The Blaufränkisch grapes used in this study were provided by the department of Grapevine Breeding at the Federal College and Research Institute for Viticulture and Pomology in Klosterneuburg, Austria (Götzhof, Rehgraben 2, 2103 Langenzersdorf). The grapes were harvested on 21 September 2023. They exhibited no signs of common vine diseases such as powdery mildew (Oïdium), downy mildew (Peronospora), or Botrytis. Immediately after harvesting, the grapes were destemmed and crushed. Must parameters were determined using Fourier-transform infrared (FT-IR) spectroscopy in accordance with OIV/OENO Resolution 390/2010 and are summarised in Table 1 ( OIV, 2010). The mash temperature was 14 °C. Potassium metabisulfite (K ₂S ₂O ₅; Lallemand, Guntramsdorf, Austria) was added at a rate of 10 g/100 kg mash.

A total of 650 kg of Blaufränkisch mash was available. Following sulphiting, the mash was homogenised in a fermentation vessel and divided into aliquots, each transferred into 50-L fermentation containers. Three fermentation replicates were prepared per treatment, resulting in a total of 15 vessels, each containing 40 kg of mash. Immediately after aliquoting, yeast strain ES488 (Enartis, Bratislava, Slovakia) was added at a rate of 60 g/100 kg after rehydration. The experimental design and treatment setup are shown in Figure 1.

Co-inoculation with lactic acid bacteria was carried out on 22 September 2023, i.e., 24 hours after yeast inoculation, using ML Prime (Lallemand, Guntramsdorf, Austria) at 10 g per 100 kg must. Although ML Prime is typically recommended for musts with pH ≥ 3.4 and total SO ₂ ≤ 50 mg/L, our must (pH 3.36; total SO ₂ ≈ 50 mg/L) fitted within these recommendations and has been shown to support rapid malolactic fermentation with minimal lag phase and no volatile acidity formation. The 24‑hour delay aligns with the recommended co‑inoculation protocol and is intended to balance nutrient competition between yeast and bacteria, ensuring robust MLF onset while preserving yeast performance. Depending on the treatment, oak chips (Incanto Natural, Enartis) were added at 80 g/100 kg, and glutathione was applied in the form of a GSH-IDY yeast product (ProBlanco, Enartis) at 30 g/100 kg. In addition, the Blaufränkisch mash was enriched to 23.5 °Brix using sucrose. According to the manufacturer, ProBlanco contains a minimum of 1.5 % reduced glutathione, as regulated by the OIV Codex (COEI-1-LEVGLU; OIV, 2017), which corresponds to an application rate of 4.5 mg/kg of reduced glutathione.

Fermentation was conducted at 25.0 ± 1.0 °C. The cap was punched down twice daily and monitored using a handheld density meter (DMA 35, Anton Paar, Graz, Austria). No nitrogen supplementation ( e.g., diammonium phosphate (DAP)) was applied, as the must’s YAN concentration was approximately 300 mg/L. Macro-oxygenation was carried out for selected treatments using a MicroOx system (Enartis) at the end of the second third of fermentation (between 1.3402 and 1.3888 g/cm ³), applying 15 mg/kg of oxygen (Linde, Vienna, Austria).

After seven days, on 28 September 2023, the mash was pressed. The analytical parameters of the resulting young wines are summarised in Table 2. All wines completed both alcoholic fermentation and malolactic conversion. The wines were transferred into 25-L demijohns and racked off the lees on 12 October 2023. Subsequent sulphiting was carried out with 16 g/100 L K ₂S ₂O ₅ (corresponding to 80 mg/L free SO ₂). Free SO ₂ was adjusted twice to 50 mg/L prior to bottling and set to 60 mg/L at bottling on 15 December 2023.

The wines were bottled in olive-green 500 mL ‘BD Exklusiv’ bottles (Vetropack, Bülach, Switzerland) and sealed with Tin-Saran screw caps (BT-Watzke GmbH, Pinggau, Austria). The bottles were stored horizontally at 16 ± 1 °C in the vinothèque of the Enology department at HBLAuBA Klosterneuburg until analysis.

Table 2. Quantified parameters of the young wines ( n = 3).

Note that the limit of detection was 0.1 g/L, and the limit of quantification was 0.2 g/L. GSH-IDY, glutathione-rich inactivated yeast; n.d., not detectable. All fermentations were completed on 28 September 2024.

Figure 1. Experimental setup.

The experimental wines were analysed between May and August 2024. Prior to sample preparation, the bottles were removed from cold storage and allowed to equilibrate to room temperature.

Quantification of basic must and wine parameters was conducted in the department of Chemistry at the Federal College and Research Institute for Viticulture and Pomology, Klosterneuburg. Measurements were performed using FT-IR spectroscopy in accordance with OIV/OENO Resolution 390/2010 ( OIV, 2010), employing a FOSS WineScan (FT 120 Reference Manual; Foss, Hamburg, Germany).

The following parameters were quantified: °Brix, α-amino nitrogen (NOPA), ammonium, density, ethanol, glucose, fructose, titratable acidity, pH, tartaric acid, malic acid, lactic acid, volatile acidity, and citric acid. YAN was calculated from the individual values for NOPA and ammonium using the following formula: YAN = NOPA + (Ammonium × 0.8225) ( Bell & Henschke, 2005).

Quantification of esters, higher alcohols, C ₆ alcohols, carboxylic acids, free monoterpenes, and C ₁₃-norisoprenoids was carried out at the department of Chemistry-Research and Isotope Analysis of the Federal College and Research Institute for Viticulture and Pomology, Klosterneuburg. Analysis of polyfunctional thiols and low-boiling-point sulfur-containing compounds was conducted at the department of Microbiology and Biochemistry, Hochschule Geisenheim University (HGU).

In total, five gas chromatography systems (Agilent Technologies, Santa Clara, CA, USA) were employed, each equipped with specific column technologies, detectors, and sample preparation procedures tailored to the physicochemical characteristics of the respective analytes. This multi-system approach ensured analytically differentiated detection of volatile aroma compounds.

