How Pre-Harvest Inactivated Yeast Treatment May Inﬂuence the Norisoprenoid Aroma Potential in Wine Grapes

: Carotenoids are important secondary metabolites in wine grapes and play a key role as potential precursors of aroma compounds (i.e., C 13 -norisoprenoids), which have a high sensorial impact in wines. There is scarce information about the inﬂuence of pre-harvest inactivated yeast treatment on the norisoprenoid aroma potential of grapes. Thus, this work aimed to study the e ﬀ ect of the foliar application of yeast extracts (YE) to Negro Amaro and Primitivo grapevines on the carotenoid content during grape ripening and the di ﬀ erence between the resulting v é raison and maturity ( ∆ C). The results showed that β -carotene and (all E )-lutein were the most abundant carotenoids in all samples, ranging from 60% to 70% of total compounds. Their levels, as well as those of violaxanthin, (9 (cid:48) Z )-neoxanthin, and 5,6-epoxylutein, decreased during ripening. This was especially observed in treated grapes, with ∆ C values from 2.6 to 4.2-fold higher than in untreated grapes. Besides this, a principal components analysis (PCA) demonstrated that lutein, β -carotene, and violaxanthin and (9 (cid:48) Z )-neoxanthin derivatives principally characterized Negro Amaro and Primitivo, respectively. Thereby, the YE treatment has proved to be e ﬀ ective in improving the C 13 -norisoprenoid aroma potentiality of Negro Amaro and Primitivo, which are fundamental cultivars in the context of Italian wine production.


Introduction
The presence of carotenoids in grape berries is well documented [1]. 5,6-epoxyxanthophylls and their 5,8-epoxy isomers were identified together with the most common carotenes and xanthophylls (i.e., β-carotene and lutein), the content of which was established to decrease during grape ripening from véraison to harvest [2][3][4]. The grape variety and viticulture practices, but also climate conditions and geographic origin, can influence the qualitative and quantitative profile of carotenoids in berries [5][6][7].
Structurally, carotenoids are C 40 tetraterpenoids with a long chromophore of conjugated double bonds; thus, they can confer from red to yellow coloration to fruit [8]. However, especially in red/black grapes, this function is ascribed to anthocyanins and polyphenols [9]. Instead, carotenoids are mainly known as precursors of volatile compounds (i.e., C 13 -norisoprenoids), with very low olfactory perception thresholds. Thus, they have a fundamental role in defining the varietal aroma of grapes and wines [10,11]. C 13 -norisoprenoids form by the direct enzymatic oxidation of carotenoids, such

Plant Materials
The experiment was conducted in 2019 on a 10 year-old commercial vineyard of Negro Amaro cv. located in the Salice Salentino wine grape area (Masseria Filippi, Apulia region, Southern Italy) and a 6 year-old commercial vineyard of Primitivo cv. located in the Gioia del Colle D.OC. (Denomination of Controlled Origin) area (Azienda Benagiano, Apulia region, Southern Italy). Both Negro Amaro and Primitivo vines, grafted onto Vitis berlandieri × Vitis rupestris 1103 Paulsen rootstock, were planted in north-south oriented rows; they were spaced 2.15 m between rows and 0.90 m on the row, trained to a Vertical Shoot Positioned (VSP) system and spur-pruned with 14 buds per vine.
The experimental design for the current study was a randomized complete block with four replicates, with three rows of each treatment (80 vines per row) separated by a buffer row. Treatment consisted in two canopy spraying times, the first at the beginning of veráison (5%) and the second 12 days later, with Lalvigne ® MATURE diluted in water without adjuvant at 1 kg/ha dose (YE) in comparison to untreated rows used as control (C). Lalvigne ® MATURE is a formulation of 100% natural inactivated wine yeast (Saccharomyces cerevisiae) derivatives, non-pathogenic, non-hazardous, food grade and non-GMO, which are specifically designed to be used with the patented foliar application technology WO/2014/024039 (Lallemand Inc., Montreal, QC, Canada).
A random sample of three bunches for each repetition was manually picked between the third and seventh node from random vines at periodic intervals from August until September. Berries (~30 g) were then chosen at random from these bunches and stored in the dark at −80 • C until analysis.

