Influence of Skin-Contact Treatment on Aroma Profile of Malvasia Aromatica Wines in D.O

The effects of prefermentative cold skin-contact technique using Malvasia aromatica were studied as a first step to adapt to the climate change related effects in order to intensify the aroma potential of white wines of the D.O. “Vinos de Madrid” keeping the organoleptic characteristics of the region. Major volatile compounds were extracted by liquid–liquid extraction and quantified by GC-FID. Minor volatile compounds were determined by HS-SPME/GC–MS. Sensory analysis were also carried out to describe and quantify attributes of the wines. A total of 37 components were identified and quantified. Volatile components showed mixed behavior depending on the skin-contact time. Skin-contact for longer helps to enhance the floral character provided by some compounds contained in the skin, especially linalool and 2-phenyl etanol and were impact odorants of Malvasia aromatica wine based on odor activity values (OAVs).


Introduction
Skin-contact treatment has been proposed as a technique to try to increase the extraction of varietal aromas from the skins in different white cultivars [1][2][3]. It is a technique extensively used in the production of young white wines with the aim of improving their intensity and aroma profile by transferring free and glycosidically bound aroma compounds from the grape skins to the must before fermentation begins. The compounds responsible for the varietal aromas of wines depend on grape variety, climate, and soil and will determine the quality and local character of wines. Early winemaking procedures such as skin contact and the amount of pressure applied during pressing together with temperature conditions applied, will affect the extraction of aroma compounds and their precursors into the grape juice and consequently their concentrations in the resulting wine [4][5][6][7]. In the course of maceration, the concentration of aromas may increase in the must but there are not always changes at the sensory level in the wines. The varietal characteristics of the wine may be enhanced with the skin contact, however, there is some risk of the and then racked. The total acidity in the must was corrected with tartaric acid to 6 g/L. The must was racked, dividing the volume equally in three stainless steel tanks. Commercial yeast was added for its fermentation which took place at 16°C and was followed daily by measuring density. The conventional way samples (C) of each assay (1 and 2) were different from each other, they came from different grapes.
For the skin contact treatment, the grapes were destemmed and crushed. The pomace (musts and skin) was mixed 5 g/hl of sulfur dioxide, kept at 10°C for 18 h (A1) and 6 h (A2). At the end were pressed in a hand-press (M18 and M6 assays). The juice was settled, racked and divided as mentioned in the conventional way. The rest of the process was equal to the conventional way.

Physical-chemical analysis and fermentation kinetics
Oenological parameters (°Brix, free and total sulfur dioxide, pH, total acidity, volatile acidity, ethanol (% v/v) and residual sugars) were analyzed following OIV official methods [14]. Yeast assimilable nitrogen (YAN) was determined following the Sörensen method.
A daily control of temperature and density was carried out to determine the influence of pre-fermentative skin contact on the kinetics of the fermentations. Fermentation velocity (V F ) was measured checking daily the sugar percentage lost during the fermentation. On the other hand, V 50 amount of sugar daily transformed by the yeasts when 50% of the sugar content had been used up was also evaluated [15].

