3.1. Dehydration Kinetics and Berries Chemical Composition
Usually, a dehydration process conducted on berries pre-treated with an alkaline treatment leads to faster weight loss when compared with the normal on-wine or off-vine grape withering process [
10,
13,
40,
41,
42]. Berries weight loss (WL) during dehydration, mainly due to water loss, was rapid during the first 8 days of dehydration (up to 50% WL, about 6% of WL for day) and then slowed down from day 8 to 13 (WL interval of 15%, about 3% of WL for day;
Table 1). The absolute berry weight decreased consistently from 4.97 g for fresh berries to 1.63 g for the maximum dehydration tested (up to 65% WL). The decreased speed of water loss found during the process is in agreement with the berries lowered water content [
43]. Indeed, drying kinetics are reported as not constant during the process because after certain dehydration values, the channels clogging limits the amount of water spread and evaporation into the atmosphere [
43,
44,
45,
46]. In particular, the dehydration phenomenon is explained by mass transfer, which is mainly dependent on water and sugar content. Water can spread in a liquid state by capillary action from an area of higher water content to another of lower content. It passes from the pulp to the skin as the speed of dehydration of the skin is greater than that of the pulp. The sugars retrieve the water via osmosis and at the same time, they partly move, with the water, as far as the cells of the skin surface that act as a barrier [
43]. Here, the sugars retain a certain quantity of water that, as it is remaining in the matrix, does not reach the atmosphere (osmotic effect). A further phenomenon is that hygroscopicity counters the action of osmosis. With the increase in temperature, there is an increase in the requirement of water in the atmosphere and the physical state of the sugars also changes [
45]. This phenomenon happens even at temperatures of almost 40 °C, similar to those reached during sun drying of Zibibbo grapes in Sicily island, from which the wine
Passito di Pantelleria is produced [
2].
The temperature at which the grapes were maintained in this study (30 °C ± 1 and 30% RH) would have prompted a considerably lower speed of dehydration had the grapes not been treated with sodium hydroxide. It is hypothesized that in an environment with basic pH, it is possible to remove part of the bloom of the berry and increase dehydration speed. As well as a change in relative humidity and the atmospheric requirement for water, the speed of diffusion would also change, especially if drying does not take place in a controlled environment. This phenomenon is also accentuated by the formation or exposure of micro-fissures on the berry surface, through which evaporation is facilitated. Other treatments (such as ethyl oleate) should also allow one to achieve such objectives. To confirm this, previous studies examined the effect of alkaline solutions, obtained with potassium carbonate (K
2CO
3), on the speed of dehydration [
21,
44,
47,
48,
49]. In our conditions and according to previous findings, the greatest water loss recorded in the first 8 days seems to be due to this phenomenon (creation of preferential hydrophilic pathways with a quicker passage of water). During the successive stages of dehydration, from 8 to 13 days, lower WL percentages were found and the wrinkling of the skin was observed. This effect could be ascribed to pores clogging and to micro-fissures created on the skin by pre-treatment with sodium hydroxide, limiting the movement of water by the creation of a hydrophobic barrier.
Considering sugar contents (
Table 1), fastest water loss corresponded to higher quantities of sugar accumulated in the outer part of the pulp. In our experimental conditions, sugar levels increased considerably from 189 g/L of fresh grapes to 266 g/L at 35% WL and 344 g/L at 50% WL (about 41% and 82% increase, respectively, compared with fresh grapes,
p < 0.001). Reducing sugars rose less in the final phase (387 g/L at 65% WL, about a 105% increase compared with fresh grapes), in accordance with the lower water loss. The titratable acidity show significant changes during water loss and the decrease in total acidity values in the first stage of the dehydration process could be ascribed to the metabolism effect of malic acid, which is in agreement with a previous study (7). The pH value was not significantly affected by dehydration (
p > 0.05) and ranged from 3.20 (fresh grape) up to 3.26 at 35% WL, whereas it barely changed between 50% WL and 65% WL (pH = 3.25 and 3.24, respectively [
7]).
