Vinification Technique Matters: Kinetic Insight into Color, Phenolics, Volatiles, and Aging Potential of Babica Wines

Abstract

Unveiling how vinification technique shapes wine identity, this study provides a comparative insight into the chemical and sensory profiles of Babica wines produced using traditional, enzyme-assisted, and thermovinification approaches. The kinetics of color parameters changes and the phenolic extraction were monitored during the first five days of maceration. Individual phenolics and volatiles were determined using high-performance liquid and gas chromatography, respectively, while the overall sensory quality of the wines was evaluated by panelists. Significant differences in the extraction kinetics of compounds of interest were observed among treatments, particularly during the first days of maceration. By the end of the study, the thermovinified wine exhibited the highest color intensity (3.80), redness (52.5%), and approximately two-fold higher concentrations of total phenolics (2205 mg gallic acid equivalents/L) compared to the other two treatments. It contained the lowest concentration of tannins (100 mg catechin equivalents/L), anthocyanins (117 mg of malvidin-3-glucoside equivalents/L), and esters and showed the highest levels of volatile alcohols. It was also characterized by the most intense blueberry aroma and astringency in sensory analysis. The applied maceration technique affects the chemical and sensory profiles of Babica wines, with thermovinification favoring young and highly colored wines, whereas conventional vinification enhances the wine’s aging potential.

1. Introduction

The importance of red wine phenolic compounds and volatiles in shaping the visual and sensory quality of wine, as well as their role during its aging, is well known to every winemaker and has recently gained recognition among consumers [1,2]. The most important prerequisite for obtaining a sensory adequate and stable wine is a high initial phenolic content, together with characteristic aroma components extracted from the grapes [2]. While grape-derived anthocyanins and tannins provide color, mouthfeel, taste, and aging potential, volatile compounds give wine its aroma and flavor [3,4,5].

Various parameters influence the overall chemical and sensory profile of wine, and vinification technology is a parameter that can be controlled. According to the findings of Cerpa-Calderón and Kennedy [6] on Merlot winemaking (considering traditional vinification, maceration duration, and the yield of crushed grapes), the authors demonstrated that only about 40% of anthocyanins and 20% of tannins are extracted from the grape skin into the wine. Therefore, different pre-fermentative techniques, such as application of pectolytic enzymes and pre-fermentation heat treatment or thermovinification, which can enhance the extraction of valuable compounds from grapes into wine, are intensively investigated and are becoming more widespread [7,8,9,10].

Thermovinification is a short, pre-fermentation heat treatment that rapidly disrupts skin cells and enhances anthocyanin extraction into the aqueous phase, without correspondingly increasing the extraction of alcohol-soluble tannins [11,12]. The accelerated color extraction allows shorter maceration duration, making thermovinification highly attractive for the production of fruit-driven red wine with vibrant color [10]. Originally developed in the 1970s as a preservation technique to inactivate laccase in botrytized grapes, it has evolved into a widely adopted technique for improving color intensity and early aromatic expression [11,13]. However, the efficacy of thermovinification depends strongly on grape cultivar, heating parameters (temperature × time), and the grape matrix composition [10,12].

The addition of pectolytic enzymes during winemaking accelerates the extraction of grape phenolics and polysaccharides, increases must yield, and enhances filtration. This practice also improves sensory and chromatic characteristics of wine by positively affecting its color, stability, structure, and volatile profile [1,14,15,16]. The enzymes not only facilitate the release of anthocyanins but also of more polymerized and galloylated polyphenols, which can improve mouthfeel and promote polymeric pigment formation [17].

Despite the large number of studies, contradictory results have been reported, and wine outcomes remain complex and unpredictable. The initial quality and potential of the grapes should be especially emphasized because the extraction of key ingredients is closely related to the cultivar, growing conditions, and harvest period [1,11,14,15,18,19,20]. In addition to the characteristics of raw material, various winemaking parameters also affect chemistry and properties of the final wines—grape crushing and skin-to-juice ratio [1,6,8], maceration duration [1,5,6,8], maceration and/or thermovinification temperature [7,8,10], use of enzyme-preparations and/or oenological tannins (e.g., type and dose) [1,7,8,14,15], and aging duration [1,7], among others.

The Croatian coastal region of Dalmatia has a centuries-old viticultural tradition and is home to numerous autochthonous grapevine varieties. For many minor grapevine varieties in Croatia, the optimal winemaking technology that would fully valorize their phenolic and volatile potential remains insufficiently understood. Among these, Babica has recently regained attention due to its favorable viticultural, chemical, and enological traits. Preliminary ampelographic observations indicate that Babica exhibits moderate vigor, good disease resistance, and yields grapes with medium sugar content and balanced acidity, supporting its suitability for varietal, light-bodied red wines. Babica is primarily used as a blending grape valued for its soft tannins and distinctive aromatic qualities [21]. It has a strong potential for enhanced extraction of color and bioactive compounds, particularly anthocyanins, which are central to the chromatic intensity, antioxidant activity, and sensory perception of red wines [22]. However, traditional winemaking techniques may not fully utilize this potential. Therefore, the aim of this study was to investigate the impact of three different vinification techniques, namely, traditional maceration, thermovinification, and enzyme-assisted maceration, on the extraction kinetics and final composition of phenolic compounds, color parameters, volatile profiles, and sensory characteristics of Babica wines. Special focus was placed on monitoring the phenolic and color changes during the first five days of maceration, as well as evaluating the compositional and sensory differences of the final wines.