Maturation Indexes of Grapes
Total soluble solids (TSS), titratable acidity (TA), and pH were determined according to protocols established by the OIV, 1990 [23]. Berries were crushed to determine the TSS (expressed as g/L) of berry juice using a portable refractometer (ATAGO PR32). TA (as g/L of tartaric acid equivalents) was also determined for the juice by titrating with 0.1 N sodium hydroxide to the bromothymol blue end point. Finally, juice pH was measured by the pH meter CRISON BASIC 20.

Extraction of Carotenoids from Grapes
The carotenoid extraction procedure was adapted from the method of Crupi et al. 2010 [4]. Approximately 30 g of freeze berries containing 25 µL of BHA (12.66 mg/mL in EtOH) were crushed in an IKA A11 basic homogenizer (IKA, WERKE GMBH & CO.KG, Staufen, Germany) for 5 min in the presence of magnesium carbonate basic (2 to 4 g for mature and green berries, respectively). The homogenate was diluted with 40 mL of distillated water and spiked with 100 µL of internal standard (150 µg/mL of β-apo-8 -carotenal). Extraction was done with 40 mL of hexane/diethyl ether (1:1, v/v) by agitating the mixture for 30 min. The resulting upper layer was separated by centrifugation at 4000 rpm and 4 • C for 1 min (EPPENDORF 5810R, Eppendorf AG, Hamburg, Germany). The extraction procedure was repeated twice for the lower phase using 20 mL of hexane/diethyl ether (1:1, v/v). The pooled extract was evaporated to dryness using a rotovapor Buchi-R-205. The residue was dissolved in 2 mL of acetone/hexane (1:1, v/v), filtered through 0.20 µm syringe PTFE filters and stored at −20 • C until the carotenoid analysis by HPLC-DAD-MS.

HPLC-DAD-MS Analyses of Carotenoids
HPLC-DAD-MS analyses were carried out with an Agilent model 1100 equipped with quaternary pump solvent delivery, a thermostated column compartment, diode array detector, and XCT-trap mass detector (Agilent Technologies, Palo Alto, CA, USA). A positive electrospray mode was used for the ionization of molecules with acquisition of mass spectra between m/z 100 and 1200, capillary voltage at −4000 V, and skimmer voltage at 30 V. The reversed stationary phase employed was a YMC pack C 30 (YMC Inc., Wilmington, NC, USA) 5 µm (250 × 3 mm i.d.) with a precolumn C 30 5 µm (20 × 3 mm i.d.). The following gradient system was used with H 2 O (solvent A), methanol (solvent B), and tert-butyl methyl ether (solvent C): 0 min, %A-%B-%C, 40-60-0; 5 min, %A-%B-%C, 20-80-0; 10 min, %A-%B-%C, 4-81-15; 60 min, %A-%B-%C, 4-11-85; 65 min, %A-%B-%C, 4-11-85; 70 min, %A-%B-%C, 40-60-0. Stop time at 70 min with a re-equilibration time of 20 min corresponding to ca 3.0 column volumes (Vc = 1.3 mL). The column was kept at 20 • C, the flow was maintained at 0.2 mL/min, and the sample injection volume was 3 µL. Diode array detection was between 250 and 700 nm and absorbance was recorded at 447 nm. Positive electrospray mode was used for the ionization of molecules with a capillary voltage at −4000 V and skimmer voltage at 40 V. The nebulizer pressure was 15 psi and the nitrogen flow rate was 5 L/min. The temperature of drying gas was 350 • C. In the full scan mode, the monitored mass range was from m/z 100 to 1200. MS 2 was performed by using helium as the collision gas at a pressure of 4.6 × 10 −6 mbar. Collision induced dissociation (CID) spectra were obtained with an isolation width of 4.0 m/z for precursor ions and a fragmentation amplitude of 0.6 V for epoxyxanthophylls, and 1.0 V for the other carotenoids.
Compound identification was achieved by combining different information: the positions of absorption maxima (λ max ), the degree of vibration fine structure (% III/II), the ratio of the absorbance of the cis peak to the absorbance of the second absorption band in the visible region, known as the Q ratio or D B /D II [8,24], and the capacity factor values k' and mass spectra were compared with those from pure standards and interpreted with the help of structural models already hypothesized in the literature (such as (9Z) or (9 Z)-lutein and the other lutein-like structures reported in Table 1, which were only tentatively identified) [4]. a %III/II is the ratio of the height of the longest-wavelength absorption peak, designated III, and that of the middle absorption peak, designated as II, taking the minimum between the two peaks as a baseline [8]; b The Q ratio is the quotient between the cis peak band and band II (normally λ max. ) [24]. The fragments corresponding to the base peak in MS 2 spectra were underlined.
To evaluate linearity, calibration curves with seven concentration points for each compound were prepared separately. Calibration was performed by a linear regression of peak-area ratios of the carotenoids to the internal standard (β-apo-8 -carotenal) versus the respective standard concentration. The precision of the method was determined by calculating the intraday and interday repeatability, expressed as standard deviation (SD) (σ) and relative standard deviation (RSD) in terms of retention times and peak width (W 1/2 ), by the injection of four different replicates of extracts on the same day or over three consecutive days. The detection limit (LOD) and quantification limit (LOQ) were calculated on the basis of the calibration curve of low concentrations of target compounds and defined as LOD = 3*(Syx/b) and LOQ = 10*(Syx/b), where b is the slope and Syx is the standard error of the calibration curve (Supplementary Materials, Table S1).

