Characterization of Brazilian Syrah winter wines at bottling and after ageing

Double pruning extended the harvest season of wine grape (Vitis vinifera L.) to dry winter, enabling production of high quality wines in the southeastern Brazil. Winter harvest allows grapes to fulfill not only technological maturation, but also phenolic ripeness. Winter wines from Syrah grapes harvested from eight vineyards in southeastern Brazil during three harvests were analyzed for their chemical and aromatic composition after bottling and after ageing for 20, 30, and 42 months in bottle. Winter wines have high content of total phenolic compounds, which remained almost constant through ageing, as well as color intensity. Malvidin 3-O-glucoside stood out among anthocyanins, remaining 5-10 % after 39 months of ageing. Moreover, malvidin 3-O-glucoside-pyruvic acid was the main pyranoanthocyanin identified in winter wine. Polymerized pigments index ranged from 54 % at bottling to 80 % after 42 months of ageing. Young winter wines are rich in ester and monoterpene, as well as alcoholic volatile compounds responsible for ethereal, fruity, flowery, fresh and sweet aromas. Aged winter wines showed higher contents of furfural, geranyl ethyl ether, isoamyl decanoate, α-muurolene and α-calacorene, contributing to sweet, fruity and woody aromas. Syrah winter wines are characterized by high content of phenolic compounds and color stability, and keep good sensorial characteristics after ageing in bottle.


Introduction
The use of double pruning enables to harvest grapes under climatic conditions that favor grape ripening Palliotti et al., 2017;Toda et al., 2019). In southeastern Brazil, this technique allowed vineyard dissemination for high quality fine wine production .
Wines elaborated from grapes harvested at winter season under low water availability and high thermal amplitude have higher alcoholic and phenols content as well as color intensity than summer wines (Mota et al., 2009). Similar results were obtained with controlled water deficits in 'Cabernet sauvignon' vines (Cáceres-Mella et al., 2018). The authors observed that wines from less irrigated vines exhibited high levels of total phenols, anthocyanins and chroma and therefore showed higher color intensity and sensorial perception of more fullness. Phenolic compounds are the main component of color and mouthfeel in wine, also affecting its ageing ability. Double pruning favors phenolic maturation mainly by high thermal amplitude and low rainfall during autumnwinter season .
Besides color, aroma is a wine characteristic immediately observed and highly appreciated by consumers. The presence of compounds, such as alcohols, acids, aldehydes, lactones, benzenes, esters, ketones, terpenes, originating from grapes or formed during the fermentation process and ageing, are responsible for the aroma profile of the wine (Vidal and Segurel, 2005;Belda et al., 2017).
Among the cultivars tested under double-pruning management in the southeastern Brazil (Mota et al., 2010, Syrah vines showed the best adaptation to this management. Winter Syrah wines have been well accepted by consumers, which increased the interest in management of this vineyard in Brazil (Salettes, 2016); however, there is little information on the composition of these wines and the effect of ageing.
As far as the authors know, there is no information in the literature about aromatic profile of Syrah wines from winter harvest. Therefore, this work investigated the volatile aroma compounds in Syrah wines from winter harvest and the effect of bottle ageing in the composition of these wines. Syrah winter wine composition Sci. Agric. v.78, n.3, e20190233, 2021 were removed to avoid harvest in the summer season. In January of the subsequent year, the lignified shoots were pruned to allow the productive cycle during the autumn-winter season .
Grapes were harvested on average with 23.1 °Brix, pH 3.61 and total acidity 5.92 g L -1 tartaric acid. Not all vineyards were represented in the three seasons due to environmental damages or erroneous management that compromised vine production.
Harvested bunches were stored at 4 °C for 24 h at the winery. For each region, 200 kg of grape clusters were destemmed, crushed and placed in 200 L inox fermentation tanks. The musts were inoculated with rehydrated wine yeast Saccharomyces cerevisiae × S. kudriavzevii and added with 80 mg SO 2 kg −1 .
Wine density was determined daily during alcoholic fermentation performed at 21 °C. When the density reached approximately 990 g L −1 , wines were racked to 100 L stainless steel tanks. Malolactic fermentation was carried out with native bacteria flora at 21 °C and the presence of malic acid was routinely followed by the paper chromatography method (Amerine and Ough, 1980). When malic acid was not detected by paper chromatography, wines were carefully racked to avoid lees, added with 35 mg SO 2 L −1 and kept at 3 °C for 15 d to allow tartaric stabilization. The wines were bottled in 750 mL green glass bottles closed with natural cork and allowed to age in lying position at 15 °C in a dark cell for 20, 30 or 42 months.