2. Materials and Methods

2.1. Grapes and Vinification

In this study, fully ripe and healthy red grapes from Vitis vinifera L. cultivar Babica (300 kg), harvested in mid-September from the Kaštela-Trogir vineyard (Dalmatia, Croatia), were used. The sugar content in grapes was 18°KMW (88°Oe), and the total acidity was 5.75 g/L (expressed as tartaric acid). The vinification procedure was modified from Generalić Mekinić et al. [14,15]. The grapes were destemmed and crushed using an electric machine (MGM-940, MIO, Osijek, Croatia), treated with 10 g/hL of potassium metabisulfite, and divided into three batches of 100 kg each. Applied winemaking techniques included traditional maceration, thermovinification, and enzyme-assisted vinification. All samples were inoculated with a commercial Saccharomyces cerevisiae yeast (15 g/hL, SIHA Hefe 8, Eaton Begerrow GmbH & Co., Langenlonsheim, Germany). Maceration and fermentation were carried out in open-top tanks with a mechanical cap submersion barrier to keep the pomace cap submerged under the juice. The fermentation temperature in all samples was 25 ± 1 °C. Maceration lasted for 6 days, after which the grape pomace was pressed using a press (VSPIX 170 L, Lancman, Vransko, Slovenia).

The first batch, traditional vinification was used as a control, and it was prepared without enzymes or heat treatment. For thermovinification, the must was decanted into a stainless steel tank equipped with a stainless-steel probe (immersion heat exchanger composed of two parallel coils with 11 turns, a diameter of 175 mm, tube ϕ21 mm). The heat source was an electric water heater for central heating (21 kW) with a hot water circulation pump (2.5 m³/h). The pomace was gradually heated from 25 °C to 50 °C over 2.5 h with constant stirring, after which heating ceased and the mass was allowed to cool passively for 24 h. Yeast inoculation was performed after cooling, i.e., one day later compared to the other two treatments. For the enzyme-treated vinification, SIHAZYM Extro (2 g/hL, Eaton Begerrow) was added to the pomace immediately after grape crushing (Figure S1).

The sampling was conducted daily throughout maceration (days 1–5), after the racking (Rack, day 20), and young wine (day 170) from the start of vinification. Sampling and analytical measurements were performed in triplicate to ensure data reliability.

2.2. Spectrophotometric Measurements of Color and Phenolics

Spectrophotometric measurements were performed using SPECORD 200 Plus, Edition 2010 (Analytik Jena AG, Jena, Germany) spectrophotometer.

Sample color parameters: color intensity (CI), hue (H), and chromatic structure (percentage of yellow at 420 nm, percentage of red at 520 nm, and percentage of blue at 620 nm) were measured spectrophotometrically and calculated according to Ribéreau-Gayon et al. [23].

Total phenolics were quantified by the Folin–Ciocalteu method [24], and the results are expressed as milligrams of gallic acid equivalents per liter (mg GAE/L). The total flavonoid content was determined according to Yang et al. [25], and the results are expressed as milligrams of rutin equivalents per liter (mg RE/L). Tannin content was measured by the vanillin-HCl method according to Julkunen-Titto et al. [26] and expressed as milligrams of catechin equivalents per liter (mg CE/L). The anthocyanin content was determined by the assay described by Amerine and Ough [27], and the results are expressed as milligrams of malvidin 3-O-glucoside equivalents per liter (mg M-3-gl/L).

2.3. High-Performance Liquid Chromatography (HPLC) Analysis of Individual Phenolics

Standard solutions of 14 phenolics were prepared in the concentration range of 0.1–50 mg/L in 80% methanol. The standards included gallic acid, protocatechuic acid, p-hydroxybenzoic acid, gentisic acid, caffeic acid, epicatechin, epigallocatechin gallate, t-p-coumaric acid, t-o-coumaric acid, resveratrol, quercetin, cinnamic acid (all purchased from Sigma-Aldrich, St. Louis, MI, USA), and ferulic acid and sinapic acid (obtained from Fluka Analytical, Buchs, Switzerland). Prior to analysis, wine samples were filtered using 0.45-µm pore size PVDF filters to remove any particulate matter that may interfere with the analysis and were diluted using ultra-pure water. Subsequently, 10 µL of each sample was injected into the analytical system for evaluation, in triplicate, to ensure accuracy and reliability.

Individual phenolics were identified and quantified using a Shimadzu Nexera LC-40 HPLC system (Shimadzu, Kyoto, Japan), coupled with a UV/Vis detector and C18 reverse-phase column (250 mm × 4.6 mm × 5 μm) (Phenomenex, Torrance, CA, USA), according to the method described by Boban et al. [28]. Briefly, the mobile phase was composed of ultra-pure water containing 0.2% (v/v) 85% o-phosphoric acid (Sigma-Aldrich) as solvent A and HPLC-grade acetonitrile as solvent B. Analyses were performed at a flow rate of 1 mL/min at 30 °C, at wavelengths of 220 and 320 nm. The total run time was 65 min, following this gradient: initially, 96% A; 10% A from 19 to 27 min; 15% A from 27.5 to 37.5 min; 25% A at 39 min until 43.5 min; 30% A from 44 to 60 min; 4% A from 61 min until the end of the analysis.

Anthocyanins were analyzed by an HPLC system, Perkin Elmer, Series 200 with a UV/VIS detection (Perkin Elmer, Waltham, MA, USA) using a Kinetex core-shell C18 column (150 mm × 4.6 mm × 5 μm, Phenomenex, Torrance, CA, USA) according to the previously reported procedure [14,15]. The individual anthocyanins were identified by their retention times (elution order) at 520 nm and quantified (mg/L) using malvidin 3-O-glucoside as the external standard.