3.2. Free Volatile Compounds
In the present study, 19 free volatile compounds were identified and quantified in fresh and dehydrated grapes
cv Muscat of Alexandria. These included 17 terpene compounds and two alcohols. In order to evaluate the evolution of volatile compounds both from a physiological (biosynthesis/degradation ratio) and technological point of view (final grapes concentration), the data are reported in μg/100 berries (
Table 2) and μg/kg of berries (
Table 3), respectively.
Considering the physiological aspects, as reported in
Table 2, several free terpene monohydroxylate alcohols were affected by the different dehydration levels. Among these compounds, known as varietal markers with aromatic character of sweet, rose-like, flowery notes [
28,
29,
30,
33,
50], linalool, and geraniol, are those quantitatively most important (532 and 269 µg/100 berries, respectively), which is in agreement with earlier studies on
cv Muscat of Alexandria [
5,
35,
51,
52]. In contrast with these studies, lower values of nerol were found (36 µg/100 berries). Other terpene alcohols were found in relevant concentrations, such as 2,6-dimethyl-3,7-octadiene-2,6-diol and 3,7-dimethyl-1,7-octadiene-3,6-diol (391 and 94 µg/100 berries, respectively),
trans- and
cis-pyran-linalool oxides (192 and 94 µg/100 berries, respectively), and
trans-geranic acid (217 µg/100 berries). Lower values of α-terpineol, both 8-hydroxy-linalool isomers, hydroxy-geraniol,
trans- and
cis-furan-linalool oxides, and hotrienol were found in fresh grapes, ranging between 15 and 35 µg/100 berries. Relevantly, hotrienol may be derived from 2,6-dimethyl-3,7-octadiene-2,6-diol as extraction artefact or by H
+ catalyzed hydrolysis of 2,6-dimethyl-3,7-Octadiene-2,6-diol [
53,
54]. Small amounts of citronellol and 2,6-dimethyl-7-octadiene-2,6-diol (3 and 5 µg/100 berries, respectively) were also found.
Several significant differences were found during the dehydration process for terpene compounds, except the two furan-linalool oxide isomers, hotrienol, citronellol, 2,6-dimethyl-3,7-octadiene-2,6-diol, cis-8-hydroxy-linalool, and hydroxy-geraniol. After 5 days of dehydration, at 35% WL, the content of linalool dropped significantly (from 532 to 53 µg/100 berries, p < 0.001), whereas geraniol remained almost unchanged (from 268 to 215 µg/100 berries; p > 0.05). Subsequently, at 50% and 65% WL, the linalool content remained unchanged (42 and 51 µg/100 berries, respectively; p > 0.05), in contrast to geraniol, which dropped significantly (80 and 28 µg/100 berries, for 50% and 65% WL, respectively). At 35% WL, the concentration of trans-pyran linalool oxide also decreased (from 192 to 100 µg/100 berries, p < 0.001), as well as at 50% and 60% WL (44 and 24 µg/100 berries, respectively, p < 0.001).
Among the other terpenols, the content of
cis-pyran-linalool oxide was reduced at 65% WL (49 µg/100 berries,
p < 0.001). This increase was also found for 2,6-dimethyl-7-octadiene-2,6-diol (ranging from 5.2 to 26 µg/100 berries between fresh and 65% WL, respectively). These compounds’ increase may be explained by linalool decrease. In fact, linalool has been proposed as the substrate for conversion to higher oxidation state compounds such as hydroxy-linalool derivatives (2,6-dimethyl-3,7-octadiene-2,6-diol and 2,6-dimethyl-1,7-octadiene-2,6-diol) [
35]. Particularly, the increases of these two diols during sun drying of Muscat of Alexandria grapes in Pantelleria island was previously reported by [
35]. On the other hand, the significant increase of content of α-terpineol during the entire dehydration process, even if small, could be due to H
+ catalysed reaction on linalool, nerol, and geraniol [
55], which are by contrast reduced in our experimental condition. The same trend was found for
trans-geranic acid, which progressively decreased from 217 µg/100 berries in fresh grapes to 19 µg/100 berries in 65% WL dehydration point.