Quantification of key aroma compounds—including selected higher alcohols, short- and medium-length carboxylic acids, C ₆ alcohols, and major esters—was performed by using a partial stable isotope dilution analysis-headspace solid-phase microextraction-gas chromatography-mass spectrometry (SIDA-HS-SPME-GC-MS) method as described by Philipp et al. (2019b), employing an Agilent 6890N GC system equipped with a ZB-Wax Plus column. Small and large ester compounds were analysed on an Agilent 7890A GC coupled to a 5975C mass selective detector (MSD) using a ZB-5MS column and a partial stable isotope dilution analysis-solid-phase microextraction-gas chromatography-single ion monitoring mass spectrometry (SIDA-SPME-GC-SIM-MS) method based on Philipp et al. (2019a) and Fauster et al. (2020).

Free monoterpenes (11 compounds) were analysed on the same system using a headspace solid-phase microextraction-gas chromatography-single ion monitoring-mass spectrometry (HS-SPME-GC-SIM-MS) method according to Philipp et al. (2020). C ₁₃-norisoprenoids—including β-damascenone, β-ionone, 1,1,6-trimethyl-1,2-dihydronaphthalene (TDN), and vitispirane—were determined using a Trace 1300 GC system coupled to a TSQ Duo mass spectrometer with automated SPME via a TriPlus RSH autosampler, following the method described by Philipp et al. (2023).

Analysis of low-boiling-point sulfur-containing compounds was conducted by using an HP 6890 GC system equipped with a sulfur-selective SPB-1 column and a cooled injection system (CIS-4). Detection was carried out using headspace gas chromatography with pulsed flame photometric detection (HS-GC-PFPD) according to Jung et al. (2021). All systems were equipped with autosamplers and were validated for the corresponding target analytes.

Qualitative and quantitative analysis of polyfunctional thiols was performed after derivatisation with ethyl propiolate (ETP), followed by solid-phase extraction (SPE) and subsequent gas chromatography coupled to tandem mass spectrometry (GC-TQMS). The analysis was carried out using an Agilent 7890B GC system coupled to an Agilent 7010B triple quadrupole mass spectrometer. Chromatographic separation was achieved using a polar Stabilwax-DA column (30 m × 0.25 mm × 0.25 µm; Restek GmbH, Bad Homburg, Germany) with a 5 m IP-deactivated guard column (0.25 mm ID).

The sample preparation and analysis method was based on protocols developed by Herbst-Johnstone et al. (2013), Coetzee et al. (2018), and Kiene et al. (2021), with modifications tailored to the available instrumentation.

For SPE, 100 µL of 3- tert-butylanisole (2 mM in absolute ethanol, ≥ 99.9 %, VWR, Vienna, Austria), 10 µL of the internal standard 4-methoxy-2-methyl-2-butanethiol (11.2 µM in ethanol), and 20 µL of ETP (99 %, Merck, Vienna, Austria) were added to 50 mL of the sample. After mixing briefly, the pH was adjusted to 10 using sodium hydroxide (≥ 98 %, Carl Roth, Karlsruhe, Germany). The samples were centrifuged for 15 min at 4,000 rpm, and the supernatant was loaded onto a conditioned SPE cartridge (Supelclean ENVITM-18, 500 mg, 6 mL; Merck). The cartridge was conditioned with 10 mL methanol and 10 mL of a 13 % aqueous–ethanolic solution (v/v).

After loading (approximately 20 min under light vacuum), the cartridge was washed with 5 mL of ultrapure water (Gen Pure System, Thermo Scientific, Waltham, USA). Then, the cartridge was dried under vacuum with a defined nitrogen flow (5.0, Nippon Gases, Düsseldorf, Germany) for approximately 25 min. Target analytes were eluted using 10 mL of dichloromethane (DCM; 99.5 %, Carl Roth), and the eluate was dried over sodium sulphate (99.5 %, Carl Roth). The extract was reduced to 0.3 mL using a Syncore Analyst R-12 evaporator (Büchi Labortechnik AG, Flawil, Switzerland), followed by further concentration under a gentle nitrogen stream to approximately 30 µL.

2 µL of the DCM extract (kept at 10 °C at a cooled tray) were injected into the GC-TQMS system using an MPS robotic pro autosampler (Gerstel, Mülheim an der Ruhr, Germany). The injector temperature was 260 °C and operated in splitless mode for 2 min, followed by a purge flow of 50 mL/min. Helium (5.0, Nippon Gases) was used as carrier gas at a constant flow of 1.2 mL/min.

The GC oven programme was as follows: initial temperature of 60 °C (1 min), ramped at 25 °C/min to 100 °C (2 min), then at 5 °C/min to 250 °C (20 min). The transfer line temperature was set to 275 °C.

Detection of ETP-derivatised thiols was performed in the electron ionisation (EI) mode at 70 eV. Data were acquired in the multiple reaction monitoring (MRM) mode. Nitrogen (5.0) served as the collision gas (1.5 mL/min), while helium was used as quench gas (4 mL/min). The ion source temperature was 230 °C, and both quadrupoles were maintained at 150 °C.

Quantification was performed using external calibration in white wine ( Vitis vinifera L. cv. Reichensteiner, 2021 vintage; 0.1 g/L residual sugar, 6.4 g/L total acidity, and 11.2 % vol. alcohol) obtained from the department of Plant Breeding, Hochschule Geisenheim University.

Data analysis was performed using Agilent MassHunter Workstation Software (version B10.2).

Statistical analyses were performed using SPSS Statistics version 29.0 (IBM, Armonk, NY, USA) and XLSTAT version 2023.3.1 (Lumivero, Denver, CO, USA). The data were first log-transformed in accordance with the approach described by Leydesdorff and Bensman (2006). The data were subjected to the Shapiro–Wilk test to check whether it followed a normal distribution, and Levene’s test to assess homogeneity of variances. In SPSS Statistics, one-way analysis of variance (ANOVA) was used to assess differences among the treatment variants, followed by Tukey’s B test for post hoc comparisons between groups. A p-value < 0.05 was considered to indicate a statistically significant difference. The main effects of the three oenological factors—oak chips, GSH-IDY, and oxygen—were analysed via a three-way ANOVA (excluding interaction terms) using XLSTAT, following the approach of Williams (1986). Additionally, a principal component analysis (PCA) was carried out in XLSTAT based on the methodology of Leyer and Wesche (2007).