2.4. Solid-Phase Microextraction (SPME) of Volatiles

Volatile compounds were extracted by SPME using a DVB/CAR/PDMS fiber (Supelco, Sigma Aldrich, Bellefonte, PA, USA), according to Sagratini et al. [29], with slight modifications. Briefly, each wine sample (10 mL) was placed in 20 mL vials containing 3 g of NaCl (to enhance the extraction efficiency by promoting the partitioning of analytes into the headspace) and sealed with PTFE–silicone septa. To enhance the release of volatile compounds, samples were equilibrated at 40 °C. After 13 min of stirring at 250 rpm, the SPME fiber was exposed to the headspace for 30 min at 40 °C. It was then removed and inserted into the GC injection port. The extract was immediately transferred to the analytical column, and analyte desorption was performed over 3 min. To prevent cross-contamination, the SPME fibers were conditioned before each run by heating for 10 min at 230 °C in the auxiliary injection port. The total analysis time was 41 min. All analyses were performed in triplicate.

2.5. Gas Chromatography–Mass Spectrometry (GC-MS) Analysis of Volatiles

Wine volatile compounds were detected using a gas chromatograph–mass spectrometry system (GC-MS; Agilent Inc., Santa Clara, CA, USA) consisting of a gas chromatograph (model 8890) and a non-polar HP-5MS column (5% phenyl methylpolysiloxane, 30 m × 0.25 mm × 0.25 μm, Agilent Inc.), coupled with a tandem mass spectrometer (model 7000D GC/TQ). Helium was used as the carrier gas at a flow rate of 1 mL/min in splitless mode. The injection temperature was set to 230 °C for the DVB/CAR/PDMS fiber. The mass spectrometer underwent calibration using perfluorotributylamine at an electron impact ionization energy of 70 eV, utilizing a full MS scan (33–350 m/z). The column temperature program included an equilibration time of 3 min, where the initial temperature was set at 35 °C, increased to 270 °C at a rate of 8 °C/min, and held for 2 min, followed by a final increase to 300 °C at a rate of 15 °C/min and isothermal holding for 5 min. Volatile compound identification was performed by comparing their mass spectra with commercial libraries (Wiley 7 MS library, Wiley, New York, NY, USA, and NIST02, Gaithersburg, MD, USA) and literature. Additional identification was performed by comparing the retention times, and retention indices (Kovats index) were determined using a standard mixture of aliphatic hydrocarbons in the C8-C40 range. The relative amounts of volatiles, expressed as percentages, were calculated by dividing the area of each detected component by the total area of all isolated components. These percentage values were averaged from three replicates for each wine sample to ensure accuracy and reliability. Data processing was performed using Agilent Mass Hunter Qualitative Analysis (v. 10.0) post-run software (Agilent Inc.).

2.6. Sensory Descriptive Analysis of Wines

Sensory analysis was carried out by a panel of eight professional winemakers (aged 32–63 years; mixed gender), all with expertise in Croatian red wine evaluation and previous experience in formal wine tasting, including descriptive analysis. The sensory evaluation was conducted in accordance with the ethical principles for research involving human participants at the Institute for Adriatic Crops and Karst Reclamation (Regulation on Occupational Safety, No. 01-284/3-17). All participants were of legal drinking age and participated voluntarily.

The evaluations were performed in the sensory laboratory at the Institute for Adriatic Crops and Karst Reclamation (Split, Croatia), maintained at an ambient temperature of 20 °C. Wine samples (25 mL) were served in standard wine tasting glasses and coded with random three-digit numbers generated using a randomization table. The serving order of samples was randomized individually for each session to minimize order effects. The identity of the wines was concealed from the panelists throughout the evaluation process. Only the panel supervisor, who did not participate in the sensory evaluation, had access to the sample codes. Samples were served at a temperature between 15 and 17 °C.

Prior to formal evaluation, the panel underwent training for both olfactory and taste attributes. For aroma assessment, panelists familiarized themselves with the wine aroma wheel [30] and reference solutions from the Le Nez du Vin Master kit (54 aromas; Brizard and Co., York, UK). During preliminary aroma evaluation, descriptors relevant to the tested wines were identified, and those recognized by at least half of the panelists were included in the final descriptive list. Panelists were also trained to evaluate key taste and mouthfeel attributes, including body, sourness, sweetness, bitterness, and astringency. Collective tasting and discussion of these standards were used to calibrate scoring and ensure consistent interpretation of the intensity scale. In the second session, the panelists were asked to rate the intensity of the attributes from the descriptive list of wines on a scale from 0 to 9 (0 = not perceived, 9 = extremely intense). In this part of the evaluation, each panelist evaluated two samples of each wine (two repetitions). Both evaluations were performed on the same day, with a break in between to minimize fatigue. The same wine samples were used for individual phenolic and volatile composition analysis. Table S1 provides a detailed list and definitions of the sensory attributes used in the wine evaluation.

2.7. Statistical Analysis

One-way analysis of variance (ANOVA) was carried out to assess the attributes that were significantly different among wines using STATISTICA v. 14.0 (TIBCO Software Inc., Palo Alto, CA, USA). Least significant differences were calculated by Fisher’s LSD test (at 95% confidence level, p < 0.05).

3. Results and Discussion

3.1. Color Parameters Evaluation During Vinification

The evolutions of color intensity (CI), color hue (H), and optical density (OD) during the first five days of maceration, after racking, and for final young wines of three different treatments are presented in Figure 1.

The CI, an indicator of total color (wine darkness), primarily reflects the concentration and composition of extracted anthocyanins [31], but it is highly influenced by the grape variety, applied vinification procedure, maceration duration and temperature, heating duration and temperature, enzyme preparations used, etc. [1,9,10,11,14,15,22]. In conventional and enzyme-treated wines, the CI exponentially increases in the first few days of maceration, reaching a maximum at the fourth day in the conventional sample (5.78) and at the fifth day in the enzyme-treated sample (4.81), respectively. In contrast, CI in thermovinified treatment remained stable during this period, showing a notable decrease between the fourth and fifth days of maceration. Interestingly, the final CI values showed an inverse trend: Both conventional and enzyme-treated wines at the end of the study showed reduced CI results compared to earlier stages, with the lowest value found in conventional wine. Conversely, thermovinified wine was the darkest with the highest final CI value (3.80). These findings are in accordance with previously reported higher CI in wines produced by the addition of pectolytic enzymes but also reflect the variations among effects obtained on different grape varieties and/or using different enzyme preparations [14,15,32,33,34]. Bautista Ortin et al. [1] reported higher CI in Monastrell wine produced by thermovinification, while enzyme-treated samples showed significantly lower values. Similar observations were reported by El Darra et al. [9] on Cabernet Sauvignon, where CI peaked at day 3 and declined sharply thereafter. Additional studies reported higher CI in thermovinified wines of Carignan, Fer, and Grenache [11], and Teran [35].

