# Insights into the evolution of certain sulfur-containing compounds during artificial ageing of Xinomavro PDO wines

Elli Goulioti, David Jeffery, Somaya Sachot, Aurélie Roland, Yorgos Kotseridis

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Elli Goulioti, David Jeffery, Somaya Sachot, Aurélie Roland, Yorgos Kotseridis. Insights into the evolution of certain sulfur-containing compounds during artificial ageing of Xinomavro PDO wines. Food Chemistry, 2025, 484, pp.144229. 10.1016/j.foodchem.2025.144229. hal-05497733

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https://institut-agro-montpellier.hal.science/hal-05497733v1

Submitted on 6 Feb 2026

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reverse transformation during wine ageing, are not fully understood (Roland et al., 2016).

Varietal thiols constitute a type of volatile sulfur compound (VSC), of which a variety of others can also influence aged wine bouquet. Dimethyl sulfide (DMS) is one such VSC that is well known for enhancing the fruity aroma in wines (Segurel et al., 2004), specifically affecting the ester family of volatile compounds (Lytra et al., 2014). DMS concentrations above 100 µg/L, however, may cause the perception of green olive characters as revealed for Shiraz and Xinomavro red wines (Anocibar Beloqui, 1996). The primary source of DMS is deemed to be the breakdown of S-methyl-L-methionine (SMM), an amino acid derivative found in grapes, during alcoholic fermentation or wine ageing (Loscos et al., 2008). According to an investigation of Shiraz wine aged for 12 months, 23 % of DMS was produced by SMM hydrolysis, with no plateauing at the end (Bekker et al., 2018). An alternative route for DMS release during bottle ageing has been described for chemical reduction of DMSO (Bekker et al., 2018), but SMM remains as the key DMS precursor to monitor during ageing.

To regulate and manage the development of a wine's bouquet throughout bottle ageing, and consequently, control wine quality, it is imperative to have a better understanding of the relationship between wine chemical composition and sensory perception (Picard et al., 2015). The typical aroma profile of Xinomavro PDO wine, an important red wine variety in Greece, was defined previously using a comprehensive sensory approach (Goulioti et al., 2023). Those findings indicated that the olfactory representation can be structured around a collection of nuances including green bell pepper, fresh red and black fruits, and a more mature olive character (Goulioti et al., 2023). The initial research offered a basis for further chemical investigations addressing the balance between youthful and evolved scents attributes.

Considering wines from Xinomavro PDO can be aged in bottle for two to over ten years (Wines of Greece, 2024), establishing a chemical process to investigate the aroma evolution throughout ageing is crucial for protecting the quality of these PDO wines. The primary goal of this study was therefore to examine how the combined effects of wine age and region of origin might influence volatiles associated with ageing for Xinomavro. In particular, the evolution of precursors and corresponding VSCs was monitored under artificial (accelerated) ageing conditions to understand chemical interconversion mechanisms that may contribute to aged aroma nuances.

## 2. Material and methods

### 2.1. Chemicals

Formic acid (99 %), methanol (99.98 %), water (99.98 %), and isopropanol (99.95 %) (ULC/MS grade) used for UHPLC analysis were purchased from Biosolve Chemicals (Holland). Ammonium bicarbonate (99 %), N-phenylmaleimide (97 %), and sodium chloride (≥ 99 %) were obtained from Sigma Aldrich (Saint-Quentin-Fallavier, France). Analytical standards available from previous research and obtained according to published protocols were as follows: 3-sulfanylhexan-1-ol-d₃ (3-SH-d₃) from Pardon et al. (2008); 3-sulfanylhexyl acetate-d₃ (3-SHA-d₃), 3-glutathionylhexan-1-ol (GSH-3-SH), and GSH-3-SH-d₂ from Roland, Schneider, Razungles, et al. (2010), Roland, Vialaret, Moniatte, et al. (2010), Roland, Vialaret, Razungles, et al. (2010); l-cysteinylglycine-3-SH (CysGly-3-SH), CysGly-3-SH-d₂, γ-L-glutamyl-L-cysteine-3-SH (GluCys-3-SH), and GluCys-3-SH-d₂ from Bonnaffoux et al. (2017); 3-cysteinylhexan-1-ol (Cys-3-SH), and Cys-3-SH-d₂ from Dagan (2006); 3-(N-acetyl-cysteinyl)-hexanol-1-ol (NAC-3-SH) from Dournes, Sachot, et al. (2023); SMM and SMM-d₃ from Deed et al. (2019). DMS and DMS-d₆ standards (>99 %) were supplied by Fluka (Charlotte, NC, USA).

### 2.2. Wine samples

As previously reported by Goulioti et al. (2023), Xinomavro PDO zone grapes were hand-harvested during the 2021 vintage from six selected vineyards in Amyndeon and five in Naoussa. Vineyard location, altitude, and soil description are provided in Table A.1 of Appendix A. The harvested parcels were transferred to the Laboratory of Enology and Alcoholic Drinks at the Agricultural University of Athens and small-scale vinifications were carried out in duplicate using a conventional red winemaking procedure with the use of a commercial *Saccharomyces cerevisiae* strain (Goulioti et al., 2023). Yeast assimilable nitrogen (YAN) and total soluble solids (TSS expressed as °Brix) were determined in the must after grape crushing according to methods detailed in International Organisation of Vine and Wine (2020). Samples from Amyndeon region were labelled A1, A2, A3, A4, A5, and A6 and from Naoussa as N1, N2, N3, N4, and N5 according to the relevant PDO zone and vineyard.