Table 3 presents the results of quantified monoterpenes. These compounds—including geraniol, linalool, nerol, and citronellol, as well as their derivative rose oxide—are volatile isoprenoids that contribute to characteristic aroma notes such as ‘rose’, ‘lilac’, ‘pine’, or ‘citrus’. These C ₁₀ compounds tend to accumulate during late ripening, particularly in Muscat and Gewürztraminer, but are also present in smaller amounts in varieties such as Riesling, Viognier, and Chenin blanc ( Robinson et al., 2014).

In the present study, trans-linalool oxide concentrations differed significantly among the variants ( p = 0.015), although none of the experimental factors (oak chips, GSH-IDY, and oxygen) had a significant effect. BF_V1 had the highest concentration, whereas the remaining variants showed concentrations similar to the control. cis-Linalool oxide also showed significant differences ( p = 0.002), with oak chips exerting a moderate effect (**). The control variant exhibited the lowest concentration, while BF_V1 displayed a significantly elevated concentration, suggesting a specific influence of oak on this compound.

There were no significant differences for linalool or hotrienol among the variants ( p = 0.303 and 0.348, respectively); the concentrations of both compounds remained stable under the applied experimental conditions. Similarly, the concentrations of β-terpineol and nerol oxide showed no substantial variation across the variants ( p = 0.098 and 0.205, respectively). These results indicate that their concentrations are primarily varietal in origin.

trans-Limonene oxide showed significant differences among the variants ( p = 0.015), with oak chips (**) and GSH-IDY (***) exerting a significant effect. BF_V2 showed the highest concentration, while BF_V4 had the lowest concentration. α-Terpineol also showed significant differences ( p = 0.038), with oak chips (**) as the only significant factor. The highest concentration was detected in BF_V1, while the other variants showed only slight increases compared with the control.

For nerol ( p = 0.002), the GSH-IDY (**) effect was significant. BF_V3 showed the highest concentration, whereas the other variants showed concentrations similar to the control. These findings highlight the role of glutathione in the preservation or promotion of this compound, likely through antioxidant mechanisms.

Citronellol and geraniol did not exhibit significant differences among the variants ( p = 0.071 and 0.073, respectively). Although oak chips exerted a slight influence on citronellol, and GSH-IDY had a slight effect on geraniol, these effects were not statistically significant.

Monoterpenes such as linalool, α-terpineol, and citronellol are key contributors to the floral and fruity aroma of many wines. Based on the results, the experimental factors had only a limited influence on their concentrations. The observed trends for GSH-IDY are partly consistent with previous studies that have attributed a protective and stabilising role to glutathione with respect to monoterpenes ( Bahut et al., 2020; Rodríguez-Bencomo et al., 2014; Gabrielli et al., 2017; Cojocaru & Antoce, 2019). The relatively modest effects observed in this study may be explained by the specific combination of treatments and the relatively short storage period of five months, as glutathione is known to exhibit more pronounced antioxidant activity over longer ageing periods ( Bahut et al., 2020).

Table 3. The content of free monoterpenes and their modulation on Blaufränkisch wines after the application of selected additives and treatment aids.

The three columns to the right of the parameter column present the results of the three-way analysis of variance, indicating the main effects of oak chips, GSH-IDY, and oxygen (** p < 0.01; *** p < 0.001; n.s., not significant). The adjacent columns to the right contain the quantified concentrations (x̄ ± σ) of individual compounds for the control group and the four experimental variants. Statistically significant differences ( p < 0.05) between the variants are indicated by different superscript letters. Bold p-values indicate statistically significant differences at p < 0.05. GSH-IDY, glutathione-rich inactivated yeast.

Table 4 presents the concentrations of quantified C ₆ alcohols. These compounds are formed by the enzymatic reduction of C ₆ aldehydes such as hexanal and hexenal, which themselves originate from the oxidation of polyunsaturated fatty acids ( e.g., linoleic and linolenic acid) due to damage to grape seeds and skins during processing. They are generally characterised by ‘fresh-green’, ‘grassy’, or ‘herbaceous’ aromas ( Dudareva et al., 2013).

In the present study, trans-2-hexen-1-ol showed significant differences among the variants ( p = 0.001), with oak chips (**) exerting a significant influence, while GSH-IDY and oxygen had no significant effect. The control variant exhibited the highest concentration, whereas BF_V3 and BF_V4 showed the lowest concentrations. These findings suggest that oak chip treatment may reduce trans-2-hexen-1-ol concentrations.

Hexanol also presented highly significant differences among the variants ( p < 0.001), with both oak chips (***) and GSH-IDY (***) showing strong effects. Oak chips led to lower concentrations in BF_V1 and BF_V4, while BF_V2 and BF_V3 exhibited slightly elevated concentrations, indicating a potentially stabilising role of GSH-IDY. Oxygen had no significant effect in either case.

These findings suggest that oak chips play a key role in modulating both trans-2-hexen-1-ol and hexanol. They partially contrast with mechanisms that have been described in the literature, which propose that glutathione reacts with trans-2-hexenal to form Glut-3MHal, a precursor to thiols such as 3-SH and 3-SHA ( Clark & Deed, 2018). The non-significant effect of GSH-IDY observed here may be attributed to the phenolic composition specific to Blaufränkisch wines or to the comparatively short ageing period, which may have limited the kinetics of glutathione–aldehyde reactions.

Harsch et al. (2013) and Kritzinger et al. (2013) have also reported that H ₂S released during fermentation can react with trans-2-hexenal to form polyfunctional thiols. The relatively high YAN (296 mg/L) in this study may have limited H ₂S release by yeast ( Kwasniewski et al., 2011), thereby reducing the availability of precursors for thiol formation.