The results of the H values had the opposite trend. In conventional and enzyme-treated wines, H decreased exponentially during the first three days. Thermovinified wines showed minimal change in H throughout the vinification process (from 0.79 at day 1 to 0.73 in the final wine). In the final wines, the H was the highest in the enzyme-treated (0.88), followed by the conventional wine. Hue values in young wines are usually low between 0.5 and 0.7 and increase throughout wine aging up to 1.2–1.3 [23,34,36]. Previous studies reported contrasting H values depending on variety and enzyme application. Enzyme-treated (Sihazym Extro) Crljenak kaštelanski wine had lower H compared to control, whereas opposite was found for Babica, which implies different effects of the enzymes used in different matrices [14,15]. Lower H values in thermovinified wines from three varieties compared to the control samples were also reported in the study by Geffroy et al. [11], while Lukić et al. [35] observed no significant difference. de Andrade Neves et al. [34] found a significant increase in hue for thermovinified Cabernet Sauvignon and Pinot Noir wines following bottle aging.

Optical density (OD) measurements provide insight into the relative contribution of basic yellow (420 nm), red (520 nm), and blue (620 nm) spectral components to the overall wine color [1,37]. According to Glories [31], the ideal distribution for balanced red wine color is 35% of yellow, 55% of red, and 10% of blue. On the first day, the OD at 420 nm was the highest in the enzyme-treated, followed by the conventional wine. Both treatments showed an exponential decrease in OD 420 during maceration and high final values, 42.3% and 42.7%, respectively. However, the thermovinified wine showed almost the same value on the first day and in the final young wine (~38). All samples showed a high increase in OD 620 at day 3 of maceration, with the highest value detected at day 5, especially for thermovinified wines. In the final young wines, the OD 620 values followed the order: enzyme-treated (10.1) > thermovinified (9.4) > conventionally produced wine (5.9), reflecting the different impact of maceration technique on pigment extraction and retention in the blue spectrum. Bautista Ortin et al. [1] reported the same values for OD 420 in enzyme-treated and control wines (31.1), while OD 620 values were higher in enzyme-treated wines (15.5) of Monastrell, which is in accordance with our findings on the enhancing effect of enzyme treatment on blue pigment extraction.

Among the OD parameters, OD 520 is the most important as it represents the contribution of the red pigments (anthocyanins in the flavylium cationic form) in the overall wine color. This wavelength corresponds to the absorption maximum for red wine chromophores [37]. The highest OD 520 was in thermovinified wine (52.5%), followed by conventional (50.4%) and enzyme-treated wines (47.7%). Lower OD 520 values in enzyme-treated wines were also reported by Bautista Ortin et al. [1] and El Darra et al. [9]. Babincev et al. [37] attributed this to differences in enzyme specificity or matrix interactions during wine aging that affect anthocyanin stability and polymerization. During wine aging, red pigment intensity decreases and yellow H increases. Although the wines in the present study were not aged, the color profiles of the final young wines suggest an early onset of pigment transformation, particularly in conventionally vinified wines.

3.2. Phenolic Compounds Evaluation During Vinification

The contents of total phenolic compounds, as well as main phenolic subgroups (flavonoids, tannins, and anthocyanins), were detected spectrophotometrically through the vinification process (Figure 2). As shown in Figure 2A, thermovinification led to a higher extraction of total phenolic compounds in the first two days of maceration compared to the other two treatments (on day 1, it was more than 5 times higher than in the enzyme-treated sample and more than 4.9 times higher than in the sample from conventional winemaking). While the total phenolics increased in the other two samples by day 2, these differences were lower. On day 3, all samples recorded a sharp decrease in phenolic content. However, at the end of the study, the thermovinified wine had approximately two-fold higher concentration of phenolics compared to the other two wines. As expected, the content of total flavonoids followed this trend (Figure 2B), with consistently higher content in thermovinified wines. On day 1, tannin content was highest in the thermovinified sample. However, a strong increase in tannins was detected on day 2 in the conventional treatment, reaching 793 mg CE/L (more than three-fold higher than in the other two samples) (Figure 2C). Although tannin content decreased after the second day, in all samples, the final content remained the highest in conventional (391 mg CE/L), followed by enzyme-treated (219 mg CE/L) and thermovinified wines (100 mg CE/L).

Similarly, a significant increase in total phenolics, flavonoids, and anthocyanins in thermovinified wines at the beginning of the alcoholic fermentation was reported by El Darra et al. [9], and it correlates with an increase in the CI. Thermovinification, compared to enzyme-treated and control wines, resulted in the highest content of phenolics in País and Lachryma Christi grapes, while preserving stability after 24 h of heating [8]. Their study also confirmed lower phenolic content in enzyme-treated wines compared to the control across all tested temperatures, with optimal extraction obtained at 50 °C. Santos Silva et al. [10] reported elevated total phenolics in thermovinified Syrah wine, and Atanacković et al. [38] in Merlot, Cabernet Sauvignon, Pinot Noir, and Prokupac wines. Moreover, prolonged heating at moderate temperatures (60 °C for 60 min) yielded higher phenolic extraction than shorter, higher temperature treatments (80 °C for 3 min). Bautista Ortin et al. [1] and Borazan and Bozan [7] reported the highest phenolic content in thermovinified wines, followed by control and pectolytic enzyme-treated Okuzgozu and Monastrell wines, which is in accordance with the present study. Similarly, a consistent increase in total phenolics and flavonoids was observed in Cabernet Sauvignon, Merlot, and Cabernet Franc wines produced by different pre-fermentation treatments [39].