Aromatic analysis of the wines
Analysis of free aroma compounds was performed by quantification of minor and mayor volatile compounds. Quantification of major volatile compounds was undertaken by GC-FID (Agilent Technologies, Santa Clara, CA, USA) with a DB-Wax column (60 mx 0.32 mm x 0.5 m) from J&W Scientific (Folsom, CA, USA) following the procedures proposed by Ortega [16]. The liquid phase extraction (LPE) of aroma compounds was performed in dichloromethane. The method conditions were: oven temperature 40°C for 5 min, then increased to 3°C/ min up to 200°C, and helium as carrier gas at 2 ml/min. Two mL of aroma extract were injected at 250°C in splitless mode. The total run time was 75 minutes per sample. Analyses were carried out in duplicate.
Minor volatile compounds (terpenoids and C 13 -norisoprenoids) were determined by HS-SPME/GC-MS following the method proposed by Yuan & Qian [17]. A 50/30 μm DVB/CAR/PDMS fiber (Supelco Inc., Bellefonte, PA) was used for volatile extraction. 20 mL vials were used for chromatography (Agilent Technologies). Two mL of the wine sample were diluted with 8 mL of a citric acid solution (0.5 g/L citric acid, pH 3 saturated with sodium chloride) and 20 μL of 4-octanol (100 μg/L) was used as internal standard were added with a small magnetic stir bar. The vials were capped and equilibrated at 50°C in a thermostatic bath for 10 min. The aromatic compounds were extracted through SPME fiber for 50 min at 50°C with stirring (1000 rpm). The fiber was inserted into the injection port of the GC (230°C) to desorb the compounds. The injection into the chromatograph was manual. An Agilent 6890 gas chromatograph equipped with an Agilent 5973 mass selective detector (Agilent, Santa Clara, CA) was used. Compound separation was achieved with a DB-WAX de J&W Scientific (Folsom, CA, USA) (60 m x 0.32 mm x 0.5 μm film thickness, Phenomenex, Torrance, CA). A constant helium column flow rate of 1.0 mL/min was used. The chromatographic program was set at 40°C for 3 min, raised to 230°C at 5°C/min for 15 min. Splitless injection mode was used.

Sensory analysis
Descriptive sensory analyses were performed by a trained panel of 8 people (4 expert tasters and 4 habitual consumers) from the IMIDRA Institute. This panel had been previously trained in the recognition of wine flavor. Sensory descriptive analysis was performed to describe and quantify attributes of the wines based on a scale from 1 (low intensity) to 10 (high intensity). A hedonic classification was also carried out establishing the order of preference of the samples presented. The final score was obtained as the mean of the wine evaluations with their respective standard deviation and interpreted by graphical representation.

Statistical analyses
The statistical processing of the data was carried out with software SPSS ver. 20.0 (SPSS, Inc., Chicago, USA). Analysis of variance (ANOVA) was applied on oenological parameters, volatile compounds and sensory attributes of the wines. Tukey HSD post-hoc tests were used to establish the significance of differences between means to assess significance (p < 0.05).

General must and wine composition
General composition of must obtained with the two skin-contact treatment and conventional way from cv. Malvasia aromatica are given in Table 1. In the skincontact treatment assays, the total acidity of the must decreases along with a slight increase in pH. This is due to the transfer of cations from the skin to the must during the previous maceration stage, and results in a decrease of acidity in the form of potassium bitartrate together with a salification of the acids [18]. The results show an increase in Yeast assimilable nitrogen (YAN) according to the time of contact with the skin, being more notable with M18; M6 did not cause variations in YAN content. These results are in agreement with the studies carried out by other authors, where a period of contact with the skin favors the enrichment of the musts in terms of amino acid content [19,20]. In general, the effect of skin-contact in both assays is not very pronounced, which could be related to the low temperature  (10°C) and the time of contact, compared to other studies on white varieties (15.5°C, 20°C and 24°C [21]). Figure 1 shows the average of the fermentative kinetics evolution of Malvasia musts at 10°C. Skin contact for 6 hours does not influence the development of fermentation (a), no differences were found in terms of fermentation time and velocity between vinifications ( Table 2). However, in the case of skin-contact for 18 hours (b), there are differences in the time and velocity of fermentation compared to the conventional one. The macerated must concludes its fermentation almost a week before the conventional one. This fact may be related to the YAN content and its high content of nutrients and fermentation activators, which seem to have a strong influence on the process (see Table 1).

Fermentation kinetics
General composition of wines obtained with skin-contact treatment and conventional way from cv. Malvasia aromatica are given in Table 3. Wines from skin contact treatments had lower values for total acidity. There was no significant difference for any quality parameter that is in accordance with research published  studies [22][23][24]. As explained in point 2.2, the conventional way samples were different from each other, hence the difference in ethanol content.