Statistical Analysis
Data were analyzed by the STATISTICA 8.0 (StatSoft Inc., Tulxa, OK, USA, 2012) software package. Specifically, after testing their normal distribution by Shapiro-Wilk's W test together with their homoscedasticity by means of the Levene test, a two-way multivariate analysis of variance (MANOVA) was performed for the HPLC-DAD-quantified carotenoids in order to evaluate the effects of the factors "treatment" and "ripening" on each cultivar. The Tukey HSD post hoc test was used to separate the means (p < 0.05) when the interaction between the factors was significant.
Finally, a principal components analysis (PCA) was performed on variables corresponding to the difference between the values of the identified carotenoids at véraison and maturity to cluster samples per treatment. The PCA was done on the correlation matrix in order to treat all variables on an equal footing, in consecutive steps (as reported by Martì et al. 2004 [25]) and starting from those variables which were shown to be significant through the MANOVA.
It is worth noting that consistency in carotenoid attribution is very complex due to their structural diversity. In particular, the double bond-conjugated system is susceptible to heat, light, oxygen, and acids, giving rise to unwanted cis-trans isomerization. Using antioxidants (i.e., BHA), an ice bath, and minimizing the light exposition of the samples during the extraction steps are mandatory to prevent the formation of cis-isomers as artifact compounds [26]. Therefore, alongside the main trans carotenoids identified, β-carotene and (allE)-lutein, accounting for 60% and 70% of total carotenoids in mature Primitivo and Negro Amaro grapes [7], we were also confident that the cis-isomers (assigned as 9Z-lutein and 9Z-β-carotene) were present in the grape tissue (Table 1).
Furthermore, the absence of flavoxanthin and chrysantemaxanthin, originating from 5,6-epoxylutein ( Figure 2), also confirmed this speculation. Although the lack of these two xanthophylls appeared to disagree with our past report [4], it is worth noting that the carotenoid composition is often influenced by climate and viticulture practices, such as trellis systems [27]. It is worth noting that consistency in carotenoid attribution is very complex due to their structural diversity. In particular, the double bond-conjugated system is susceptible to heat, light, oxygen, and acids, giving rise to unwanted cis-trans isomerization. Using antioxidants (i.e., BHA), an ice bath, and minimizing the light exposition of the samples during the extraction steps are mandatory to prevent the formation of cis-isomers as artifact compounds [26]. Therefore, alongside the main trans carotenoids identified, β-carotene and (allE)-lutein, accounting for 60% and 70% of total carotenoids in mature Primitivo and Negro Amaro grapes [7], we were also confident that the cis-isomers (assigned as 9Z-lutein and 9Z-β-carotene) were present in the grape tissue (Table 1).