### 2.3. Artificial ageing

The artificial ageing experiment took place almost two years after winemaking, in November 2023, using amber glass bottles of 0.75 mL that were stored horizontally in cellar conditions (18 ± 2 °C, 55-60 % humidity) without light exposure. One bottle from each biological duplicate was used. Bottles were opened (day 0) and 50 mL aliquots of wine were used immediately for chemical analyses (in duplicate). Wine from each bottle was also separated into six 20 mL glass vials, which were flushed with N₂ prior to sealing. Vials were incubated in an oven at 50 °C for 24 days with sampling performed at 7, 17, and 24 days. At each time point, two vials were removed from the oven and allowed to reach room temperature, with one was used for DMS quantification, and the other for analysis of varietal thiols and their precursors and SMM. The experimental design is presented graphically in Fig. A.1 of Appendix A, along with the details of the experiment.

### 2.4. Analysis of thiol precursors by SIDA-UHPLC-MS/MS

Thiol precursors (GSH-3-SH, CysGly-3-SH, GluCys-3-SH, Cys-3-SH, NAC-3-SH as 3-SH precursors, and GSH-4-MSP, Cys-4-MSP as 4-MSP precursors) were quantified by stable isotope dilution analysis (SIDA) using UHPLC-MS/MS (1290 Infinity II LC hyphenated with a 6470B triple quadrupole mass spectrometer, Agilent Technologies, Santa Clara, CA, USA) as previously reported by Roland, Schneider, Razungles, et al. (2010), Roland, Vialaret, Moniatte, et al. (2010), Roland, Vialaret, Razungles, et al. (2010) with modifications by Dournes, Dufourcq, et al. (2023).

### 2.5. Analysis of varietal thiol derivatives by SIDA-UHPLC-MS/MS

3-SH and 3-SHA were analysed by SIDA-UHPLC-MS/MS (instrumentation as in Section 2.4) adopting a previously developed method (Roland et al., 2016) based upon derivatisation with N-phenylmaleimide.

### 2.6. Analysis of S-methyl-L-methionine by SIDA-UHPLC-MS/MS

Samples were diluted five-fold with water (Biosolve, UHPLC grade), spiked with SMM-d₃ aqueous solution as internal standard (IS) at a final concentration of 1033 µg/L, and analysed for SMM by SIDA-UHPLC-MS/MS (instrumentation as in Section 2.4) according to the method of Deed et al. (2019) with slight modification. A Hypersil gold C18 column (2.1 mm × 100 mm, 1.9 µm particle size, ThermoFisher, Waltham, MA, USA) was used. Mobile phases were (A) water with 0.1 % formic acid and (B) methanol with 0.1 % formic acid. The gradient for solvent B was as follows: 0 min, 0 %; 3 min, 0 %; 5 min, 98 %; 6 min, 98 %; 6.1 min, 0 %; 9 min, 0 % (2.9 min equilibration). The flow rate was 0.3 mL/min and injection volume was 1 µL. Mass spectra were collected in positive-ion
mode with electrospray ionisation (ESI). Source parameters were as follows: nitrogen was used as desolvation gas at 12 L/min at 200 °C; the nebuliser set at 207 kPa, and the sheath gas temperature was 260 °C at 6 L/min. The nozzle voltage was set to 0 V and ESI capillary was 2500 V. Detection was performed using multiple reaction monitoring (MRM) as detailed in Table 1. SMM quantification was performed using a 14-level calibration curve in the range 0 to 10 mg/L, prepared in duplicate.

## 2.7. Dimethyl sulfide quantification by SIDA with dynamic headspace-GC-MS

DMS was quantified immediately after opening wine bottles or the incubated vials at room temperature. A dynamic headspace system with Gerstel MPS autosampler (Mülheim an der Ruhr, Denmark) and Tenax TA (Gerstel) sorbent material was used. A 10-mL glass vial had 5 mL of the sample added, along with 1.5 g of NaCl and DMS-d₆ ethanol solution as IS (final concentration 25 µg/L). Analysis was performed with a 7890 A GC system coupled with a 5975B mass spectrometer (Agilent Technologies) according to the method described by Jimenez-Lorenzo et al. (2021).

## 2.8. Statistical analysis

One-way and repeated measures ANOVA followed by Tukey's HSD post-hoc test (α = 0.05), Spearman correlation test, and principal component analysis (PCA) were performed using XLSTAT (version 2018, Addinsoft, New York, NY, USA). Two-way repeated measures ANOVA was undertaken to analyse the effect of origin of the wines and the ageing level for the analytes quantified at each time point using IBM SPSS Statistics (version 20.0, Chicago, USA). A significant difference was ascribed for *p* < 0.05.