Monomeric Anthocyanin extraction
Anthocyanins and polymeric anthocyanins identification were performed in Syrah winter wines produced in São Bento, Três Pontas, Indaiatuba, Santo Antônio do Amparo and São Sebastião do Paraíso at bottling and after 19 and 36 months of ageing.
Identification and quantification of monomeric anthocyanins were performed by the HPLC-DAD and LC-ESI-MS/MS analyses. Wines samples (5 mL) were concentrated under vacuum at 40 °C on a rotary evaporator to remove the alcohol, and filled up to 10 mL with ultrapure water prior to application to a solid-phase extraction polyamide column (1 g) previously conditioned with methanol and ultrapure water. The de-alcoholized sample (2 mL) was loaded into the column and washed with ultrapure water and eluted with 0.3 % HCl in methanol. The eluates were completely dried using a rotary evaporator under vacuum at 40 °C, resuspended in 5 % acetic acid in methanol and filtered through a 0.45 μm PTFE filter. For the polymeric anthocyanin analysis, wine was filtered through a 0.45 μm PTFE and injected directly.

LC-ESI-MS/MS and HPLC-DAD conditions
Anthocyanins were identified by LC-ESI-MS/MS using a Prominence Liquid Chromatograph attached to an ion trap Esquires-LC mass spectrometer with an electrospray ionization (ESI) interface. We used a 5-μ Prodigy ODS3 column (4.60 × 250 mm) with a flow rate of 1 mL min -1 at 25 °C. The mobile phase consisted of solvent A, water ultrapure, formic acid and acetonitrile (95:1:3, v/v/v) and solvent B, water, formic acid and acetonitrile (48:1:51, v/v/v). Anthocyanins were detected at 525 nm (Teixeira et al., 2015). For application to the mass spectrometer after DAD detection, the flow rate was reduced to 0.2 mL min -1 Mass spectrometer operated with collision energies of -3500 V and N 2 like dry gas with ESI in the positive mode using a full scan from m/z 100 to 1500. Compounds were identified according to comparison with the retention time of authentic standards when possible, as well as by absorption spectrum similarity, mass spectral characteristics and by comparison with the literature data. The calibration curve was performed by injecting the standards malvidin 3-O-glucoside, peonidin 3-O-glucoside, petunidin 3-O-glucoside, delphinidin 3-O-glucoside three times at five different concentrations. The acylated form of anthocyanins with coumaroyl and acetyl groups were quantified using the calibration curve of their respective O-glucoside form. The results were expressed as mg 100 mL -1 .

Pyranoanthocyanins analysis by LC-qTOF-MS/MS and HPLC-DAD conditions
The identification of the polymeric anthocyanins was performed using a Prominence Liquid Chromatograph linked to a qTOF mass spectrometer Compact model. The LC condition was reverse phase Luna 3 μ C18 (150 × 3.0 mm) at 25 °C. The solvent gradient condition was: phase A: 0.5 % formic acid in ultrapure water and phase B: 0.5 % formic acid in acetonitrile at a flow rate of 0.5 mL min -1 . The mass spectrometer conditions were: positive mode, N 2 like dry gas, capillary -3500 V, scan m/z 50-1500. The polymeric anthocyanins were identified comparing the mass spectra with data available in the literature (Blanco-Vega et al., 2014).