Effect of Pre-Harvest YE Application on Carotenoid Content
According to the choice of the main figures of merit reported elsewhere [28], the adopted HPLC-DAD method was validated for the target analysis of carotenoids in terms of linearity, LOD, LOQ, and precision (Supplementary Materials, Table S1). The calibration curves were obtained by the internal standard method to give determination coefficients (R 2 ) which were higher than 0.99; moreover, the detection and quantification limits were found to be under 2 and 5 μg/kg, respectively. As regards precision tests, intra-assay and inter-assay coefficients of variation were calculated, with retention times ranging between 0.11% and 0.13%, and 0.37% and 0.50%, respectively, while corresponding measures for W1/2 were between 2.9% and 15%, and 2.8% and 12%, respectively. These results confirmed the accuracy and reproducibility of data obtained by the analytical method for carotenoid analysis. Tables 2 and 3 listed the changes in carotene and xanthophyll concentrations (expressed in μg/kg of berries) during the grape ripening of the two varieties, Negro Amaro and Primitivo, in control and YE-treated samples. As expected, total carotenoids significantly decreased from véraison to maturity, even more consistently in Negro Amaro (F = 34.06, p < 0.001), due to the higher accumulation of the compounds in the first sampling date, respective to Primitivo (F = 19.54, p < 0.001) (Figure 4). Regarding the effect of the elicitor (YE), the treatment factor (T) as well as its interaction with ripening (TxR) resulted in a more pronounced effect in the variation of total carotenoid content in Primitivo (T: F = 29.11, p < 0.001; TxR: F = 7.06, p < 0.001) than Negro Amaro (T: F = 5.55, p = 0.0047; TxR: F = 6.05, p = 0.0021). This difference was mostly influenced by the anomalous behavior of the P0813 control sample; the lowest carotenoid value could be ascribed to an experimental drawback that occurred during the sampling and/or extraction phase, as suggested by the extraction yields in the four replicates, which were low (Supplementary Materials, Figure S1).