Influence on aroma compounds
Varietal aromas from grapes, terpenols and C-13 and those from fermentation were determined. The aromatic compounds have been grouped by aromatic families: terpenols, C13, alcohols, lactones, acids, esters, aldehydes and ketones ( Table 4). These were 37 aromatic compounds studied from the three processing methods together with an analysis of variance to determine the influence of two maceration times (18 hours and 6 hours) on the total volatile content. In addition, the real contribution of each compound to the aroma of the wine was measured by the corresponding perception thresholds. Table 5 shows the odor threshold values (OTH) and their sensory descriptors for those compounds with odor activity values (OAVs) >1, which actively contribute to the aroma of the wines.
In both assays, skin contact treatment increased the total concentration of volatiles in wines compared to the control wine. From the A1, the control and M18 wines contained 303.9 and 413.9 mg/L and from A2, the control and M6 309.9 and 318.1 mg/L of volatiles, respectively. Similar results were found by other authors [6,28] on different varieties. Also, in a study carried out using a period of contact between the skins and the must of the Narince grape variety resulted in an increase of the aromatic content of the wines subjected to maceration [29].
Higher alcohols were the most abundant family of volatile compounds in the four winemaking processes, contributing more than 90% of the total volatile   content analyzed. Higher alcohols, in quantities below 300 mg/L can contribute to improving the aromatic complexity of white wines, however are considered to be a negative factor in terms of aromatic quality when they exceed 400 mg/l [30]. Isobutanol, isoamyl alcohol and 2-phenylethanol were the most abundant in the four wines analyzed. Among the higher alcohols, M18 has increased the levels of 2-phenylethanol being 5.3 ( Table 5). This compound is related to floral aromas with attributes of roses and is considered to contribute positively to wine aroma [31]. There has been a significant decrease of 1-hexanol y cis-3-hexen-1-ol in M18 wines in comparison to the control. These compounds are related to herbaceous aromas and bitter taste so are unfavorable to wine quality. Skin contact treatment for 18 h resulted in significant increase in the concentration of the esters ethyl 3-hydroxybutyrate and ethyl hexanoate esters, however, the concentrations of ethyl butyrate, isoamyl acetate and hexyl acetate decreased with the maceration time.
Esters are very important for the aroma of wine, they are related to fruity aromas [32]. Due to their high OAVs ( Table 5), ethyl butyrate (apple), ethyl isovalerate (orange), isoamyl acetate (banana), ethyl hexanoate (green apple) and 2-phenylethyl acetate (flowers) should be considered as important contributors to the typical aroma of Malvasia wines. In the case of M6 no differences were found on any of the esters studied so we can conclude that maceration for a reduced period of time has not affected the ester content of the resulting wines. Eight terpenes were identified in the wines, among them, linalool, β-citronelol and geraniol increased significantly with M18 while with M6 only β-citronelol increased significantly. Ninety percent of geraniol is in the skins, while linalool is distributed 50% between the skin and 50% in the pulp [33,34]. Other authors [35] reported high concentrations of geraniol and its derived products throughout the ripening process in Malvasia grapes. Only linalool reached concentrations above its odor threshold in all wines, with the highest significant extraction in M18 wines.  This terpene gives the wine floral and citrus notes ( Table 5) typical of Muscat because it is one of the main compounds involved in the typical aromas of this variety [36]. Similar results were found by other authors in wines from white varieties for this family of compounds [23,37]. β-damascenone was the only compound from the C13-norisoprenoid family found in Malvasia wines. The concentration of this compound decreases with skincontact time, showing a significant decrease in M18 wine. C13 come from the carotenoids degradation and the hydrolysis of their glycosylated forms. In young wines they are usually present in the form of glycoconjugates [38,39]. According to the OAVs, in all wines β-damascenone is above its perception threshold and should be considered as an important compound in the aroma of Malvasia wines ( Table 5). Provides floral aromas with lilac attributes [17]. Other authors agree with these results for this variety [40].
The most abundant fatty acids in the wines were hexanoic and octanoic acid ( Table 4). These results are in agreement with those found by other authors [3,41,42]. The maceration seems to have different effects depending on the compound and the contact time between the skin and the must. In the case of M18 wines, the total concentration of fatty acids decreases, being particularly significant in octanoic acid. In M6 wines the total concentration of fatty acids increased significantly for hexanoic,  [25,26]. c [27]. octanoic and decanoic acid regards to the conventional way. In all wines, regardless of the increase or decrease produced as a result of skin-contact, isovaleric, hexanoic and octanoic acids have OAVs >1 so must to be accounted in the aroma of Malvasia wines ( Table 5). Regarding the group of aldehydes and ketones, it is known that alterations due to oxidation processes, imply the appearance of unpleasant aromas (cooked vegetables) related to the presence of compounds such as benzaldehyde, acetoin, hexanal, methional etc. [43]. Acetoin and benzaldehyde were detected in the control and M18 wines, with a significant increase in both with the maceration process (p < 0.05 and p < 0.001 respectively). According to [44] on the Verdejo grape variety, the presence of acetoin in white wines is considered negative for the flavor. In both cases, acetoin and benzaldehyde concentrations are below their perception threshold 150 mg/L [45] and 5 mg/L [46]. The two treatments (M18 and M6) significantly increased the concentration of γ-butyrolactone respect to the conventional way but in all cases it was far from its OTH (35 mg/L [47]).

