it reached the same concentration level. TDN has a kerosene-like aroma, the concentration in wine was reported to be influenced by grape sun exposure, wine age, pH, and storage temperature [
49,
50]. Our data suggested that TDN concentration in wine was not affected by grape withering, the level of TDN formed in withered wine could depend on the TDN precursors accumulated just before the start of the withering process, harvest or peduncle twist.
The occurrence of aroma notes related to TDN is often associated with aged wines; however, in the control and on-vine samples, an OAV of 1.32 and 1.39, respectively, was observed already in the young wine, indicating a possible sensory contribution.