Effect of Pre-Harvest YE Application on Carotenoid Content
According to the choice of the main figures of merit reported elsewhere [28], the adopted HPLC-DAD method was validated for the target analysis of carotenoids in terms of linearity, LOD, LOQ, and precision (Supplementary Materials, Table S1). The calibration curves were obtained by the internal standard method to give determination coefficients (R 2 ) which were higher than 0.99; moreover, the detection and quantification limits were found to be under 2 and 5 µg/kg, respectively. As regards precision tests, intra-assay and inter-assay coefficients of variation were calculated, with retention times ranging between 0.11% and 0.13%, and 0.37% and 0.50%, respectively, while corresponding measures for W 1/2 were between 2.9% and 15%, and 2.8% and 12%, respectively. These results confirmed the accuracy and reproducibility of data obtained by the analytical method for carotenoid analysis. Tables 2 and 3 listed the changes in carotene and xanthophyll concentrations (expressed in µg/kg of berries) during the grape ripening of the two varieties, Negro Amaro and Primitivo, in control and YE-treated samples. As expected, total carotenoids significantly decreased from véraison to maturity, even more consistently in Negro Amaro (F = 34.06, p < 0.001), due to the higher accumulation of the compounds in the first sampling date, respective to Primitivo (F = 19.54, p < 0.001) (Figure 4). Regarding the effect of the elicitor (YE), the treatment factor (T) as well as its interaction with ripening (TxR) resulted in a more pronounced effect in the variation of total carotenoid content in Primitivo (T: F = 29.11, p < 0.001; TxR: F = 7.06, p < 0.001) than Negro Amaro (T: F = 5.55, p = 0.0047; TxR: F = 6.05, p = 0.0021). This difference was mostly influenced by the anomalous behavior of the P0813 control sample; the lowest carotenoid value could be ascribed to an experimental drawback that occurred during the sampling and/or extraction phase, as suggested by the extraction yields in the four replicates, which were low (Supplementary Materials, Figure S1).   β-carotene and (allE)-lutein were the main compounds in terms of concentration in all analyzed samples. The xanthophyll level was reduced more drastically than the carotene level, reaching values down to 60%-70% in mature grapes compared to the setting (Tables 2 and 3). Moreover, our data clearly showed that the at-harvest β-carotene content was greater than lutein in both varieties, which was in accordance with our previous report [7]. Geographic origin is recognized to affect the relative (allE)-lutein and β-carotene contents [2]. In some regions (northern Spain and southern Italy), βcarotene prevailed in grapes at maturity [7,20] in contrast to other cultural situations (northern Italy and southern France) in which the xanthophyll level was higher than the carotene level [1,3].
The interaction between treatment and ripening was very significant for lutein variation (p < 0.001) in both varieties (Figure 5a). YE-treated grapes showed an enhanced content of lutein only at véraison; then, they had lower levels in the following samples until reaching 200 μg/kg and 249 μg/kg of berries in Negro Amaro and Primitivo, respectively (Tables 2 and 3). An oscillating trend was observed in the case of β-carotene (Figure 5b). YE treatment conditioned the level of variation of this carotenoid less homogenously in Negro Amaro (TxR: F = 5.04, p = 0.008) and, especially, in Primitivo (TxR: F = 4.43, p = 0.013). However, a common reduction of the compound between YE (270 μg/kg and 370 μg/kg) and control samples (470 μg/kg and 426 μg/kg) was recorded in both cultivars (Tables  2 and 3). These findings were in disagreement with those of Gutierrez-Gamboa et al., 2018 [20], who reported that YE foliar application triggered β-carotene and lutein accumulation in mature grapes; however, they studied its effect on different varieties (Tempranillo, Graciano, and Garnacha). β-carotene and (allE)-lutein were the main compounds in terms of concentration in all analyzed samples. The xanthophyll level was reduced more drastically than the carotene level, reaching values down to 60%-70% in mature grapes compared to the setting (Tables 2 and 3). Moreover, our data clearly showed that the at-harvest β-carotene content was greater than lutein in both varieties, which was in accordance with our previous report [7]. Geographic origin is recognized to affect the relative (allE)-lutein and β-carotene contents [2]. In some regions (northern Spain and southern Italy), β-carotene prevailed in grapes at maturity [7,20] in contrast to other cultural situations (northern Italy and southern France) in which the xanthophyll level was higher than the carotene level [1,3].
The interaction between treatment and ripening was very significant for lutein variation (p < 0.001) in both varieties (Figure 5a). YE-treated grapes showed an enhanced content of lutein only at véraison; then, they had lower levels in the following samples until reaching 200 µg/kg and 249 µg/kg of berries in Negro Amaro and Primitivo, respectively (Tables 2 and 3). An oscillating trend was observed in the case of β-carotene (Figure 5b). YE treatment conditioned the level of variation of this carotenoid less homogenously in Negro Amaro (TxR: F = 5.04, p = 0.008) and, especially, in Primitivo (TxR: F = 4.43, p = 0.013). However, a common reduction of the compound between YE (270 µg/kg and 370 µg/kg) and control samples (470 µg/kg and 426 µg/kg) was recorded in both cultivars (Tables 2 and 3). These findings were in disagreement with those of Gutierrez-Gamboa et al., 2018 [20], who reported that YE foliar application triggered β-carotene and lutein accumulation in mature grapes; however, they studied its effect on different varieties (Tempranillo, Graciano, and Garnacha). Finally, concerning the identified 5,6-epoxyxanthophylls, the data confirmed the difficulty of defining the border between the end of storage and the beginning of degradation, mainly in the case of 5,6-epoxylutein (Figure 6c) [7]. Nevertheless, the elicitor treatment caused a significant reduction in the concentrations of violaxanthin, (9′Z)-neoxanthin, and 5,6-epoxylutein, as particularly evident in the two last sampling dates of Primitivo grapes ( Figure 6; Tables 2 and 3). Finally, concerning the identified 5,6-epoxyxanthophylls, the data confirmed the difficulty of defining the border between the end of storage and the beginning of degradation, mainly in the case of 5,6-epoxylutein (Figure 6c) [7]. Nevertheless, the elicitor treatment caused a significant reduction in the concentrations of violaxanthin, (9 Z)-neoxanthin, and 5,6-epoxylutein, as particularly evident in the two last sampling dates of Primitivo grapes ( Figure 6; Tables 2 and 3). Finally, concerning the identified 5,6-epoxyxanthophylls, the data confirmed the difficulty of defining the border between the end of storage and the beginning of degradation, mainly in the case of 5,6-epoxylutein (Figure 6c) [7]. Nevertheless, the elicitor treatment caused a significant reduction in the concentrations of violaxanthin, (9′Z)-neoxanthin, and 5,6-epoxylutein, as particularly evident in the two last sampling dates of Primitivo grapes ( Figure 6; Tables 2 and 3).