Volatile extraction and analysis
For the isolation and concentration of volatiles, the headspace solid-phase microextraction technique (HS-SPME) was used according to Gürbüz et al. (2006) with some modifications. All extractions were carried out using a DVB/CAR/PDMS fiber, of 50/30 μm film thickness. An aliquot of 10 g of wine was placed in 20 mL vials closed with Teflon cap. Vials were heated to 30 °C under agitation with a magnetic stir bar for 10 min for headspace equilibrium. Adsorption time was 45 min at the same temperature. The SPME fiber was then injected directly into a gas chromatograph mass spectrometer operating with ChemStation software. The SPME fiber was held for 10 min at 250 °C for desorption of volatile compounds, which were separated in HP-5MS (30 m × 0.25 mm × 0.25 μm) capillary column with helium as carrier gas at constant flow of 1 mL min -1 . Initial oven temperature was 40 °C held for 5 min, then increased to 160 °C at 3 °C min -1 and to 250 °C at 10 °C min -1 and kept for 10 min before returning to 40 °C, in a total cycle of 64 min; transfer line temperature at 250 °C; MS detector in SCAN mode 30-500 m/z.
Volatile compounds were tentatively identified by comparison with the NIST library considering 70 % similarity as the cut-off, further confirming the results with the retention indexes calculated according to the Kovats Index and compared to data reported on Nist Webbook (https://webbook.nist.gov), Chemspider (www. chemspider.com) or PubChem (www.pubchem.ncbi. nlm.nih.gov) websites. Only aromatic compounds with difference in Kovats Retention Indices lower than 70 units up or down were accepted. All analyses were carried out in triplicate.

Statistical analysis
The Partial Least Squares Discriminant Analysis (PLS-DA) was performed to investigate the trends or group formations of wines from different ageing times on all volatile compounds in the wine samples analyzed by the MetaboAnalyst Program (www.metaboanalyst.ca).
Ageing decreased the total monomeric anthocyanin content and increased color hue and the polymerized pigments index, with no changes in color intensity (Tables 2 and 3 (Table 4), remaining 5-10 % of the concentration at bottling after 36 months of ageing.
Contribution of yellow component to the overall wine color was on average 38 % at bottling reaching 41 % after 42 months of ageing, while red color changed from 49 % to 46 % in the same period. Blue component (OD 620) was almost constant during ageing (Tables 1 and 3).
Polymerized pigments ranged from 54 % at bottling to 80 % after 42 months of ageing (Tables 1 and  3). Although not all wine samples were analyzed at 30 and 42 months after ageing, it seems that polymerized pigments reached maximum values after 30 months of ageing and remained constant afterwards.
The Partial Least Squares Discriminant Analysis (PLS-DA) accounted for 63 % of the total data variance. The two-dimensional graph showed a clear separation of aromatic compounds from bottled wines to those over 30 months ageing (Figure 1). Table 7 summarizes all the aromatic volatile compounds tentatively identified in the samples, regardless of vineyard and ageing. Esters represented the principal class of compounds with 40 aromatic volatile compounds identified, followed by terpenes (17 compounds), benzene (14), and alcohol (12). Esters were found mainly at bottling, but their concentration increased slightly at 30 months ageing.