# 3. Results and discussion

## 3.1. Evolution of analytes during artificial ageing

### 3.1.1. Thiol precursors

Varietal thiol precursors are partially degraded during alcoholic (Tominaga et al., 1998) and malolactic (Takase et al., 2018) fermentations due to enzymes, but no details about their chemical stability were apparent in the literature. GSH-3-SH is a pro-precursor of 3-SH via stepwise degradation to Cys-3SH under different oenological conditions (Roland, Schneider, Charrier, et al., 2011; Roland, Schneider, Razungles, et al., 2011). The pathway from GSH-3-SH to Cys-3-SH might primarily occur via intermediate CysGly-3-SH, or alternatively through the less abundant GluCys-3-SH progenitor (Wang, 2022). From a quality and ageability standpoint, it is important to explore the possible transformation of thiol precursors into varietal thiols during wine ageing in bottle. To address this gap and investigate the potential effects of terroir and wine age on thiol precursors, the evolution profiles of GSH-3-SH, CysGly-3-SH, GluCys-3-SH, and Cys-3-SH were monitored throughout 24 days of artificial ageing for Xinomavro red wines from different PDO locations.

Total thiol precursor concentrations are presented in Table 2, with Amyndeon wines having 5.5-fold higher initial concentrations than

<table><caption>**Table 1**
MRM transitions and collision energies used for UHPLC-MS/MS analysis of SMM in Xinomavro wines.</caption><thead><tr><th></th><th>SMM</th><th>SMM-d<sub>3</sub></th><th>Collision voltage (V)</th></tr></thead><tbody><tr><td>Quantifier</td><td>164.0 → 102.0</td><td>167.0 → 102.0</td><td>8</td></tr><tr><td rowspan="3">Qualifiers</td><td>164.0 → 84.1</td><td>167.0 → 84.1</td><td>16</td></tr><tr><td>164.0 → 85.3</td><td>167.0 → 85.0</td><td>16</td></tr><tr><td>164.0 → 131.9</td><td>167.0 → 134.9</td><td>0</td></tr></tbody></table>

Naoussa wines. Precursors degraded during the artificial ageing experiment, with Amyndeon wines having almost 30 % less than initial after 24 days, whereas Naoussa wines experienced a loss of 50 %. Final concentrations were seven times higher in Amyndeon PDO wines compared to Naoussa, with the residual level of precursors generally being consistent with previous research (Tonidandel et al., 2021).

The initial concentration of GSH-3-SH, the most abundant form of precursor, was significantly higher in Amyndeon wines, being almost seven times that of Naoussa (46 ± 26 vs 7 ± 4 µg/L at 0 days, Table 2 and Fig. A.2a and b of Appendix A for individual wines) and remaining so at all time points. On the other hand, mean concentrations within a zone remained relatively stable, with no significant difference arising during artificial ageing (Table 2).

CysGly-3-SH was detected in all wine samples, with Amyndeon PDO wines presenting significantly higher concentrations ranging from 13 ± 5 to 17 ± 7 µg/L compared to Naoussa with 2.1 ± 0.3 to 3.7 ± 0.5 µg/L (Table 2 and Fig. A.2c and d of Appendix A for individual wines). Amyndeon wine mean concentrations remained stable during the artificial ageing experiment, whereas Naoussa wines had a significant increase in mean concentration during the first 7 days of ageing, from 2.1 ± 0.3 to 3.7 ± 0.5 µg/L (Table 2), followed by a diminution to 2.8 ± 0.8 µg/L (17 days), then remaining stable and finishing at 3.0 ± 0.9 µg/L (24 days).

Concentrations of GluCys-3-SH were of the same magnitude but somewhat lower than CysGly-3-SH, with Amyndeon wines presenting significantly higher levels (7.4 ± 4.4 to 13 ± 7 µg/L) than Naoussa wines (1.2 ± 1.0 to 3.2 ± 1.6 µg/L) at each time point (Table 2 and Fig. A.2e and f of Appendix A for individual wines). There was a relatively constant decline in GluCys-3-SH across the experiment and significant differences within both regions according to repeated measures ANOVA, with Amyndeon wines revealing an approximate 40 % decrease during the 24 days of artificial ageing, and those from Naoussa being about 60 % lower (Table 2).

Concentrations of Cys-3-SH in Amyndeon wines presented significantly higher levels (5.9 ± 3.8 to 8.1 ± 3.2 µg/L) than in Naoussa wines (not detected to 3.1 ± 0.8 µg/L) at each time point (Table 2 and Fig. A.2g and h of Appendix A for individual wines). According to repeated measured ANOVA results, there was a relatively stable concentration of Cys-3-SH across the experiment for Amyndeon wines, while significant differences were presented for Naoussa wines, with an initial increase and then declining until undetectable by the end (Table 2). NAC-3-SH is a variant of Cys-3-SH recently reported in fermented synthetic must (Dournes, Sachot, et al., 2023), with the present work being the first time this compound has been detected in red wine. Although absent in Naoussa wines, NAC-3-SH was detected in Amyndeon zone wines at all sampling points (data not shown), albeit at concentrations below 0.3 µg/L, which was the limit of quantification.

GSH-4-MSP is a precursor of 4-MSP, a thiol that imparts box tree and blackcurrant aromas in wines (Fedrizzi et al., 2009; Roland, Schneider, Razungles, et al. (2010), Roland, Vialaret, Moniatte, et al. (2010), Roland, Vialaret, Razungles, et al. (2010)). This precursor is generally much less abundant in contrast to GSH-3-SH in grape must, which leads to a corresponding difference in the concentration range of the varietal thiols (Roland, Schneider, Charrier, et al., 2011; Roland, Schneider, Razungles, et al., 2011). GSH-4-MSP was not detected in wines from either zone on any of the sampling days.