The effect of GSH-IDY on hexanol may be explained by its function as a reducing agent that inhibits oxidative degradation processes, thus preserving hexanol stability. This view is consistent with reports indicating that hexanol is formed via the lipoxygenase/hydroperoxide lyase (LOX/HPL) pathway from enzymatic oxidation of linoleic acid ( Hatanaka, 1993). Glutathione may further contribute indirectly to hexanol formation by protecting polyfunctional thiols and other C ₆ compounds such as hexanol from oxidation ( Kritzinger et al., 2013). As noted earlier, GSH also plays a central role in thiol release during early fermentation via H ₂S-mediated mechanisms ( Harsch et al., 2013; Araujo et al., 2017).

Table 4. The C ₆ alcohol content and its modulation in Blaufränkisch wines after the application of selected additives and treatment aids.

The three columns to the right of the parameter column present the results of the three-way analysis of variance, indicating the main effects of oak chips, GSH-IDY, and oxygen (** p < 0.01; *** p < 0.001; n.s., not significant). The adjacent columns to the right contain the quantified concentrations (x̄ ± σ) of individual compounds for the control group and the four experimental variants. Statistically significant differences ( p < 0.05) between the variants are indicated by different superscript letters. Bold p-values indicate statistically significant differences at p < 0.05. GSH-IDY, glutathione-rich inactivated yeast; n.d., not detectable.

Table 5 presents the concentrations of the quantified thiols. Polyfunctional thiols such as 3-sulfanylhexan-1-ol (3-SH), 3-sulfanylhexyl acetate (3-SHA), 4-methyl-4-sulfanylpentan-2-one (4-MSP), 2-furfurylthiol (2-FM), and benzenemethanethiol (BM) contribute to distinct aroma characteristics, including notes of blackcurrant, citrus, roasted coffee, and smoke. 3-SH and 4-MSP are present in grapes in non-volatile, bound forms, predominantly as cysteine and glutathione conjugates ( e.g., Cys-3SH, Glut-3SH, Cys-4MSP, Glut-4MSP), which are enzymatically cleaved by yeast during alcoholic fermentation, provided the strain expresses sufficient β-lyase activity ( Tominaga et al., 1998; Fedrizzi et al., 2009; Swiegers et al., 2007; Subileau et al., 2008; Ruiz et al., 2019). The release of 4-MSP from its cysteinylated precursor is catalysed by enzymes encoded by the IRC7 gene ( Cordente et al., 2017), while 3-SH can be further acetylated to 3-SHA via ATF1-mediated acetyltransferase activity ( Swiegers et al., 2005).

There were significant differences in the 4-MSP concentrations among the variants ( p < 0.001). Three-way ANOVA indicated that oak chips (***) and oxygen (***) had a highly significant effect, while GSH-IDY showed no significant impact. The control variant had the highest 4-MSP concentration, whereas BF_V1 and BF_V3 (both without oxygen) exhibited the lowest concentrations. This suggests that the combined application of oak chips and oxygen may moderately increase 4-MSP. The intermediate concentrations in BF_V2 and BF_V4 further confirm the joint influence of these two factors.

The 3-SH concentrations also varied significantly among the variants ( p < 0.001). Oak chips had a highly significant impact (***), while GSH-IDY and oxygen were not significant. The control variant again showed the highest concentration, followed by BF_V4. All other variants (BF_V1, BF_V2, and BF_V3) demonstrated a significant reduction in 3-SH, underlining the dominant effect of oak chips, with only marginal contributions from GSH-IDY or oxygen.

The E3-SP concentrations differed significantly among the variants ( p = 0.005), with GSH-IDY showing a significant effect (**), while oak chips and oxygen had no significant impact. BF_V1 showed the highest concentration, while control and BF_V3 exhibited the lowest concentrations. BF_V2 and BF_V4 showed intermediate concentrations, indicating that GSH-IDY may exert a stabilising or enhancing effect on E3-SP, with little contribution from the other two factors.

There were also significant differences in the 2-FM concentrations ( p < 0.001). Oak chips had a highly significant effect (***), and GSH-IDY showed a significant effect (**), but oxygen had no significant influence. The control variant displayed the lowest concentration, while BF_V1 (oak chips only) exhibited a significantly elevated concentration, highlighting the dominant role of oak. BF_V2 and BF_V4 reached similarly high concentrations, whereas BF_V3 showed a slightly reduced concentration, suggesting that the effect of GSH-IDY may depend on the presence of oxygen. Overall, oak chips remained the strongest influencing factor, with GSH-IDY contributing moderately and oxygen showing no relevant effect.

There were significant differences for BM among the variants ( p = 0.021). Specifically, oak chips had a significant effect (**), while GSH-IDY and oxygen showed no significant impact. The control variant showed the highest BM concentration. BF_V1 and BF_V2 (both with oak chips) showed moderate reductions, while BF_V3 and BF_V4 had the lowest concentrations, suggesting a modest reductive effect of oak chips, without direct influence from GSH-IDY or oxygen.

In this study, 4-MSP, associated with blackcurrant and exotic fruit aromas, seemed to be particularly influenced by the addition of oak chips. This may be explained by interactions between ellagitannins from oak and thiol precursors, as described by Petit et al. (2015). However, the present findings contrast with those of Papadopoulou and Roussis (2008) and Bahut et al. (2020), who reported an antioxidant effect of GSH-IDY on polyfunctional thiols such as 4-MSP. Such an effect was not confirmed in this study, potentially due to the specific conditions associated with the Blaufränkisch variety. Ultimately, the concentrations of 4-MSP ranged between 1.8 and 2.9 ng/L across all variants, including 2.8 ng/L in the control, potentially exceeding the odour detection threshold and making this compound perceivable in the wines ( Muhl et al., 2022; Carlin et al., 2022).