Concerning the benzenoids found, the evolution of 2-phenylethanol did not show a regular trend because of the possible interference of yeasts in the production of this compound, due to micro-fermentation which may occur during the process [
56], while the trend of benzyl alcohol showed an inconstant behaviour during dehydration (
Table 2).
Free volatile compounds expressed in µg/kg of berries showed a relevant effect of the concentration given by the water loss by increasing volatile aroma compounds during the dehydration process (
Table 3). In fact, reporting the data in μg/kg of berries is conditioned by the fact that the number of berries needed to form 1 kg of grapes increases with increasing dehydration and this aspect allows us to better represent the actual winemaking condition. Considering terpenes, data expressed in μg/kg showed a significant decrease in linalool,
trans-pyran linalool oxide, and geranic acid during dehydration (all
p < 0.001), which is in accordance with the reduction found when expressed as μg/100 berries (
Table 2). By contrast, the contents of
trans-furan linalool oxide, hotrienol, α-terpineol, 2,6-dimethyl-3,7-octadiene-2,6-diol, and 2,6-dimethyl-7-octadiene-2,6-diol increased (all
p < 0.001). Considering other terpenes detected, an uneven trend was reported. In particular, the μg/kg contents of nerol and geraniol showed a trend increase at 35% WL (
p < 0.05 for nerol;
p > 0.05 for geraniol) and then a reduction at 65% WL. The opposite behaviour was found for linalool (the most abundant terpene alcohol found and typical aroma marker), with an initial strong decrease during the dehydration up to 35% WL that remained almost unchanged at 50% WL and then an increase at 65% WL, while the concentration of
cis-piran linalool oxide increased at 50 WL% and decreased at 65% WL. This behaviour is in accordance with previous data found on
cv Muscat of Alexandria drying in both sun-drying and controlled-room conditions [
35].
The geraniol trend showed that this compound was slightly involved in the degradation reactions occurring just after NaOH pre-treatment, but not in the subsequent reactions during prolonged dehydration. On the other hand, linalool was very sensitive, leading to an initial drop of concentration in both 35% and 50% WL samples. The behaviour of these two alcohols suggested that radical oxidation may occur during the first stage of dehydration, speeded up by reactive oxygen species (ROS) [
57]. Consequently, linalool, which is a tertiary alcohol, would be more sensitive to these reactions than geraniol, justifying its more rapid decrease. A second mechanism of terpene alcohols degradation may be imputable to catalysed H
+ hydrolytic reactions [
57], which could be responsible for the geraniol decrease. The hydrolysis is probably responsible for the increases of α-terpineol and hotrienol, while the ROS catalysed reactions may be involved in the increase of 2,6-dimethyl-3,7-octadiene-2,6-diol during dehydration (all
p < 0.001).
Notably, considering the μg/kg berries data, it is possible to understand the final concentration of odorant compounds with respect to their sensory threshold. At the end of the dehydration (65% WL), linalool, geraniol, and hotrienol were found to be above their odour threshold (expressed as μg/kg berries) and they are among the mainly volatile varietal markers of
cv Muscat of Alexandria [
4,
58].
In general, total content of terpenes expressed in µg/100 berries (
Table 2) underwent a reduction of -28.0%, -47.1%, and -52.9% for 35, 50, and 65% WL, respectively, with respect to the fresh grapes (
p < 0.01). However, the data expressed in µg/kg of berries (
Table 3) showed an increased concentration during the dehydration process (+18.0%, +25.4% and +56.5% for 35%, 50%, and 65% WL respectively, with respect to fresh grapes,
p < 0.05). Therefore, the aroma compounds concentration effect is higher than degradation reactions, although final grapes’ aromatic profile is changed depending on the susceptibility of individual compounds to several oxidative and hydrolytic reactions and on their reaction products accumulation. As well, the effects of pre-treatment and dehydration had an impact on the terpenes, considerably changing the aroma profile of dehydrated grapes compared to the fresh ones. The results obtained are in agreement with [
4], whereas a slight contrast was found with the results of
cv Muscat of Alexandria drying at different levels of dehydration reported by [
5], demonstrating the complexity of subsequent reactions that are involved in water loss process. However, a shared result is that if the dehydration rate is limited (35% WL, “
Passolata” type), with the exception of linalool, an increase in content can be seen with certain terpenes, such as geraniol and nerol (data in μg/kg berries), resulting in a terpene profile with a predominance of geraniol.