This was the first time that thiol precursor concentrations have been determined in Xinomavro wines, and to the best of our knowledge, the first report of thiol precursors being monitored during simulated ageing. GSH-3-SH concentration in Xinomavro wines was reported in a similar range to Chardonnay wines (27–40 µg/L (Tonidandel et al., 2021)), while lower than the Sauvignon blanc (59–102 µg/L (Tonidandel et al., 2021)) and Australian Shiraz wines (70 µg/L (Wang et al., 2022)). Cys-3-SH concentrations presented similarly to those detected in Chardonnay wines (2–11 µg/L) and lower than Sauvignon blanc wines (0–25 µg/L (Tonidandel et al., 2021)), but higher than Australian Shiraz wines,
<caption>**Table 2**</caption>

Mean values (± standard deviation) of thiol precursors, varietal thiols, S-methyl-1-methionine, and dimethyl sulfide for Xinomavro wines from Amyndeon (A) and Naoussa (N) PDO zones. One-way ANOVA results for each analyte show differences between zones for a given sampling day, and repeated measures ANOVA shows differences for an analyte according to artificial ageing for a single zone, with different lower-case letters for an analyte representing significant differences (α = 0.05) between the sampling time means within Amyndeon zone (A) and different upper-case letters pertaining to Naoussa zone (N). n.s.: not significant; \*: p < 0.05; \*: p < 0.01; \*: p < 0.001.

<table><thead><tr><th rowspan="2"></th><th colspan="3">0 days</th><th colspan="3">7 days</th><th colspan="3">17 days</th><th colspan="3">24 days</th><th colspan="2">Repeated measures ANOVA</th></tr><tr><th>A</th><th>N</th><th>Sig.</th><th>A</th><th>N</th><th>Sig.</th><th>A</th><th>N</th><th>Sig.</th><th>A</th><th>N</th><th>Sig.</th><th>A</th><th>N</th></tr></thead><tbody><tr><td colspan="15" style="text-align: center; font-weight: bold;">Thiol precursors (µg/L)</td></tr><tr><td>GSH-3-SH</td><td>46 ± 26</td><td>7.0 ± 4.4</td><td>***</td><td>33 ± 20</td><td>5.4 ± 4.4</td><td>***</td><td>41 ± 22</td><td>5.4 ± 3.4</td><td>***</td><td>31 ± 15</td><td>4.4 ± 3.2</td><td>***</td><td>n.s.</td><td>n.s.</td></tr><tr><td>CysGly-3-SH</td><td>14 ± 6</td><td>2.1 ± 0.3</td><td>***</td><td>13 ± 5</td><td>3.7 ± 0.5</td><td>***</td><td>17 ± 7</td><td>2.8 ± 0.8</td><td>***</td><td>15 ± 5</td><td>3.0 ± 0.9</td><td>***</td><td>n.s.</td><td>***</td></tr><tr><td></td><td></td><td>C</td><td></td><td></td><td>A</td><td></td><td></td><td>B</td><td></td><td></td><td>B</td><td></td><td></td><td></td></tr><tr><td>GluCys-3-SH</td><td>13 ± 7</td><td>3.1 ± 1.3</td><td>***</td><td>9.5 ± 4.6</td><td>3.2 ± 1.6</td><td>***</td><td>9.4 ± 5.5</td><td>2.1 ± 1.1</td><td>***</td><td>7.4 ± 4.4</td><td>1.2 ± 1.0</td><td>***</td><td>***</td><td>***</td></tr><tr><td></td><td>a</td><td>A</td><td></td><td>ab</td><td>A</td><td></td><td>ab</td><td>AB</td><td></td><td>b</td><td>B</td><td></td><td></td><td></td></tr><tr><td>Cys-3-SH</td><td>6.6 ± 1.7</td><td>2.2 ± 0.6</td><td>***</td><td>7.3 ± 1.6</td><td>3.1 ± 0.8</td><td>***</td><td>8.1 ± 3.2</td><td>0.4 ± 0.4</td><td>***</td><td>5.9 ± 3.8</td><td>n.d.<sup>a</sup></td><td>***</td><td>n.s.</td><td>***</td></tr><tr><td></td><td></td><td>B</td><td></td><td></td><td>A</td><td></td><td></td><td>C</td><td></td><td></td><td>D</td><td></td><td></td><td></td></tr><tr><td colspan="15" style="text-align: center; font-weight: bold;">Varietal thiols (ng/L)</td></tr><tr><td>3-SH</td><td>106 ± 17</td><td>74 ± 24</td><td>***</td><td>88 ± 11</td><td>57 ± 7</td><td>***</td><td>48 ± 10</td><td>24 ± 5</td><td>***</td><td>41 ± 15</td><td>10 ± 6</td><td>***</td><td>***</td><td>***</td></tr><tr><td></td><td>a</td><td>A</td><td></td><td>b</td><td>A</td><td></td><td>c</td><td>B</td><td></td><td>c</td><td>C</td><td></td><td></td><td></td></tr><tr><td>3-SHA</td><td>0.1 ± 0.3</td><td>1.8 ± 3.3</td><td>*</td><td>n.d.</td><td>n.d.</td><td></td><td>n.d.</td><td>n.d.</td><td></td><td>n.d.</td><td>n.d.</td><td></td><td></td><td></td></tr><tr><td colspan="15" style="text-align: center; font-weight: bold;">Sulfur compounds (µg/L)</td></tr><tr><td>SMM</td><td>180 ± 29</td><td>224 ± 61</td><td>*</td><td>150 ± 53</td><td>266 ± 78</td><td>***</td><td>287 ± 9</td><td>312 ± 27</td><td>**</td><td>189 ± 5</td><td>196 ± 9</td><td>*</td><td>***</td><td>***</td></tr><tr><td></td><td>bc</td><td>BC</td><td></td><td>c</td><td>AB</td><td></td><td>a</td><td>A</td><td></td><td>b</td><td>C</td><td></td><td></td><td></td></tr><tr><td>DMS</td><td>61 ± 11</td><td>88 ± 22</td><td>***</td><td>113 ± 28</td><td>165 ± 49</td><td>**</td><td>108 ± 31</td><td>116 ± 36</td><td>n.s.</td><td>-</td><td>-</td><td></td><td>***</td><td>***</td></tr><tr><td></td><td>b</td><td>C</td><td></td><td>a</td><td>A</td><td></td><td>a</td><td>B</td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>