Influence of YE Treatment on the Norisoprenoid Aroma Potential (ΔC)
As accepted, C13-norisoprenoid varietal aroma in wine grapes is strictly related to carotenoid degradation during ripening [5,29]. Therefore, determining the difference of total carotenoids between véraison and harvest points (ΔC) together with their decreasing kinetics can be a useful tool for defining the aroma potentiality of grapes for wine making [7]. On the other side, elicitor treatment by foliar application in vineyards has proved to influence the development of aroma compounds in wines [19]; in particular, grapevines treated with benzothiadiazole have been shown to increase the concentrations of some C13-norisoprenoids, such as β-damascenone and β -ionone, while the use of methyl jasmonate did not alter their content [30].
In our work, a similar change in norisoprenoid aroma potential was shown by control samples of Negro Amaro and Primitivo, with ΔC values of 270 μg/kg and 290 μg/kg, respectively (Tables 2  and 3). This finding was in agreement with data from a 3 year-long study conducted on the same cultivars in Apulia [7]. Moreover, the YE treatment significantly affected the aroma potential of the grapes, increasing the ΔCs 2.6 and 4.2-fold in Primitivo (740 μg/kg) and Negro Amaro (1130 μg/kg), respectively (Tables 2 and 3). In the literature, it is well documented that the use of elicitors (i.e., methyl jasmonate, chitosan, yeast extracts) stimulates plant defense mechanisms and consequently activates the enzymes responsible for the biosynthesis of secondary metabolites, such as phenolic compounds [16,31]. YE caused an accumulation of anthocyanins, stilbenes, and flavonoids in wine grapes without varying their phenological maturation [18,32,33]. Since there was no significant difference in maturation indexes between YE and C samples (Tables 2 and 3), the effect of treatment

Influence of YE Treatment on the Norisoprenoid Aroma Potential (∆C)
As accepted, C 13 -norisoprenoid varietal aroma in wine grapes is strictly related to carotenoid degradation during ripening [5,29]. Therefore, determining the difference of total carotenoids between véraison and harvest points (∆C) together with their decreasing kinetics can be a useful tool for defining the aroma potentiality of grapes for wine making [7]. On the other side, elicitor treatment by foliar application in vineyards has proved to influence the development of aroma compounds in wines [19]; in particular, grapevines treated with benzothiadiazole have been shown to increase the concentrations of some C 13 -norisoprenoids, such as β-damascenone and β -ionone, while the use of methyl jasmonate did not alter their content [30].
In our work, a similar change in norisoprenoid aroma potential was shown by control samples of Negro Amaro and Primitivo, with ∆C values of 270 µg/kg and 290 µg/kg, respectively (Tables 2  and 3). This finding was in agreement with data from a 3 year-long study conducted on the same cultivars in Apulia [7]. Moreover, the YE treatment significantly affected the aroma potential of the grapes, increasing the ∆Cs 2.6 and 4.2-fold in Primitivo (740 µg/kg) and Negro Amaro (1130 µg/kg), respectively (Tables 2 and 3). In the literature, it is well documented that the use of elicitors (i.e., methyl jasmonate, chitosan, yeast extracts) stimulates plant defense mechanisms and consequently activates the enzymes responsible for the biosynthesis of secondary metabolites, such as phenolic compounds [16,31]. YE caused an accumulation of anthocyanins, stilbenes, and flavonoids in wine grapes without varying their phenological maturation [18,32,33]. Since there was no significant difference in maturation indexes between YE and C samples (Tables 2 and 3), the effect of treatment on ∆C variation could be attributed to the activation of enzymes catalyzing either the biosynthesis of carotenoids (as proved by the highest values being in YE grapes at véraison) or their degradation (as confirmed by the lowest values being in YE grapes at harvest) ( Figure 4).
The knowledge of the carotenoid profile of grapes is linked to the type of norisoprenoid compound which will form in wine. β-ionone is derived from β-carotene oxidation, whereas 3-hydroxy-α-ionone and 3-oxo-α-ionol originate by lutein cleavage [11,12]. The factor score plot (accounting for 82.06% of total variance) from the PCA performed on the variables corresponding to the difference of the identified carotenoid level between véraison and harvest in both YE and C grape samples is shown in Figure 7. This clearly shows that YE-treated grapes of Negro Amaro and Primitivo were different from C samples along PC 1 and PC 2 , respectively, and they were more correlated to carotenoid-derivative variables. Furthermore, NYE was mainly characterized by lutein, β-carotene, and violaxanthin derivatives (factor coordinates: −0.9535, −0.8657, and −0.8643, respectively, onto PC 1 ), while PYE was principally characterized by a higher concentration of (9 Z)-neoxanthin derivatives (factor coordinate: −0.8492 onto PC 2 ) (Figure 7).
Appl. Sci. 2020, 10, x FOR PEER REVIEW 2 of 16 on ΔC variation could be attributed to the activation of enzymes catalyzing either the biosynthesis of carotenoids (as proved by the highest values being in YE grapes at véraison) or their degradation (as confirmed by the lowest values being in YE grapes at harvest) ( Figure 4). The knowledge of the carotenoid profile of grapes is linked to the type of norisoprenoid compound which will form in wine. β-ionone is derived from β-carotene oxidation, whereas 3hydroxy-α-ionone and 3-oxo-α-ionol originate by lutein cleavage [11,12]. The factor score plot (accounting for 82.06% of total variance) from the PCA performed on the variables corresponding to the difference of the identified carotenoid level between véraison and harvest in both YE and C grape samples is shown in Figure 7. This clearly shows that YE-treated grapes of Negro Amaro and Primitivo were different from C samples along PC1 and PC2, respectively, and they were more correlated to carotenoid-derivative variables. Furthermore, NYE was mainly characterized by lutein, β-carotene, and violaxanthin derivatives (factor coordinates: −0.9535, −0.8657, and −0.8643, respectively, onto PC1), while PYE was principally characterized by a higher concentration of (9′Z)neoxanthin derivatives (factor coordinate: −0.8492 onto PC2) (Figure 7).