3.3. Glycosylated Volatile Compounds
The glycosylated volatile composition of fresh and dried grapes is shown in
Table 4 (µg/100 berries) and
Table 5 (µg/kg berries), accounting for 36 compounds detected (27 terpenes, 4 norisoprenoids, and 5 benzenoids). With respect to free volatile compounds reported in
Table 2 and
Table 3, other compounds have been identified in the enzymatic hydrolysis product of fresh and dehydrated grapes’ glycoconjugate precursors. Relevantly,
trans-8-hydroxy-nerol,
trans- and
cis-8-hydroxy-geraniol, and 2,6-dimethyl-6-hydroxy-2,7-Octadienoic acid are products of H
+ catalyzed transformation of 8-hydroxy linalool [
59,
60]. Moreover, compounds belonging to the norisoprenoids class (4-oxo-α-damascone, 3,4-dihydro-3-oxo-actinidol isomer 1, 3-oxo-α-ionol, and vomifoliol) and benzenoids (4-vinylguaiacol, dihydrocoliferyl alcohol, and vanillin) were identified and quantified.
Terpenols (µg/100 berries) were the most abundant class of compounds identified as glycoconjugates in fresh and dehydrated berries. Among these, linalool, 2,6-dimethyl-3,7-octadiene-2,6-diol, geraniol, the two 8-hydroxy linalool isomers, nerol, and
trans-geranic acid were quantitatively the most important compounds found (
Table 4). A slight decrease was generally observed in the concentration of most of the terpene compounds during dehydration. Nevertheless, the difference between the contents of such compounds were significant only for linalool, hydroxy nerol, and hydroxy geraniol (
p < 0.001) and for geranial, hydroxy citronellol,
p-menth-1-ene-7,8-diol,
trans-8-hydroxy geraniol, and
trans-geranic acid (
p < 0.05). The content of terpene compounds (from enzymatic hydrolysis of heterosidic fraction) such as linalool and
trans-geranic acid decreased from the fresh berries to the 65% WL (
p < 0.05), while geraniol, 3,7-dimethyl-1,7-octadiene-3,6-diol, and
cis-8-hydroxy-linalool marked non-significant decreases for the same dehydration process (
p > 0.05). The biggest drop was found in linalool from fresh to 35% WL, but with this decrease, a significant (
p < 0.05) increase of hydroxy geraniol corresponded and a not significant (
p > 0.05) increase of 2,6-dimethyl-3,7-octadiene-2,6-diol,
trans- and
cis-furan-linalool oxide, hotrienol, and α-terpineol. Nevertheless, at 50% and 65% WL, these compounds also decreased. The evolution of the other terpene compounds was less regular, even if the content of almost all decreased at 65% WL.
In contrast, the content of the norisoprenoids recovered after enzymatic hydrolysis of the heterosidic fraction generally increased from fresh to the dehydrated berries, as well as total benzenoids, although both increases were not significant as total compounds (
p > 0.05,
Table 4).
Considering the concentration in µg/kg berries, the content of total terpenes, norisoprenoids, and benzenoids compounds increased significantly during the dehydration process (
Table 5). This significant increase of aroma precursors has been reported elsewhere during both grape ripening [
58] and dehydration [
4]. Considering the individual glycosylated terpenes, the dehydrated 65% WL berries were significantly richer with respect to the fresh grapes in several terpenes, with values at least tripled in the case of α-terpineol,
cis-furan-linalool oxide, hydroxy-citronellol, hydroxy-nerol,
trans-8-hydroxy-linalool, hydroxy-geraniol,
p-menth-1-ene-7,8-diol,
trans-8-hydroxy-nerol, and
trans-8-hydroxy-geraniol.