3-SH imparts citrus and tropical fruit aromas ( Tominaga et al., 1996). The results indicate that oak chip treatment leads to a marked reduction of 3-SH in Blaufränkisch wines. This may again be related to ellagitannin interactions, as suggested by Petit et al. (2015). These findings contradict reports by Papadopoulou and Roussis (2008) and Bahut et al. (2020), who observed a protective effect of glutathione on 3-SH. Notably, the control variant—produced without oak—showed the highest 3-SH concentration. Given that glutathione naturally reacts with trans-2-hexenal to form Glut-3MHal ( Clark & Deed, 2018), the significant reduction in trans-2-hexenal by oak observed in this study may explain the lower 3-SH concentrations in the oak chip-treated variants. Across all variants, 3-SH concentrations ranged from 139.4 to 187.3 ng/L, clearly exceeding the reported odour detection threshold of 60 ng/L ( Tominaga et al., 1996), and are therefore likely to contribute perceptibly to the wine’s aroma profile.

The ester 3-SHA, formed through acetylation of 3-SH by yeast enzymes, was not detected in either the control or experimental variants. This is likely due to the use of a single yeast strain that potentially does not express the ATF1 gene ( Swiegers & Pretorius, 2007). Alternatively, matrix effects specific to red wine or post-fermentation losses during bottle storage may have inhibited its formation ( Herbst-Johnstone et al., 2011).

2-FM is associated with roasted coffee aromas and is formed by the addition of H ₂S to furfural, a compound extracted from oak ( Blanchard et al., 2001). This reaction highlights the pivotal role of oak chips in 2-FM formation and supports the findings of this study. In the present work, 2-FM concentrations ranged from 3.1 to 4.0 ng/L, clearly exceeding the reported odour detection threshold of 0.4 ng/L, indicating a likely perceptible contribution to the wine’s aroma profile.

BM is associated with smoky, mineral notes ( e.g., flint) and is considered a stylistic element in varieties such as Chardonnay and Sauvignon blanc, rather than a fault ( Tominaga et al., 2003). Although its formation is not fully understood, it is hypothesised to result from the addition of H ₂S or other reductive compounds to benzaldehyde ( Tominaga et al., 2003). This mechanism was recently supported by Cordente et al. (2024), who observed increased BM formation in fermentations spiked with benzaldehyde—however, only when high-H ₂S-producing yeast strains were used. The observed reduction in BM due to oak may be attributed to interactions with ellagitannins ( Petit et al., 2015). In the present study, BM concentrations ranged from 5.4 to 7.4 ng/L, well above the reported odour detection threshold of 0.3 ng/L, suggesting that it likely contributed perceptibly to the wines’ aromatic profile.

Table 5. The content of polyfunctional thiols and their modulation in Blaufränkisch wines after the application of selected additives and treatment aids.

The three columns to the right of the parameter column present the results of the three-way analysis of variance, indicating the main effects of oak chips, GSH-IDY, and oxygen (** p < 0.01; *** p < 0.001; n.s., not significant). The adjacent columns to the right contain the quantified concentrations (x̄ ± σ) of individual compounds for the control group and the four experimental variants. Statistically significant differences ( p < 0.05) between the variants are indicated by different superscript letters. Bold p-values indicate statistically significant differences at p < 0.05. GSH-IDY, glutathione-rich inactivated yeast; n.d., not detectable; ODT, Odour Detection Threshold.

Table 6 presents the quantified concentrations of low-boiling-point sulfur-containing compounds. Volatile sulfur compounds such as H ₂S, MeSH, and ethanethiol are known to contribute to off-flavours reminiscent of rotten eggs, cheese, leeks, onions, and garlic at elevated concentrations. However, at sub-threshold levels, they may enhance wine complexity ( Siebert et al., 2009).

H ₂S concentrations differed significantly among the variants ( p = 0.011), with oak chips (**) and GSH-IDY (***) exerting significant to highly significant effects, while oxygen had no influence. The control variant exhibited a moderate H ₂S concentration. There were slight reductions in BF_V1 and BF_V4 (oak chips + oxygen), whereas BF_V2 and BF_V3 showed slightly elevated levels.

The MeSH concentrations did not differ significantly between the variants ( p = 0.083). GSH-IDY exhibited a significant effect (**), whereas oak chips and oxygen had no significant impact. The concentrations remained relatively similar across all treatments, suggesting minimal modulation by the experimental factors.

The dimethyl sulphide (DMS) concentrations also showed no significant differences among the variants ( p = 0.090), although oxygen exerted a highly significant effect (***), while oak chips and GSH-IDY showed no influence. The concentrations indicated no clear trends among the variants.

The carbon disulfide (CS ₂) concentrations varied significantly among the variants ( p = 0.006), with oak chips (***) and GSH-IDY (***) showing highly significant effects, while oxygen showed no influence.

These findings suggest that GSH-IDY, particularly when combined with oak chips, may promote H ₂S formation. Despite these moderate concentrations—likely mitigated by the sufficiently high YAN concentration of 296 mg/L in the must ( Bell & Henschke, 2005)—H ₂S remains of sensory concern due to its low perception threshold and potential to cause off-aromas ( Siebert et al., 2009).

MeSH, associated with cheese and cooked vegetable notes, was detected at concentrations below its odour threshold, and is therefore unlikely to have contributed perceptibly to the aroma profile of the wines ( Solomon et al., 2010).

At low concentrations, DMS may contribute to positive aromas such as blackcurrant, red fruit, and truffle ( Escudero et al., 2007; Vidal & Aagaard, 2008). However, it may become problematic at higher levels due to vegetal or unpleasant notes ( Mestres et al., 2000). In this study, DMS concentrations ranged between 7.5 and 9.5 µg/L, remaining well below the reported perception threshold of 27–60 µg/L. This suggests that any sensory impact would likely be subtle and potentially positive.

Recent research indicates that moderate oxygen addition during fermentation acts primarily as a preventive measure to reduce the formation of malodorous sulfur compounds such as H ₂S and mercaptans ( Nelson et al., 2025). In the case of Blaufränkisch, where nitrogen availability was high, H ₂S production was minimal from the outset, resulting in only trace levels of the downstream compound carbon disulfide (CS ₂). Consequently, the single oxygen addition of 15 mg/L had no measurable effect on the already low H ₂S and CS ₂ concentrations. Moreover, CS ₂ is relatively unreactive and is not degraded by moderate amounts of oxygen ( Zeng et al., 2019). These findings are consistent with other studies, which report that while oxygen additions can reduce reductive off-flavours overall, absolute H ₂S and CS ₂ levels remain unchanged in the low µg/L range ( Lyu et al., 2021).