Conclusions
Fifteen carotenoids, including (allE)-lutein and β-carotene together with their 9Z isomers and 5,6-/5,8-epoxyxanthophylls, were tentatively identified by matching UV-Vis characteristics, MS spectra, and elution order, and were accurately quantified by the used analytical method. As expected, a decrease of carotenoids during ripening was observed in both the two cultivars, but this was even more noticeable in grapes treated with YE. β-carotene and (allE)-lutein were present as the main compounds in all the analyzed samples, and the level of xanthophyll appeared to be more drastically reduced than the carotene level, especially in mature Negro Amaro and Primitivo treated grapes. Similarly, the elicitor treatment caused a more consistent decrease of violaxanthin, (9′Z)neoxanthin, and 5,6-epoxylutein.

Conclusions
Fifteen carotenoids, including (allE)-lutein and β-carotene together with their 9Z isomers and 5,6-/5,8-epoxyxanthophylls, were tentatively identified by matching UV-Vis characteristics, MS spectra, and elution order, and were accurately quantified by the used analytical method. As expected, a decrease of carotenoids during ripening was observed in both the two cultivars, but this was even more noticeable in grapes treated with YE. β-carotene and (allE)-lutein were present as the main compounds in all the analyzed samples, and the level of xanthophyll appeared to be more drastically reduced than the carotene level, especially in mature Negro Amaro and Primitivo treated grapes. Similarly, the elicitor treatment caused a more consistent decrease of violaxanthin, (9 Z)-neoxanthin, and 5,6-epoxylutein.
Thereby, because carotenoids serve as precursors of C 13 -norisoprenoids, the YE treatment proved to be determinant in enhancing the aroma potential (∆C) of both varieties up to four-fold compared to untreated grapes. Besides this, Negro Amaro and Primitivo were principally characterized by lutein, β-carotene, and violaxanthin derivatives, and (9 Z)-neoxanthin derivatives, respectively. However, further research is ongoing to confirm the real characteristics of these grapes in obtaining wines which are richer in sensorial impact C 13 aroma compounds, such as β-ionone and β-damascenone.
Funding: This research received no external funding.