The percentage variation of the total content of glycosylated terpene compounds (µg/100 berries), compared to fresh grapes during drying, is about −4.2, −16.6, and −28.6% at 35%, 50%, and 65% WL, respectively (
Table 4). On the contrary, when data are expressed in µg/kg berries, the variation from fresh grapes is about +45.2%, +71.2%, and 104.3% at each of the three levels of dehydration considered (
Table 5).
Glycosylated norisoprenoids and benzenoid compounds significantly increased their content (µg/kg berries) during the dehydration process up to almost four fold at the end of dehydration with respect to their initial concentration in fresh grapes (
Table 5). This increase is mainly related to the loss of water associated with the dehydration process, which in turn justifies the higher volatile conjugates content when expressed in μg/kg berries, but a not significant (
p > 0.05) increase for these compounds’ classes was also found for the data expressed as µg/100 berries.
Generally, glycosylated aromatic compounds are less affected by the dehydration process with respect to the corresponding free aromas, due to the protection of sugar (glucose or disaccharides) against degradation or transformation reactions. Nevertheless, even if to a lesser extent, the decrease in free linalool content (µg/100 berries), as well as in other free terpene compounds, from fresh to dehydrated berries, especially at 35% WL, may be due to free radical oxidation reactions, induced by the presence of ROS, such as hydrogen peroxide, during the dehydration [
57]. Besides oxidative reactions, catalysed H
+ hydrolysis may occur and in the case of glycosylated forms, would first lead to the production of the respective free forms and then to their transformation into H
+ catalysed forms [
57]. In our findings, the low presence of the respective free form transformation products (e.g., hotrienol, α-terpineol, and diols derived from the hydration of linalool, nerol, and geraniol) suggests that oxidation reactions prevailed over H+ catalysed reactions. Considering the compounds’ decrease kinetics, these oxidation reactions reached the maximum rate in the first phase of the dehydration process (35% WL) and then continued more slowly, which may be given by total hydrogen peroxide consumption. A previous work [
35] showed that these oxidation reactions can occur independently from the dehydration process, to which the grapes have been subjected. Among the oxidation products, 2,6-dimethyl-3,7-Octadiene-2,6-diol increase was previously reported [
61,
62]. In our case, its concentration, although found to increase, was not significantly different during dehydration (
p > 0.05), whereas other oxidation products such as 2,6-dimethyl-7-Octadiene-2,6-diol significantly increased during the dehydration (
p < 0.05;
Table 5). On the other hand, catalysed H
+ reactions can also be confirmed in the glycosides fraction by the presence of 8-hydroxy nerol and 8-hydroxy geraniol derived from the attack of H
+ on the -OH group in position 6 of the glycosides of 8-hydroxy linalool [
58].
Considering norisoprenoids, their low glycosylated contents are consistent with the characteristics of the aromatic varieties Muscat of Alexandria and Moscato bianco [
4,
28,
29] and their increasing behaviour is barely affected (
p > 0.05 for all compounds except 3-oxo-α-damascenone) during dehydration when the content of 100 berries is considered. The same trends were found for glycosylated benzenoid compounds. Both classes’ contents were significantly increased by the water loss, leading to a significantly higher concentration when the concentration in µg/kg berries is considered, to a different extent depending on the sampling points. As a consequence, due to the lower degradation suffered by volatile glycosylated forms, their content generally increased from fresh berries to those at different levels of dehydration. Finally, the content of the individual compounds detected in 1 kg of dehydrated grapes was higher than that present in 1 kg of fresh grapes. Therefore, in our experimental conditions, a gain of potential aroma precursors was found. This increase was also important for the glycosylated forms of linalool, the compound most affected by oxidative degradation reactions, underlying the effectiveness of the dehydration process in increasing the aromatic potential besides degradative reactions.