Discussion
Winter wines composition resemble that of Syrah wines from traditional regions, such as Australia (Antalick et al., 2015), Italy (Condurso et al., 2016), California (Brillante et al., 2018), and South Africa (Hunter and Volschenk, 2018) confirming the great potential of this technique for Brazilian viticulture.
The anthocyanins identified in the Brazilian Syrah winter wines were 3-O-glucoside and acylated forms of malvidin, peonidin, petunidin, and delphinidin also described in Syrah wines from Spain (Blanco-Vega et al., 2014).    It is well known that a fraction of the anthocyanin pigments disappears rapidly few months after fermentation. These pigments may be either broken down by external factors (temperature, light, oxygen), precipitated in colloidal coloring matter, combined and condensed with tannins, and also forming stable anthocyanin derived pigments, named pyranoanthocyanins. These pigments are produced by reaction between anthocyanins and acetaldehyde, pyruvic acid or vinylphenols, by-products of yeast, in young wine and also from condensation between anthocyanin and/or flavan-3-ols in aged wine (He et al., 2012). The elimination of anthocyanins leads to color loss and is detrimental to wine while pyranoanthocyanins production form more stable molecules responsible for color maintenance (Cheynier et al., 2006).
A reduction in total anthocyanins from 490 to 60 mg L -1 followed by an intense reduction in color intensity from bottling to 17 months aged was observed in Italian Syrah wines (Condurso et al., 2016). Color intensity in Brazilian Syrah winter wines, on the other hand, was not affected during ageing as well as total phenolic compounds and flavanols, which remained almost constant through ageing. Moreover, there was an increase in hydroxyphenyl pyranoanthocyanins and   flavan-3-ol pyranoanthocyanins in aged wine, pigments probably responsible for the red color of wine. The polymerized pigment index, applied to define the percentage of free and combined anthocyanins producing color in wine (Harbertson and Spayd, 2006), increased from 54 % at bottling to 80 % after 42 months of ageing, corroborating the contribution of copigmentation reactions to preserve color of winter wines.
The literature does not report aroma compounds in Syrah winter wines. According to Condurso et al. (2016), tipicity and quality of the wine are closely related to volatile aroma compounds from grape and those formed during the vinification process. Syrah wine has been described with spicy, dark fruit, or berry like flavors depending on the terroir. Therefore, studies report different volatile data. For example, rotundone, the sesquiterpene compound responsible for the peppery character of Syrah wines, requires an optimized procedure of extraction and therefore is not always found in Syrah wines samples (Siebert et al., 2008;Cincotta et al., 2015;Condurso et al., 2016). Freshly fermented wines from vineyards in southeastern Brazil were combined in the left part of the PCA plot while aged wines were displaced to the right part, with positive scores (Figure 1). Loscos et al. (2010) also observed such tendency. The second component reflects vineyard site importance, which will be discussed in another study.
Diethyl succinate (123-25-1), an ester mentioned as a chemical marker of wine ageing (Fabani et al., 2013) was found after 30 and 42 months ageing, mainly in winter wines aged for 30 months. Isopentyl acetate (123-92-2) another ester, responsible for the banana bouquet, was present at bottling and after 30 months of ageing. Fabani et al. (2013) reported a tendency to find lower levels with ageing in Syrah wines. As our results are qualitatively, we were not able to measure its content in the samples; however, it was found from bottling throughout ageing, with lower amounts after 42 months in the bottle.
Furfural (98-01-1), an aldehyde responsible for almond and caramel aroma, was found in Syrah winter wines over 30 months of age. This volatile compound is formed from carbohydrates during wine ageing; however, it can also be generated from hemicelluloses of the barrels (Condurso et al., 2016).
The presence of leafy and herbaceous aromas from C6 compounds such as cis-3-hexen-1-ol (928-96-1) released from the enzymatic degradation of lipids from grape cell membrane (Brillante et al., 2018) is related to fresh grape processing. Indeed this compound was found mainly at bottling in Syrah winter wines. Volatile compounds belonging to alcohol, alkyl sulfide, and acids classes were found mainly in young wines.
Terpenes are synthesized during grape maturation. They have pleasant flavor perceived even at low concentrations due to its very low olfactory threshold. The fermentation process has little contribution on terpene levels and therefore their content depends on vineyard management (Condurso et al., 2016). Syrah winter wines showed an increase in monoterpenes until 30 months ageing with a sharp decrease at 42 months, as observed by Loscos at al. (2010) under accelerated ageing process. Citronellol (106-22-9), the rose-like aroma, was found at bottling while D-limonene (5989-27-5) and p-cymene (99-87-6), both contributing to fresh, citrus-like aroma, increased at 30 months ageing. Pepper and peppermint aromas of terpinen-4-ol (562-74-3) and levomenthol (5989-27-5) were found mainly at bottling and at 30 months of ageing, while l-menthone (14073-97-3) content was higher in wines for 30 months aged.