where it was undetected (Wang et al., 2022).

Two-way repeated measures ANOVA (Table A.2, Appendix A) was conducted to investigate the effect of PDO zone, ageing time, and their interaction for the measured analytes (Fig. 1a–d). GSH-3-SH (p < 0.0001), CysGly-3-SH (p < 0.0001), GluCys-3-SH (p = 0.0003), and Cys-3-SH (p < 0.0001) were significantly affected by the two-way interaction of zone × time (Table A.2). In parallel, the impact of the individual factors of zone and ageing time were found to be significant for all the analytes. Spearman correlation analysis between GluCys-3-SH concentration at 0 days and Cys-3-SH at 7 days, and between GluCys-3-SH at 7 days and Cys-3-SH at 17 days, yielded values of r = 0.752 (p < 0.001)

and r = 0.759 (p < 0.001), respectively. This strong relationship between the two precursors implied that the conversion of GluCys-3-SH to Cys-3-SH in the thiol formation pathway could take place chemically during wine ageing. This could arise from acid hydrolysis of precursors in wine, as suggested by Alegre et al. (2020), with chemical degradation of GSH-3-SH into GluCys-3-SH being demonstrated previously (Muhl et al., 2024) and hypothesised for the transformation of CysGly-3-SH into Cys-3-SH in barley malt (Chenot et al., 2023).

Regarding the different levels of thiol precursors between the PDO wines, it is important to note that several factors can influence thiol precursors, including grape ripeness (Capone et al., 2011; Chen et al.,

<figure><img src="image_2.png" /><figcaption>**Fig. 1.** Plots of significant interactions from two-way repeated measures ANOVA for Xinomavro wines from Amyndeon (A) and Naoussa (N) zones during artificial ageing showing (a) GSH-3-SH, (b) CysGly-3-SH, (c) GluCys-3-SH, (d) Cys-3-SH, (e) SMM, and (f) DMS. Error bars represent the standard error.</figcaption></figure>

<footnote>a n.d., not detected (<LOD).</footnote>
2019) and handling (Capone et al., 2012), and winemaking treatments (Roland, Schneider, Charrier, et al., 2011; Roland, Schneider, Razungles, et al., 2011). The consistent vinification process used for wine production and the controlled level of maturity in harvested grapes (based on TSS, Table A.1, Appendix A) points to differences in terroir and microclimate characteristics of each zone, as found for varieties such as Sauvignon blanc (Chen et al., 2019; Roland, Schneider, Razungles, et al., 2010, Roland, Vialaret, Moniatte, et al. (2010), Roland, Vialaret, Razungles, et al. (2010)) and Melon B (Roland, Schneider, Razungles, et al. (2010), Roland, Vialaret, Moniatte, et al. (2010), Roland, Vialaret, Razungles, et al. (2010)). Higher total thiol precursor concentrations in Amyndeon wines (Table 2) were possibly influenced by the higher nitrogen status of the zone's grapes as expressed in the YAN concentration (Table A.1). Amyndeon region's soil type and geography could explain the higher grape YAN content, as it is known that grape nitrogen status influences the concentration of glutathionylated and cysteinylated precursors (Choné et al., 2006; Helwi et al., 2016). Moreover, precursors of 3-SH decrease in grapes with increasing temperature and water deficit (de Almeida et al., 2024). In the Naoussa zone, higher temperatures during the ripening months, along with the vineyards being located on the mountain slopes (Yassoglou et al., 2017), could also account for the differences in thiol precursors between the PDO zones for these Xinomavro wines.

### 3.1.2. Varietal thiols

The association between the evolution of thiol precursors under artificial ageing and the release of aroma compounds, 3-SH and 3-SHA, was determined based on four sampling points (Table 2). Amyndeon wines had an initial concentration that was 1.4 times higher than Naoussa, whereas the final concentration was four times higher in Amyndeon samples. 3-SH accounted for the vast majority of total varietal thiols in comparison with 3-SHA, which was barely detected initially and absent at the final sampling point.