Table 6. The content of volatile sulfur-containing components and their modulation in Blaufränkisch wines after the application of selected additives and treatment aids.

The three columns to the right of the parameter column present the results of the three-way analysis of variance, indicating the main effects of oak chips, GSH-IDY, and oxygen (** p < 0.01; *** p < 0.001; n.s., not significant). The adjacent columns to the right contain the quantified concentrations (x̄ ± σ) of individual compounds for the control group and the four experimental variants. Statistically significant differences ( p < 0.05) between the variants are indicated by different superscript letters. Bold p-values indicate statistically significant differences at p < 0.05. GSH-IDY, glutathione-rich inactivated yeast; n.d., not detectable; ODT, Odour Detection Threshold.

Table 7 presents the quantified concentrations of higher alcohols, which are formed during yeast metabolism and contribute significantly to the sensory profile of wine ( Valero et al., 2002; Garde-Cerdán et al., 2021). They are associated with aroma descriptors such as rose, lilac, ripe fruit, herbs, and whisky ( Francis & Newton, 2005; Perestrelo et al., 2020). While concentrations below 300 mg/L are generally considered to enhance aromatic complexity, levels exceeding 400 mg/L may have a detrimental effect on wine quality by contributing to harsh or solvent-like notes ( Rapp & Versini, 1995; Ribéreau-Gayon et al., 2006; Garde-Cerdán et al., 2021).

The propanol and isobutanol concentrations did not differ significantly among the variants ( p = 0.259 and 0.459, respectively). None of the three experimental factors exerted a significant influence. Isoamyl alcohol ( p = 0.022) was significantly influenced by oak chips, with all oak-treated variants exhibiting higher concentrations than the control. Hence, oak chips may promote isoamyl alcohol formation during fermentation. Butanol also differed significantly among the variants ( p = 0.005), with GSH-IDY emerging as the main influencing factor, while oak chips and oxygen showed no significant effects. BF_V3 showed the highest concentration, while the control variant and BF_V4 showed the lowest concentrations.

These stable concentrations of propanol and isobutanol are consistent with the findings of Gabrielli et al. (2017), who observed no clear effect of glutathione on these alcohols. At moderate levels, both compounds contribute to subtle lilac and herbal notes and enhance the aromatic complexity of wine without reaching concentrations that negatively affect sensory perception ( Ribéreau-Gayon et al., 2006).

Isoamyl alcohol contributes to the aromas of ripe fruit and herbs ( Francis & Newton, 2005) and may have enhanced the aromatic complexity of the Blaufränkisch wines evaluated in this study. However, these findings contrast with those of Gabrielli et al. (2017), who reported a positive effect of glutathione on the isoamyl alcohol concentration.

The findings regarding butanol underscore the role of glutathione in maintaining redox balance and stabilising volatile compounds, as described in the literature ( Gabrielli et al., 2017). Butanol was present at levels unlikely to produce sensory faults but may contribute to subtle, alcohol-related aroma nuances ( Francis & Newton, 2005).

The total higher alcohol concentration ranged from 304 mg/L (control) to 328 mg/L (BF_V3), which falls within the typical range reported in the literature ( Francis & Newton, 2005; Gabrielli et al., 2017). Overall, these results suggest that higher alcohols in Blaufränkisch wines may contribute positively to aromatic complexity and mouthfeel. The findings further indicate that oak chips play a significant role in modulating isoamyl alcohol, while GSH-IDY appears to enhance butanol formation.

Table 7 also presents the concentrations of quantified carboxylic acids. These compounds are associated with aroma descriptors such as fruity, fatty, cheesy, sweaty, buttery, and rancid ( Francis & Newton, 2005), and contribute to the sensory profile of wine depending on their concentration and composition. Carboxylic acids, particularly those responsible for buttery or rancid notes, can be modulated by different vinification treatments ( Carpena et al., 2020; Garde-Cerdán et al., 2021).

Propionic acid remained largely stable under the tested conditions, with only oak chips exerting a moderate influence, while GSH-IDY and oxygen had no significant effects. In contrast, isobutyric acid was significantly reduced by oak chip treatment. Neither GSH-IDY nor oxygen had a notable effect.

There were significant differences in butyric acid among the variants, but there were no clear effects of the three experimental factors. Some variants exhibited slightly higher concentrations than the control, but the differences did not reach statistical significance. Hexanoic acid showed a similar pattern: the concentrations varied among the variants, but oak chips, GSH-IDY, and oxygen did not show a significant influence. BF_V3 and BF_V4 had the highest concentrations, while the control and BF_V1 showed the lowest concentrations.

Octanoic acid emerged as a clear exception, with oak chips having a highly significant impact. The control variant showed the lowest concentration, while BF_V4 had the highest, suggesting that oak chips may promote octanoic acid formation. There was a similar trend for decanoic acid, for which oxygen exerted a significant influence. However, the differences between the variants were minor and may have been affected by analytical variability.

These results align with Gabrielli et al. (2017), who noted that treatments such as GSH-IDY addition can differentially affect the concentrations of specific fatty acids, including hexanoic, octanoic, and decanoic acid.

These findings highlight the partially significant role of oak chips in modulating fatty acid concentrations, while GSH-IDY and oxygen had less consistent effects. Previous studies, such as the one by Gabrielli et al. (2017), have reported that glutathione increases the concentrations of certain fatty acids, including hexanoic and octanoic acid. The discrepancies may be explained by different experimental conditions, such as nitrogen availability and/or storage time. The present results suggest that further research is needed to better understand the specific interactions between oak chips, GSH-IDY, and oxygen and their influence on wine aroma development.

Table 7. The content of higher alcohols and carboxylic acids and their modulation in Blaufränkisch wines after the application of selected additives and treatment aids.