3.4. Berry Phenolic Compounds and Mechanical Properties of the Berry Skin
As described in the introduction section, in the production of Sicilian DOC special wines, “Passolata”, “Bionda”, and “Malaga” dehydrated berry grapes are added to a base wine obtained from fresh grapes (< 10% of alcohol) to continue the fermentation process, however in different ratios according to the function of the type and style of wine. Therefore, whole berries are subjected to a maceration process in a medium rich in ethanol, leading to phenolic compounds extraction.
Table 6 summarizes the effect of the dehydration process on total flavonoids content (TFI) in skin, pulp, and seeds and the textural modifications of the skin. Significant differences among fresh grapes and dehydrated grapes at different levels were observed. In particular, when data are expressed as mg/100 berries, an important decrease TFI in grape skins was observed in the first phase (35% WL), followed by an increased concentration. Also, when the data are expressed in mg/kg of berries, a strong loss of flavonoids was detected from fresh and “
Passolata” dehydrated grapes (35% WL). This decrease (from 308 to 217 mg/kg berries) is mainly to be ascribed to metabolites oxidation, as observed in other studies [
5,
13,
40]. On the contrary, a substantial and significant increase of flavonoid content was observed for 50% WL (“
Bionda” dehydrated grapes type) and 65% WL (“
Malaga”), which is related to lower berry weight (i.e., 2.31 g and 1.63 g for 50% and 65% WL, respectively;
Table 1). Therefore, the increase in flavonoid compounds can be attributed to the prevalence of the concentration effect on account of water loss over decomposition oxidisation phenomena, although a possible flavonoid biosynthesis cannot be excluded.
However, it is conceivable that part of the flavonoid losses that occurred in the skins are given by some of these compounds passing from the skin to the pulp. The TFI in the berry juice did not report significant (
p > 0.05) differences from fresh grapes (584 mg/L) to 35% WL and 50% WL (719 and 745 mg/L, respectively), although an increasing trend was evidenced. At 65% WL, the analyses were not performed due to the semisolid and crystalline firmness of the pulp. Higher pulp TFI content is also justified by water loss rather than oxidative degradation; anyway, an extraction from skin to pulp is also given by a decrease in skin hardness. In this sense, break skin force values (Fsk) decreased significantly (
p < 0.001) during the dehydration process from 0.694 N (fresh grapes) to 0.193 N (dehydrated berries at 65% WL). If on the one hand the reduction in skin hardness led to an acceleration of the dehydrating kinetics of the grapes, as widely demonstrated by scientific literature [
13,
16], on the other hand, the phenomena of extractability of the phenolic substances are accelerated [
63].
During
Passito winemaking, the dehydrated “
Passolata”, “
Bionda”, and “
Malaga” berries are added as whole berries to a base wine. In this specific context, from a technological point of view, the polyphenols’ contribution given by the seeds can be considered less relevant due to the lower direct contact that they may have with liquid base wine, with respect to a traditional maceration. However, in long macerations, berries degradation may lead to seeds releasing, coming into contact with the base wine (10%
v/v ethanol), promoting polyphenols extraction. In this sense, the seeds belonging to berries dehydrated at 35% and 50% WL showed similar contents to fresh grapes when flavonoids are expressed as mg/kg berries (about 1200 mg/kg berries). Only at 65% WL did the seeds’ TFI content increase significantly when expressed as concentration on berry weight (> 4000mg/kg berries; 250 mg/100 seeds,
p < 0.001) due to extreme weight loss sustained by the berries. Therefore, the addition of this dehydration type to the base wine may lead to a higher risk of seeds’ polyphenols extraction, potentially impacting on the chromatic and sensory characteristics [
2].
During grape ripening, seeds’ histological and histochemical modifications occurs, with intensive lignification and hardening of the medium integument and the presence of phenolic compounds in the inner integument that can affect phenols release [
64]. To our knowledge, histological studies of dehydrated grape seeds that support the evidence of this highest extractability of flavonoids of berries at 65% WL are not still present in literature, although an increase of oligomer and polymer tannin content of seeds during the withering process was already noticed [
65].