Anthocyanin content was the highest in thermovinified samples at the beginning of wine production (Figure 2D), followed by the conventional sample, while at day 3, the content of these pigments increased in the enzyme-treated sample. Similar observations in which anthocyanins peaked during the first three days of maceration in Merlot and Pinot Noir with a dramatic decrease thereafter were also reported by Dimitrovska et al. [40]. In the current study, anthocyanin content continued to increase on the other two days of maceration in conventional and enzyme-treated samples, while it decreased in thermovinified samples. A significant decrease in anthocyanin content was also noted after the fourth day of thermovinification in Cabernet Sauvignon by Koyama et al. [41]. This trend was also followed by the control wine. The parabolic trend of the anthocyanin extraction during vinification can be due to an increase in ethanol at the beginning of fermentation, after which anthocyanin concentrations decrease rapidly due to polymerization, adsorption by yeast cell walls, and losses caused by lees separation. Later stage increases in anthocyanin content are usually connected with mechanical interventions such as punchdowns, pumpovers, and pressing, as well as different chemical reactions that result in their degradation, modification, and/or stabilization [2,34,42].

Table 1. Kinetics of evaluation of anthocyanins (mg/L) during vinification in Babica wines produced by (A) conventional, (B) enzyme, and (C) thermovinification.

(A)
Compound	Day 1	Day 2	Day 3	Day 4	Day 5	Rack
Delphinidin-3-O-glucoside	n.d.	0.17 ± 0.02	2.19 ± 0.04	3.39 ± 0.01	1.81 ± 0.01	3.23 ± 0.10
Cyanidin-3-O-glucoside	n.d.	0.03 ± 0.00	0.15 ± 0.01	0.20 ± 0.00	0.13 ± 0.00	0.09 ± 0.00
Petunidin-3-O-glucoside	n.d.	0.37 ± 0.02	3.81 ± 0.02	6.12 ± 0.01	3.83 ± 0.03	6.27 ± 0.20
Peonidin-3-O-glucoside	n.d.	0.61 ± 0.02	1.88 ± 0.01	2.66 ± 0.01	1.91 ± 0.02	1.62 ± 0.06
Malvidin-3-O-glucoside	n.d.	13.01 ± 0.23 a	48.10 ± 0.02 b	79.94 ± 0.15 c	61.59 ± 0.07 d	72.24 ± 2.05 e
Delphinidin-3-O-acetylglucoside	0.56 ± 0.00	8.71 ± 0.10	5.09 ± 0.12	5.46 ± 0.00	2.99 ± 0.06	1.43 ± 0.00
Cyanidin-3-O-acetylglucoside	n.d.	0.10 ± 0.00	0.26 ± 0.01	0.74 ± 0.02	0.98 ± 0.01	0.51 ± 0.08
Petunidin-3-O-acetylglucoside	n.d.	0.07 ± 0.00	0.32 ± 0.00	0.78 ± 0.00	0.78 ± 0.00	0.57 ± 0.06
Peonidin-3-O-acetylglucoside	0.11 ± 0.00	1.00 ± 0.03	1.10 ± 0.06	0.96 ± 0.40	0.20 ± 0.01	0.39 ± 0.04
Malvidin-3-O-acetylglucoside	n.d.	0.83 ± 0.02	3.41 ± 0.03	5.53 ± 0.19	4.32 ± 0.01	5.33 ± 0.11
Delphinidin-(6-O-coumaryoyl)glucoside	n.d.	0.13 ± 0.00	0.52 ± 0.00	0.94 ± 0.00	0.56 ± 0.00	0.97 ± 0.00
Cyanidin-(6-O-coumaryoyl)glucoside	n.d.	n.d.	0.04 ± 0.01	0.14 ± 0.00	0.04 ± 0.02	0.16 ± 0.00
Petunidin-3-(6-O-coumaroyl)glucoside	n.d.	0.03 ± 0.00	0.37 ± 0.02	0.62 ± 0.04	0.29 ± 0.01	0.82 ± 0.06
Peonidin-3-(6-O-coumaroyl)glucoside	n.d.	0.09 ± 0.02	0.05 ± 0.00	0.18 ± 0.01	0.19 ± 0.00	0.15 ± 0.04
Malvidin-3-(6-O-coumaroyl)glucoside	n.d.	1.23 ± 0.13	6.21 ± 0.05	9.67 ± 0.04	6.28 ± 0.02	11.19 ± 0.00
(B)
Compound	Day 1	Day 2	Day 3	Day 4	Day 5	Rack
Delphinidin-3-O-glucoside	n.d.	n.d.	2.45 ± 0.00	3.17 ± 0.01	2.65 ± 0.01	3.40 ± 0.01