The three columns to the right of the parameter column present the results of the three-way analysis of variance, indicating the main effects of oak chips, GSH-IDY, and oxygen (** p < 0.01; *** p < 0.001; n.s., not significant). The adjacent columns to the right contain the quantified concentrations (x̄ ± σ) of individual compounds for the control group and the four experimental variants. Statistically significant differences ( p < 0.05) between the variants are indicated by different superscript letters. Bold p-values indicate statistically significant differences at p < 0.05. GSH-IDY, glutathione-rich inactivated yeast; n.d., not detectable.

Table 8 presents the quantified concentrations of esters. These volatile aroma compounds are synthesised during alcoholic fermentation through the reaction of alcohols with carboxylic acids and play a central role in the aromatic expression of wine by contributing fruity and floral notes. Ethyl and acetate esters, in particular, are considered key contributors to sensory quality due to their typically high concentrations, which often exceed olfactory thresholds ( Carpena et al., 2020; Garde-Cerdán et al., 2021).

The ester concentrations in the analysed Blaufränkisch wines were partially influenced by oak chips, GSH-IDY, and oxygen. However, compounds such as ethyl benzoate, ethyl lactate, ethyl propanoate, ethyl decanoate, and ethyl palmitate showed no significant differences among the variants, suggesting that their formation is less susceptible to oxidative or fermentative modulation. This observation is consistent with Gabrielli et al. (2017), who reported similar stability for ethyl esters of long-chain fatty acids under varying oenological conditions.

In contrast, esters such as ethyl phenylacetate, diethyl succinate, and ethyl butanoate exhibited significant variations, particularly due to oak chips, GSH-IDY, and oxygen. Oak chips appeared to promote the formation of ethyl phenylacetate and diethyl succinate, possibly due to indirect effects on yeast metabolism, such as modulating the fermentation environment ( e.g., oxygen availability or adsorption of inhibitory compounds), which may favour enhanced ester biosynthesis during alcoholic fermentation ( Sánchez-Palomo et al., 2017). Diethyl succinate was also enhanced by oxygen. GSH-IDY showed a predominantly stabilising effect and significantly influenced compounds such as ethyl butanoate and ethyl octanoate, consistent with its known antioxidant properties ( Papadopoulou & Roussis, 2008; Roussis et al., 2009). Ethyl octanoate, known to contribute significantly to the fruity aroma profile of wine, increased in response to both oak chips and GSH-IDY ( Francis & Newton, 2005; Swiegers et al., 2005).

Hexyl acetate showed significant variations across the variants but no consistent correlation with the three experimental factors. In contrast, isobutyl acetate concentrations, which also varied significantly, were reduced by the addition of oak chips. There were similar trends for methyl hexanoate and methyl laurate, which presented significant differences, although the practical impact was minor. These variations may be attributable to analytical fluctuations or unaccounted factors, as has been reported in studies involving comparable grape varieties ( Andújar-Ortiz et al., 2014).

Interestingly, isoamyl acetate, a key contributor to fruity aromas, remained stable under the applied experimental conditions, despite its known sensitivity to fermentation parameters ( Valero et al., 2002). In contrast, esters such as ethyl hexanoate and ethyl laurate showed significant changes, particularly following the addition of oak chips and GSH-IDY, suggesting that the formation of these esters may be particularly influenced by specific oenological factors. ( Papadopoulou & Roussis, 2008).

With regard to esters, the results of this study highlight the central role of GSH-IDY in their stabilisation and enhancement, while oak chips promoted the formation of compounds such as diethyl succinate. These findings are consistent with previous studies highlighting the antioxidant and aroma-stabilising properties of glutathione ( Roussis et al., 2009; Bahut et al., 2020) and underline the potential to improve wine sensory quality through targeted oenological strategies. The overall limited influence of the experimental factors on ester concentrations may be partly due to the relatively short bottle ageing period of five months. Esters are known to degrade over time via oxidation or hydrolysis, processes that can be mitigated by substances such as glutathione, which preserve ester stability and sensory integrity ( Roussis et al., 2009). Oxygen exhibited only minor effects under the present conditions but appeared to contribute to the formation of certain esters, such as diethyl succinate and ethyl octanoate. These observations are consistent with previous studies, which report that oxygen additions during red wine fermentation can lead to modest increases in specific esters, particularly short-chain ethyl and acetate esters ( Day et al., 2021). However, the magnitude of these changes is typically limited, with some reports even indicating slight reductions depending on grape variety and oxygen dosage ( Picariello et al., 2020).

Table 8. The ester content and its modulation in Blaufränkisch wines after the application of selected additives and treatment aids.

The three columns to the right of the parameter column present the results of the three-way analysis of variance, indicating the main effects of oak chips, GSH-IDY, and oxygen (** p < 0.01; *** p < 0.001; n.s., not significant). The adjacent columns to the right contain the quantified concentrations (x̄ ± σ) of individual compounds for the control group and the four experimental variants. Statistically significant differences ( p < 0.05) between the variants are indicated by different superscript letters. Bold p-values indicate statistically significant differences at p < 0.05. GSH-IDY, glutathione-rich inactivated yeast; n.d., not detectable.

Table 9 presents the quantified concentrations of C ₁₃-norisoprenoids. These chemically diverse compounds—including ketones, aldehydes, and alcohols—have a 13-carbon backbone. Among the most prominent representatives are β-damascenone, associated with apple, rose, dried fruit, honey, and exotic floral notes, and β-ionone, which contributes to raspberry-like, violet, and other floral or fruity aromas ( Sefton et al., 2011; Robinson et al., 2014). These compounds enhance the fruity character of wines and can mask herbaceous or green-grassy notes originating from methoxypyrazines or C ₆ alcohols ( Pineau et al., 2007; Escudero et al., 2007).

The highly significant effects of oak chips, GSH-IDY, and oxygen on vitispirane concentrations are consistent with the literature: researchers have described complex interactions between wine matrix components and the release or preservation mechanisms of norisoprenoids. Rodríguez-Bencomo et al. (2011) reported that tannins and the polymerisation of phenolic compounds may influence the release of norisoprenoid ketones, including vitispirane. The elevated vitispirane concentration observed in BF_V4 may be attributed to phenolic compounds present in oak and their potential interactions.