In detail, 3-SH significantly degraded during the artificial ageing period in both zones according to repeated measures ANOVA (Table 2), with Amyndeon PDO wines presenting significantly higher amounts than Naoussa wines at all sampling points. Amyndeon wine 3-SH concentrations declined by around 60%, ranging from 106 ± 17 ng/L at the beginning to 41 ± 15 ng/L at the end of ageing, with statistically lower concentrations for the first three time points, highlighting a significant degradation effect. During the last week of ageing, however, the concentration appeared to plateau, with no significant difference among the mean concentrations at 17 and 24 days (Table 2). Naoussa zone 3-SH concentrations ranged from 74 ± 24 ng/L initially to 11 ± 6 ng/L after 24 days, and presented a different behaviour to Amyndeon wines, with the concentration being stable after 7 days and then undergoing a statistically significant decline (Table 2). Considering the individual wines on a regional basis as presented in Fig. A.2i and j (Appendix A), 3-SH concentrations declined in the same way during artificial ageing for each region.

3-SH concentrations in these Xinomavro PDO wines presented similar results to those reported for South Africa Cabernet Sauvignon (77–147 ng/L (Mafata et al., 2018)), but lower concentrations than Bordeaux reds (100–634 ng/L (Picard et al., 2015)) and Chile Carmenère wines (422–760 ng/L (Pavez et al., 2016)). With a detection threshold for 3-SH of 60 ng/L (Tominaga et al., 1998), it seemed likely that this compound could directly impact the aroma profile of Xinomavro wines prior to and until 7 days of artificial ageing. Furthermore, considering the analysis of 3-SH was realised for two year old wines, the initial concentrations after the end of alcoholic fermentation would presumably be somewhat greater than the specified detection threshold.

3-SHA was quantifiable only in the bottled wines before the ageing process began (0 days), at levels of <2 ng/L, with Naoussa wines presenting significantly higher mean concentration than Amyndeon wines (Table 2). Acetate esters undergo hydrolysis during ageing and their concentrations quickly decrease some months after fermentation,

especially at elevated storage temperatures (Makhotkina et al., 2012). The fact that the wines were two years old before the artificial ageing experiment could explain the low levels of 3-SHA encountered. In any case, with an odour threshold of 4.2 ng/L (Tominaga et al., 1998), 3-SHA was unlikely to impart any characteristics to these Xinomavro wines.

According to the two-way ANOVA results, varietal thiol 3-SH was not affected by the interaction of zone × time (p = 0.369), while affected by the zone (p < 0.0001) and time (p < 0.0001) factors (Table A.2). 3-SH concentration is strongly related to the terroir (Roland, Schneider, Charrier, et al., 2011; Roland, Schneider, Razungles, et al., 2011; van Leeuwen et al., 2022; Wang, 2022) and microclimate (van Leeuwen et al., 2022) where grapes are produced, whereas the decline in 3-SH noted during artificial ageing (Table 2) could be explained by oxidation of thiols to disulfides (Roland et al., 2016) and/or reaction with oxidised polyphenol intermediates (Nikolantonaki et al., 2012; Suc et al., 2021). In relation to the change in thiol precursors presented in Section 3.1.1, the decrease in 3-SH concentrations may indicate that precursors did not act as a thiol source during the experiments. Indeed, thiol precursors can be transformed but not necessarily into varietal thiols, with the release of thiols known to be catalysed by β-lyase enzymes (Ferreira & Lopez, 2019; Sahay, 2018; Wang, 2022). There could be an alternative formation pathway for varietal thiols, however, as proposed by Tran et al. (2015) for beer, with degradation reactions involving Cys-3-SH and dicarbonyls during storage. Clearly, there are a number of factors at play and future research could address whether disulfide or thiol-polyphenol adduct formation can provide a link between chemical degradation of thiol precursors and 3-SH formation.

### 3.1.3. S-Methyl-L-methionine

SMM is the main source of DMS post-bottling (Deed et al., 2019), with Bekker et al. (2018) determining a conversion yield of over 20% for SMM being transformed into DMS during 12 months of bottle ageing for a red and a white wine. Table 2 summarises the results of SMM mean concentration at the four time points during the artificial ageing experiment (with individual wines shown in Fig. A.2k and l, Appendix A). Naoussa zone's initial concentration was 1.2 times higher than Amyndeon (224 ± 61 and 180 ± 29 µg/L, respectively). Significant differences were detected between sample points within zones according to repeated measures ANOVA. Amyndeon wines had an initial concentration (0 days) that remained stable during 7 days of ageing, followed by an increase until 17 days. At this point it reached a concentration high of 287 ± 9 µg/L that differed significantly from the others, before decreasing by the end of the experiment (24 days) to a final concentration that matched the initial level (Table 2). Naoussa PDO wines followed a similar pattern, increasing to an SMM concentration of 312 ± 27 µg/L at 17 days, which differed significantly from the initial and final time points, then declining to the original level by the end of the ageing period (24 days). Two-way repeated measures ANOVA (Table A.2, Appendix A and Fig. 1e) revealed that SMM concentration was affected by the interaction of zone × time (p < 0.0001) and by both the individual factors of the zone (p = 0.0002) and ageing time (p < 0.0001).

Research into wine ageing aroma has typically reported the degradation of SMM into DMS (Argentero et al., 2024; Deed et al., 2019; Jiménez-Lorenzo et al., 2022; Samaniego Solis et al., 2024; Segurel et al., 2004; Ye et al., 2016), but the present results appear to be the first time an increase in SMM concentration has been observed in bottled wine undergoing artificial ageing. According to prior studies, SMM in grapes can range in concentration from a few µg/L to 5 mg/L (Loscos et al., 2008; Segurel et al., 2004). Apart from SMM, a variety of DMS precursors involved in wine fermentation have been investigated, such as cysteine, methionine (de Mora et al., 1986), DMSO, and glutathione (Bekker et al., 2018); however, any link between them has yet to be determined during wine ageing.