Cyanidin-3-O-glucoside	n.d.	n.d.	0.18 ± 0.00	0.22 ± 0.00	0.16 ± 0.00	0.11 ± 0.00
Petunidin-3-O-glucoside	n.d.	n.d.	4.30 ± 0.03	6.01 ± 0.07	5.30 ± 0.04	6.57 ± 0.02
Peonidin-3-O-glucoside	n.d.	n.d.	2.42 ± 0.01	2.92 ± 0.07	2.33 ± 0.04	2.06 ± 0.01
Malvidin-3-O-glucoside	0.02 ± 0.00 a	0.03 ± 0.00 a	55.63 ± 0.03 b	83.69 ± 0.79 c	77.51 ± 0.23 d	78.95 ± 0.14 e
Delphinidin-3-O-acetylglucoside	0.34 ± 0.01	1.28 ± 0.01	6.18 ± 0.01	4.79 ± 0.03	3.09 ± 0.02	2.00 ± 0.01
Cyanidin-3-O-acetylglucoside	n.d.	n.d.	0.28 ± 0.00	0.55 ± 0.00	0.59 ± 0.00	0.33 ± 0.08
Petunidin-3-O-acetylglucoside	n.d.	n.d.	0.38 ± 0.00	0.67 ± 0.00	0.71 ± 0.00	0.52 ± 0.04
Peonidin-3-O-acetylglucoside	0.07 ± 0.00	0.28 ± 0.02	1.43 ± 0.01	0.93 ± 0.06	0.59 ± 0.00	0.39 ± 0.08
Malvidin-3-O-acetylglucoside	n.d.	n.d.	3.90 ± 0.00	5.85 ± 0.09	5.29 ± 0.01	5.94 ± 0.03
Delphinidin-(6-O-coumaryoyl)glucoside	n.d.	n.d.	0.60 ± 0.00	0.96 ± 0.00	0.84 ± 0.00	0.64 ± 0.52
Cyanidin-(6-O-coumaryoyl)glucoside	n.d.	n.d.	0.05 ± 0.02	0.15 ± 0.01	0.14 ± 0.00	0.09 ± 0.04
Petunidin-3-(6-O-coumaroyl)glucoside	n.d.	n.d.	0.38 ± 0.04	0.64 ± 0.01	0.57 ± 0.05	0.84 ± 0.01
Peonidin-3-(6-O-coumaroyl)glucoside	n.d.	n.d.	0.06 ± 0.00	0.10 ± 0.00	0.15 ± 0.00	0.10 ± 0.00
Malvidin-3-(6-O-coumaroyl)glucoside	n.d.	n.d.	6.95 ± 0.01	11.09 ± 0.03	9.74 ± 0.08	11.61 ± 0.02
(C)
Compound	Day 1	Day 2	Day 3	Day 4	Day 5	Rack
Delphinidin-3-O-glucoside	0.41 ± 0.04	0.64 ± 0.00	3.00 ± 0.02	2.21 ± 0.01	1.14 ± 0.00	2.84 ± 0.01
Cyanidin-3-O-glucoside	0.80 ± 0.01	0.13 ± 0.01	0.28 ± 0.01	0.22 ± 0.00	0.18 ± 0.01	0.18 ± 0.00
Petunidin-3-O-glucoside	1.40 ± 0.03	1.32 ± 0.00	4.81 ± 0.02	3.87 ± 0.02	2.63 ± 0.01	4.64 ± 0.02
Peonidin-3-O-glucoside	4.19 ± 0.08	1.67 ± 0.00	3.17 ± 0.01	2.68 ± 0.01	2.20 ± 0.00	2.46 ± 0.01
Malvidin-3-O-glucoside	41.09 ± 0.67 a	30.09 ± 0.15 b	55.68 ± 0.13 c	51.64 ± 0.07 d	42.61 ± 0.06 e	53.83 ± 0.09 f
Delphinidin-3-O-acetylglucoside	1.72 ± 0.02	5.61 ± 0.03	4.40 ± 0.00	4.10 ± 0.01	3.19 ± 0.07	2.66 ± 0.01
Cyanidin-3-O-acetylglucoside	0.32 ± 0.01	0.32 ± 0.00	0.60 ± 0.01	0.81 ± 0.01	0.45 ± 0.01	1.01 ± 0.00
Petunidin-3-O-acetylglucoside	0.31 ± 0.01	0.30 ± 0.00	0.62 ± 0.00	0.70 ± 0.00	0.39 ± 0.00	0.67 ± 0.00
Peonidin-3-O-acetylglucoside	0.19 ± 0.01	0.60 ± 0.04	0.49 ± 0.00	0.53 ± 0.05	0.25 ± 0.03	0.36 ± 0.12
Malvidin-3-O-acetylglucoside	2.72 ± 0.15	2.04 ± 0.14	3.52 ± 0.01	3.45 ± 0.03	2.65 ± 0.18	3.96 ± 0.03
Delphinidin-(6-O-coumaryoyl)glucoside	0.42 ± 0.00	0.28 ± 0.01	0.64 ± 0.00	0.57 ± 0.01	0.38 ± 0.01	0.68 ± 0.00
Cyanidin-(6-O-coumaryoyl)glucoside	0.04 ± 0.02	0.01 ± 0.00	0.12 ± 0.00	0.10 ± 0.00	0.04 ± 0.02	0.04 ± 0.00
Petunidin-3-(6-O-coumaroyl)glucoside	0.18 ± 0.01	0.09 ± 0.01	0.38 ± 0.00	0.28 ± 0.00	0.15 ± 0.02	0.38 ± 0.01
Peonidin-3-(6-O-coumaroyl)glucoside	0.07 ± 0.00	0.06 ± 0.00	0.11 ± 0.00	0.15 ± 0.01	0.07 ± 0.01	0.21 ± 0.00
Malvidin-3-(6-O-coumaroyl)glucoside	4.04 ± 0.11	2.34 ± 0.02	4.88 ± 0.02	4.22 ± 0.02	2.75 ± 0.03	4.64 ± 0.01

n.d.—not detected. Significant differences (at 95% confidence level, p < 0.05) of malvidin-3-O-glucoside concentration among samples taken during the vinification are marked using different letters (a–f).