Influence on sensory profile of wines
Wines were evaluated using descriptive and preference tests. The olfactory phase of the Malvasia wines from assay 1 (A) and assay 2 (B) is shown in Figure 2.
The macerated wine (a) 18 hours had a higher score in the descriptors of altered aroma due to problems during the conservation process of the M18 wine. Tasters also indicated oxidation aromas in M18 sample with a significance level of p < 0.001. The conventional wine in assay 1 was scored positively on overall aroma quality and fruity character (p < 0.01). In spite of the above-mentioned defects, the M18 wine received the highest score in floral character, being significantly superior to the control wine. This fact is in consonance with the results obtained in the aroma profile of these wines (see Table 4). In Figure 2(b), M6 wines score higher in terms of fruit and floral aromatic intensity (p < 0.05). The rest of the parameters obtained similar scores regarding their control. Figure 3 contain graphs of the taste using different winemaking methods. The results of the taste evaluation of in assay 1 (a), show significant differences in favor of C wine in overall taste quality (p < 0.001), bitterness (p < 0.01) and fruity character (p < 0.01). This could be related to the oxidation suffered by the M18. In case of assay 2, M6 wine (b) received the highest score in the fruity character with respect to the control (p < 0.01). This fact may be related to the release of varietal aromas through the hydrolysis of aromatic precursors by the enzymatic activity over the period of conservation in the bottle.
In the preference test, in A1 the preferences were shared between the M18 and C wines. The most preferred wine was the one produced with a 6 h skin contact treatment in the A2.

Conclusions
This first study in order to combat climate change related effects, the aromatic profile of Malvasia wines winemaking with different skin-contact time shows some relevant conclusions. Volatile components showed mixed behavior depending on the skin-contact time. Some compounds increased in concentration with time, while others decreased. Skin-contact for longer helps to enhance the floral character provided by the terpenols contained in the skin, especially linalool, major alcohols such a 2-phenylethanol. It also helps the increase of some esters (ethyl 3-hydroxy butyrate, ethyl hexanoate and 2-phenylethyl acetate) and the loss of others (isoamyl acetate, ethyl isovalerate and ethyl butyrate), all related to the fruity character of the wines. Short skin-contact does not cause significant effect on the content of terpenols, or ester content. The β-damascenone remains constant during M6 period, on the contrary, decreases significantly in case of M18. In general, the results of the sensory analysis show a preference for wines macerated for 6 hours. The wines macerated for 18 hours highlighted their floral character. The skin-contact process needs more studies at different time periods to optimize the aromatic potential of the grape and wine and oenological and conservation conditions of the wine.