The increase in SMM in the present research suggests that a DMS precursor may have been present in the Xinomavro wines, with SMM
being a degradation intermediate in the formation of DMS. It would seem that any pro-precursor form must be more heat-labile than SMM, given that SMM accumulated, but its presence and identity require confirmation. A cyclic derivative of SMM may be formed in heat-sterilised melon juice, seemingly in a reversible reaction controlled by heat and alkaline pH (Pan et al., 2021). As alkaline conditions are far from oenological ones, the formation of cyclic SMM would not be fav-oured in wine. That might point to an *N*-acetyl derivative of SMM, with hydrolysis of the acetate ester possibly explaining the production of SMM during artificial wine ageing. This could potentially be formed by yeast as is the case with NAC-3-SH (Dournes, Sachot, et al., 2023), with genes encoding for methionine *N*-acetylation being known in *S. cerevisiae* (Kulkarnis & Shermans, 1994). However, further in-vestigations would be necessary to verify this prospect or to identify the compound(s) responsible for the apparent increase in SMM.

### 3.1.4. Dimethyl sulfide

DMS is a low molecular weight VSC present in wine (Zhang et al., 2020) that has received considerable attention due to its complex chemistry, biochemistry, and potential sensory contribution, especially in aged red wines (de Mora et al., 1987; Samaniego Solis et al., 2024). DMS concentration was measured during the 17 days of ageing trial for Xinomavro PDO wines. Paralleling the SMM concentrations, DMS was determined to be higher in Naoussa wines than those from Amyndeon at initial (0 days) and final (17 days) sampling points (Table 2).

Amyndeon wines ranged between 61 ± 11 and 113 ± 28 µg/L of DMS and Naoussa wines were between 88 ± 22 and 165 ± 49 µg/L (Table 2 and Fig. A.2m and n of Appendix A for individual wines). DMS increased significantly after 7 days of artificial ageing in Amyndeon wines and then remained stable, with no difference in concentration between 7 and 17 days. The peak SMM concentration after 17 days of ageing was not mirrored in the evolution of DMS. The concentration of DMS in Naoussa wines also increased significantly after 7 days of ageing, before undergoing a significant decrease, but finishing higher than the initial amount (Table 2).

DMS levels detected in the present research were in agreement with those reported in aged Bordeaux wines (23 to 146 µg/L (Picard et al., 2015)), but higher than aged Syrah and Grenache wines from Rhone valley (6 to 46 µg/L (Segurel et al., 2004)). Notably, even relatively high DMS level in wines (e.g., 100 µg/L) may enhance the aroma profile complexity rather than affecting it negatively, emphasizing its crucial function in the overall complexity and balance of wine aroma (Picard et al., 2015; Silva Ferreira et al., 2003). Considering the likely sensory effect, DMS concentrations in the present findings accorded with typical varietal aromas described as fruity and olive for Xinomavro red wine (Goulioti et al., 2023).

Comparing the Amyndeon zone's SMM concentration at 0 days with the corresponding DMS concentration at 7 days of artificial ageing identified an increase of DMS that was inconsistent with the SMM degradation (Table 2). However, the strong positive correlation ($r = 0.786, p < 0.001$) between the two might further justify the presence of SMM-related compounds that increase DMS levels. Naoussa zone's simultaneous increase of both SMM and DMS between 0 and 7 days of ageing could similarly be linked with the notion presented in Section 3.1.3 about the presence of pro-precursor allowing accumulation of SMM. The DMS formation mechanism is not fully understood (Deed et al., 2019; Silva Ferreira et al., 2003), but a chemical process has been proposed to involve progressive hydrolysis of SMM in young wines during ageing (Argentero et al., 2024; Jiménez-Lorenzo et al., 2022; Ye et al., 2016). Other chemical routes such as DMSO reduction could not fully explain the increased DMS concentration, given the maximum reported yield from DMSO is 9 % (Bekker et al., 2018).

DMS levels were affected by the two-way interaction of zone × time ($p < 0.0001$). Moreover, the effects of individual factors of zone ($p = 0.007$) and ageing time ($p < 0.0001$) were significant (Table A.2 and Fig. 1f). The interaction of geographical origin and time on DMS levels in

wines has also been revealed by Samaniego Solis et al. (2024), with the influence of terroir factors being worthy of further exploration. DMS precursor research has also focused on the timing of degradation. The formation of aroma during bottle ageing of wine was shown to follow nonlinear kinetics, accompanied by an indirect equilibrium involving DMS. Samaniego Solis et al. (2024) quantified DMS content and discovered a complex formation pattern, indicating the presence of several precursors with varying reactivity, which accords with the present results.