presents the concentrations of individual anthocyanins in conventional (A), enzyme-treated (B), and thermovinified (C) wine samples during the vinification process (days 1 to 5 of maceration and at the racking stage). In thermovinified wines, at the first two days of maceration, all individual anthocyanins were detected (Table 1C) in contrast to the other two wines in which more gradual extraction is seen with lower initial concentration of all anthocyanins (Table 1A,B). As expected, malvidin-3-O-glucoside was the dominant form, followed by malvidin-3-O-acetylglucoside, malvidin-3-(6-O-coumaroyl) glucoside, and delphinidin-3-O-acetylglucoside. Significant extraction of malvidin-3-O glucoside in thermovinified wine at day 1 (41.09 mg/L) and day 2 (30.09 mg/L) can be noted. At day 2, the concentrations of delphinidin-3-O-acetylglucoside were also high in conventional (8.71 mg/L) and enzyme-treated samples (1.28 mg/L), respectively. The increase in anthocyanin content, especially of malvidin-3-O-glucoside and its derivatives in samples between days 2 and 3, in accordance with spectrophotometric data presented in Figure 2D. Significant and exponential increase in this compound at day 3 in Merlot and Pinot Noir wines, followed by a decrease in young wines after 6 months of aging, was reported by Dimitrovska et al. [40]. Similar trend on malvidin-3-O-glucoside and its derivatives in the first few days of conventional maceration of Dornfelder and Portugieser grapes was reported by Fischer et al. [43]. Conventional and enzyme-treated samples had the highest content of malvidin-3-O-glucoside at day 4, after which a decline was observed by day 5, and again an increase was observed in samples taken after racking. Dimitrovska et al. [40] also reported the highest amounts of delphinidin-, petunidin-, and peonidin-3-O-glucosides at day 4 for both investigated varieties and a significant decrease in their concentration in final products. Among other compounds in samples after racking, two-fold higher amounts of malvidin-3-(6-O-coumaroyl) glucoside were found in the control and enzyme-treated sample (over 11 mg/L), compared to the thermo-treated sample (4.64 mg/L), as well as higher concentrations of malvidin-3-O-acetylglucoside and peonidin-3-O-glucoside (Figure 2 and

Table 2. Individual phenolic compounds (mg/L) in Babica wines produced by conventional, enzyme, and thermovinification.

Phenolic Class/Component	Conventional	Enzyme	Thermovinification
Phenolic acids
Gallic acid	25.81 ± 0.04 a	31.75 ± 0.08 b	73.28 ± 0.14 c
Protocatehuic acid	9.66 ± 0.02 a	9.53 ± 0.09 a	16.91 ± 0.01 b
p-Hydroxybenzioc acid	54.81 ± 0.17 a	40.92 ± 0.09 b	84.47 ± 0.74 c
Gentisic acid	29.94 ± 0.04 a	18.77 ± 0.04 b	51.44 ± 0.10 c
Caffeic acid	23.03 ± 0.37 a	13.67 ± 0.50 b	14.16 ± 0.25 b
Cinnamic acid	3.25 ± 0.51 a	3.86 ± 0.03 a	1.21 ± 0.04 b
t-p-Coumaric acid	9.65 ± 0.03 a	10.19 ± 0.01 b	5.10 ± 0.11 c
Ferulic acid	0.73 ± 0.00 a	1.24 ± 0.00 b	1.92 ± 0.01 c
Sinapic acid	3.73 ± 0.03 a	1.63 ± 0.02 b	1.37 ± 0.00 c
t-o-Coumaric acid	1.87 ± 0.01 a	5.32 ± 0.07 b	0.38 ± 0.00 c
Stilbene
Resveratrol	3.82 ± 0.00 a	4.49 ± 0.05 b	2.03 ± 0.00 c
Flavonoids
Quercetin	20.20 ± 0.29 a	17.00 ± 0.02 b	20.70 ± 0.17 a
Epicatechin	21.46 ± 0.02 a	24.24 ± 0.11 b	18.74 ± 0.04 c
Epigallocatechin galate	8.47 ± 0.01 a	11.26 ± 0.10 b	19.64 ± 0.00 c
Anthocyanins
Delphinidin-3-O-glucoside	5.81 ± 0.27 a	3.33 ± 0.09 b	2.54 ± 0.16 c
Cyanidin-3-O-glucoside	0.06 ± 0.02 a	0.24 ± 0.04 b	0.13 ± 0.04 c
Petunidin-3-O-glucoside	9.87 ± 0.08 a	6.87 ± 0.07 b	3.35 ± 0.08 c
Peonidin-3-O-glucoside	0.88 ± 0.03 a	0.82 ± 0.02 a	1.47 ± 0.07 b
Malvidin-3-O-glucoside	97.62 ± 0.61 a	70.54 ± 0.92 b	33.09 ± 0.15 c
Delphinidin-3-O-acetylglucoside	0.74 ± 0.18 a	1.51 ± 0.16 b	2.34 ± 0.11 c
Cyanidin-3-O-acetylglucoside	0.12 ± 0.03 a	0.38 ± 0.10 b	1.05 ± 0.08 c
Petunidin-3-O-acetylglucoside	0.44 ± 0.07 a	0.41 ± 0.06 a	0.68 ± 0.09 b
Peonidin-3-O-acetylglucoside	0.30 ± 0.06 a	0.44 ± 0.15 a	0.46 ± 0.08 a
Malvidin-3-O-acetylglucoside	6.77 ± 0.08 a	5.49 ± 0.20 b	2.47 ± 0.00 c
Delphinidin-(6-O-coumaryoyl)glucoside	2.13 ± 0.10 a	1.54 ± 0.03 b	0.84 ± 0.13 c
Cyanidin-(6-O-coumaryoyl)glucoside	0.43 ± 0.05 a	0.38 ± 0.08 a	0.22 ± 0.04 b
Petunidin-3-(6-O-coumaroyl)glucoside	1.58 ± 0.12 a	0.99 ± 0.28 b	0.47 ± 0.06 c
Peonidin-3-(6-O-coumaroyl)glucoside	0.42 ± 0.00 a	0.26 ± 0.06 b	0.31 ± 0.07 b
Malvidin-3-(6-O-coumaroyl)glucoside	16.45 ± 0.06 a	10.22 ± 0.08 b	2.76 ± 0.34 c

Significant differences (at 95% confidence level, p < 0.05) in compound concentrations among samples (n = 3) are marked using different letters (a–c).