The strong effect of GSH-IDY on β-damascenone is particularly noteworthy, as glutathione may protect norisoprenoids from oxidation through its free thiol group ( Papadopoulou & Roussis, 2008). BF_V3 had the highest β-damascenone concentration, highlighting the stabilising role of GSH-IDY, while oak chips and oxygen had no significant influence. These results contrast with those of Rodríguez-Bencomo et al. (2011), who reported reduced norisoprenoid release at higher tannin levels. This discrepancy may be due to differences in phenolic composition or storage conditions.

The β-ionone concentrations differed significantly among the variants, although none of the tested factors showed a significant effect. Therefore, these variations may be attributed to uncontrolled variables or analytical uncertainty.

The consistent concentrations of TDN across all variants and the absence of significant factor effects confirm the chemical stability of this norisoprenoid under the present experimental conditions. TDN is commonly associated with kerosene-like aromas and typically increases in aged wines, suggesting a distinct dynamic of release and preservation ( Gök, 2015).

In summary, the results underscore the central role of GSH-IDY in stabilising β-damascenone and the multifactorial influences of oak chips, GSH-IDY, and oxygen on the vitispirane concentration, while β-ionone and TDN remain largely unaffected.

Table 9. The content of free C ₁₃-norisoprenoids and their modulation in Blaufränkisch wines after the application of selected additives and treatment aids.

The three columns to the right of the parameter column present the results of the three-way analysis of variance, indicating the main effects of oak chips, GSH-IDY, and oxygen (** p < 0.01; *** p < 0.001; n.s., not significant). The adjacent columns to the right contain the quantified concentrations (x̄ ± σ) of individual compounds for the control group and the four experimental variants. Statistically significant differences ( p < 0.05) between the variants are indicated by different superscript letters. Bold p-values indicate statistically significant differences at p < 0.05. GSH-IDY, glutathione-rich inactivated yeast.

To provide a comprehensive visual representation of the dataset, a PCA was performed on the analytical data. The Kaiser–Meyer–Olkin (KMO) measure of sampling adequacy was 52.35 %, which supports the suitability of PCA for the data ( Leyer & Wesche, 2007).

Figure 2 illustrates the relationship between the control and experimental variants and the quantified aroma compounds, with several variables presented as grouped parameters. The first two principal components (F1 and F2) accounted for nearly 53 % of the total variance, with F1 and F2 explaining 23.58 % and 28.78 %, respectively. The PCA revealed distinct positive correlations between specific analytical compounds and the individual treatment variants.

In the upper right quadrant, BF_V2 and BF_V3, both combining oak chips and GSH-IDY, showed strong positive correlations with butanol, acetate esters, even-chain ethyl esters, isoamyl esters, and aromatic esters, as well as with isoamyl alcohol, ethyl propanoate, DMS, and diethyl succinate. These compounds are known contributors to fruity, floral, and complex wine aromas ( Rapp & Versini, 1995; Francis & Newton, 2005). As fermentation-derived volatiles, esters and higher alcohols are critical to wine flavour and aroma ( Valero et al., 2002; Ribéreau-Gayon et al., 2006). Their positive association with these treatments may be attributed to the synergistic effects of oak and GSH-IDY, the latter potentially enhancing ester stability and concentration via antioxidant mechanisms ( Rodríguez-Bencomo et al., 2014; Bahut et al., 2020). Linalool, α-terpineol, citronellol, and hotrienol, all monoterpenes associated with citrus and rose notes, also showed positive correlations with these variants ( Kritzinger et al., 2013). Notably, β-damascenone, which contributes to red fruit aromas, was positively associated with these treatments, suggesting a potential protective effect of GSH-IDY ( Escudero et al., 2007; Pineau et al., 2007).

In the upper left quadrant, a biological replicate of BF_V3 correlated positively with odd-chain ethyl esters, esters of higher alcohols, and medium-length fatty acid esters, as well as with geraniol, nerol, hexanol, and trans-Limonene oxide compounds known to enhance floral and fruity notes and contribute to sensory complexity ( Francis & Newton, 2005). These correlations may also be attributed to the antioxidant capacities of GSH-IDY and oak ( Papadopoulou & Roussis, 2008; Andújar-Ortiz et al., 2012). Additionally, volatile sulfur-containing compounds such as MeSH, H ₂S, and CS ₂ also correlated positively. These compounds can enhance complexity at low levels but cause sensory faults at higher concentrations ( Siebert et al., 2009; Solomon et al., 2010).

In the lower left quadrant, all biological replicates of the control were associated with compounds such as β-ionone, 3-SH, 4-MSP, BM, and trans-2-hexen-1-ol. Both 3-SH and 4-MSP are thiols linked to blackcurrant, grapefruit, and tropical fruit aromas ( Tominaga & Dubourdieu, 2003). The positive correlation between these thiols and trans-2-hexen-1-ol is particularly noteworthy. During fermentation, trans-2-hexen-1-ol can react with H ₂S and glutathione to form intermediates such as Glut-3MHal ( Clark & Deed, 2018). The strong correlation of the control with 3-SH and 4-MSP may be partly explained by higher levels of trans-2-hexen-1-ol, which is reduced significantly in treatments involving oak chips—potentially limiting thiol precursor availability.

In the lower right quadrant, the biological replicates of BF_V1 and BF_V4 (both without GSH-IDY) were associated with ethyl lactate, vitispirane, nerol oxide, E3SP, and 2-FM. The absence of reduced glutathione, known for its antioxidant properties, may have facilitated oxidative processes, leading to increased formation of oxidised monoterpene derivatives such as nerol oxide ( Papadopoulou & Roussis, 2008; Rodríguez-Bencomo et al., 2014). The presence of E3SP suggests that its formation may not be exclusively modulated by glutathione but could also be influenced by H ₂S and other factors ( Clark & Deed, 2018; Harsch et al., 2013). The positive correlation with 2-FM is likely attributable to the use of oak chips ( Blanchard et al., 2001).

Figure 2. Principal component analysis biplot correlating the individual analytical results of Blaufränkisch wines treated with oak chips, glutathione-rich inactivated yeast (GSH-IDY), and oxygen.