Factors such as grape cultivar, viticultural and winemaking techniques, vineyard nitrogen status, wine pH, and storage temperature influence the presence of DMS and its precursors in wine (Samaniego Solis et al., 2024). The significantly different levels of DMS in wines from the different PDO zones presented herein could relate to the terroir characteristics of each region, as in the work of De La Burgade et al. (2023) on Syrah wines from the South of France. Naoussa region soils impart a wine water deficit, which reportedly results in higher levels of "potential" DMS in berries and in the corresponding wine (De Royer Dupré et al., 2014; Ye et al., 2016), which is linked with DMS production during ageing (De Royer Dupré et al., 2014). Moreover, the YAN content of must can relate to the DMS concentration in wines (Argentero et al., 2024; Samaniego Solis et al., 2024; Siebert et al., 2010). Yeast can synthesise or degrade compounds like SMM, and the availability of alternative nitrogen sources has a significant regulating impact (Samaniego Solis et al., 2024; Ugliano et al., 2008). Naoussa region grape must YAN concentrations were reported to be around half of those in the musts from Amyndeon (Table A.1, Appendix A), which reinforces a possible connection between YAN and DMS.

### 3.2. Principal component analysis

PCA was performed to visualise the relationship between the analytes measured (excluding DMS due to less data points) with wine origin and duration of artificial ageing. The first two principal components represented 64.23 % of the variation, with PC1 accounting for 47.27 % and PC2 for 16.98 % (Fig. 2). Amyndeon wines are located to the right along PC1 (Fig. 2a) and characterised by their higher concentration of thiol precursors and 3-SH (Fig. 2b), whereas Naoussa wines are located to the left and associated with 3-SHA (especially 0 days) and SMM, and a lack of thiol precursors. The concentration of precursors correlated well with 3-SH concentration ($r = 0.571$), whereas SMM and 3-SH were negatively correlated ($r = -0.312$).

PC2 separates wines from within the regions, with the upper two quadrants consisting of sample N_0d at top left (Fig. 2a), where 3-SHA is located (Fig. 2b), and A_0d and A_7d to the right, characterised especially by 3-SH and GluCys-3-SH concentrations and opposite to SMM. The lower two quadrants consisted of N_7d, N_17d, and N_24d to the lower left, and A_17d and A_24d on the lower right, with Naoussa wines strongly related to SMM, and Amyndeon wines to the remainder of the thiol precursors. Artificial ageing seemed to affect Naoussa PDO wines earlier than Amyndeon wines, as the thiol precursor and varietal thiol concentrations decreased from the first week of ageing, while the same behaviour was more evident in Amyndeon samples after two weeks.

PCA of all the analytes measured for 17 days of artificial ageing (i.e., including DMS) was also undertaken. Fig. A.3 (Appendix A) presents the biplot of PC1 vs PC2 (85.30 % explained variance), with wine samples separating along PC1 much like with Fig. 2. As such, Amyndeon wines are located on the right along with thiol precursors and 3-SH, with Naoussa samples to the left along with 3-SHA and SMM, but also DMS. Notably, SMM was strongly correlated with DMS concentration ($r = 0.627$). As in Fig. 2, PC2 separates wines N_0d, A_0d, and A_7d, located above, from N_7d, N17d, and A_17d located below, with the Naoussa wines being characterised by higher levels of DMS.
<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Principal component analysis plots (PC1 and PC2) showing (a) scores with 95 % confidence ellipses and (b) loadings based on the chemical analysis of replicate Xinomavro wines from Amyndeon (A1–A6) and Naoussa (N1–N5) during 24 days of artificial ageing (assessed at 0, 7, 17, and 24 days).</figcaption></figure>

## 4. Conclusion

This study highlighted the difference in composition between Xinomavro wines from PDO Amyndeon and Naoussa zones during artificial bottle ageing, investigating the effect of terroir and ageing time on thiols and their precursors, and DMS and its main putative precursor SMM. Thiol precursors either remained stable or slightly declined, but that appeared not to have any effect on thiol concentrations. The varietal thiols were continuously decreased under artificial ageing conditions, likely due to oxidation related reactions. The total concentration of thiol precursors and 3-SH was determined to be higher in the Amyndeon PDO wines, whereas Naoussa wines contained higher amounts of SMM and DMS. The complicated relationship between the expression of terroir and the concentrations of SMM and DMS in regional wines during ageing could be reflected in these differences. There were indications that unidentified DMS sources may have been pro-precursors, that is, precursors to SMM as an intermediate, which then breaks down into DMS. This was indicated by the increase in SMM concentration during artificial ageing. Overall, the present research provides useful understanding of the chemical changes that can occur in Xinomavro PDO bottled wines. The degradation and evolution of sulfur compounds could have a strong impact on the sensory profile of aged regional wines, differentiating between the two most important zones of Xinomavro in Greece.

### CRediT authorship contribution statement

**Elli Goulioti:** Writing – review & editing, Writing – original draft, Visualization, Methodology, Investigation, Formal analysis, Conceptualization. **David W. Jeffery:** Writing – review & editing, Validation, Supervision, Project administration, Methodology, Conceptualization. **Somaya Sachot:** Writing – review & editing, Validation, Methodology, Investigation. **Aurélie Roland:** Writing – review & editing, Validation, Supervision, Resources, Project administration, Methodology, Conceptualization. **Yorgos Kotseridis:** Writing – review & editing, Supervision, Resources, Project administration, Methodology.

### Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

### Declaration of competing interest

The authors declare that they have no known competing financial

interests or personal relationships that could have appeared to influence the work reported in this paper.

### Acknowledgements

The authors would like to express their acknowledgment to Alpha Estate and Boutari Winery for providing access to their vineyards and donating the harvested grapes.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.foodchem.2025.144229.

### Data availability

All data is reported in the article and provided in the supporting information file.

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