).

Among the analyzed wine samples, conventionally produced wine had the highest (175 mg M-3-gl/L), while thermovinified wine had the lowest content of total anthocyanins (117 mg M-3-gl/L) (Figure 2D). Similarly, a significant decrease in total phenolics, flavonoids, and anthocyanins in thermovinified Cabernet Sauvignon and Pinot Noir wines after 6 months of aging was reported by de Andrade Neves et al. [34]. While Pinot Noir had higher extraction of anthocyanins after heat treatment, the final anthocyanin content was significantly reduced after fermentation, likely due to heat-induced degradation and limited stability of these compounds in low tannin matrices [12]. A progressive decrease in anthocyanin content during wine aging was confirmed by Borazan and Bozan [7], who attributed this to complex chemical reactions of oxidative degradation, polymerization, precipitation, or hydrolysis. Lukić et al. [35] reported significantly lower content of anthocyanins in thermovinified Teran wines. On the contrary, higher concentrations of anthocyanins in thermovinified wines were reported for Cabernet Sauvignon, Merlot, and Cabernet Franc by Wang et al. [39], and for Monastrell by Bautista Ortin et al. [1], with the lowest results for enzyme-treated wines. Comparable trends are also seen on Carignan, Grenache, and Fer wines investigated by Geffroy et al. [11].

Geffroy et al. [13] investigated the impact of fermentation variables (maceration temperature and duration, yeast species, and pectolytic enzymes addition) and winemaking techniques, including thermovinification, on red wine phenolics. A significant increase in phenolic and a decrease in anthocyanin content were observed in thermovinified compared to enzyme-treated and control wine. However, the authors pointed out that the analyses were carried out only one week after the end of alcoholic fermentation, so these changes might not hold over a longer period. The anthocyanin content in thermovinified wines is strongly influenced by the applied temperature. Moderate temperatures (55–65 °C) favor anthocyanin extraction, while those above 75 °C promote thermal degradation. Thermovinification of Syrah at 75 °C caused a 58.8% reduction in total anthocyanins compared to thermovinification at 65 °C, which led to a 28.7% decrease [10].

Our results confirm that anthocyanin content continues to decrease during wine stabilization and aging, which is in accordance with Bautista Ortin et al. [1] and Borazan and Bozan [7]. In contrast, total phenolics remained stable during the study period, suggesting that thermovinified wines should not be intended for long-term aging and are suitable for consumption as young wines [1].

3.3. Wine Individual Phenolics

Table 1 presents the individual phenolic compounds (mg/L) in Babica wines produced by conventional, enzyme, and thermovinification, detected by means of HPLC. In total, 10 phenolic acids, 1 stilbene (resveratrol), and 18 flavonoids (including 15 anthocyanins) were identified and quantified.

Thermovinified wine had the highest content of total phenolic acids (250 mg/L), almost two-fold higher than conventional wine (137 mg/L). This is in accordance with previous findings by Koyoma et al. [41] and Borazan and Bozan [7] who reported the significant impact of winemaking technique on the extraction of phenolic acids. Among phenolic acids, hydroxybenzoic acids predominated across all treatments. Thermovinified wines had the highest concentration of gallic acid at ~73 mg/L, p-hydroxybenzoic acid at ~84 mg/L, gentisic acid at ~51 mg/L, and protocatechuic acid at ~17 mg/L. These findings are consistent with Bautista Ortin et al. [1] and Lukić et al. [35], who reported higher content of these compounds in thermovinified wines. However, gallic acid was an exception in enzyme-treated wine, where it was detected in a higher concentration than in conventional Babica wine. Among hydroxycinnamic acids, caffeic acid was the most abundant across all wines (ranging from 13.7 to 23.0 mg/L). Together with sinapic acid, its content was around two-fold higher in conventional compared to other samples. Coumaric acids, both ortho- and para-isomers, were present in the highest concentrations in enzyme-treated wines, while the thermovinified wine had the lowest concentrations of all cinnamic acids except ferulic acid. This is in accordance with the results obtained by Bautista Ortin et al. [1].

Enzyme-treated wine sample also had the highest concentration of stilbene resveratrol (4.5 mg/L), while the thermovinified wine contained a more than two-fold lower concentration. Atanacković et al. [38] investigated the influence of winemaking techniques and grape variety on the resveratrol content in red wine and reported the absence of a correlation between the applied technique and its content, while Santos Silva et al. [10] detected slightly higher concentrations of resveratrol in thermo-treated wines compared to the control wine produced by classic maceration. Orbanić et al. [44] in their study on the application of different vinification techniques in Teran production also reported higher stilbene content in samples obtained by pre-fermentative heating. Enzyme-treated wines had the lowest concentration of flavonol quercetin and the highest amounts of both flavanols, epicatechin and epigallocatechin galate. These findings suggest selective extraction of certain flavonoid subclasses, probably due to enhanced cell wall degradation and improved compound solubilization.

Among the anthocyanins, derivatives of the five main anthocyanidins in the form of 3-O-glucosides, 3-O-acetylglucosides, and (6-O-coumaroyl) glucosides were found in different proportions in dependence on the used maceration technique. Monoglucosides were the most abundant, with a dominance of malvidin-3-O-glucoside with the highest concentration found in conventional (97.6 mg/L), and more than three-fold lower concentration in thermovinified wine.

These results are consistent with previous findings by Borazan and Bozan [7] and Santos Silva et al. [10], who reported the lower content of malvidin-3-O-glucoside in thermovinified wines due to thermal degradation or reduced extraction efficiency at elevated temperatures. In the current study, a similar trend was observed for delphinidin-3-O-glucoside (2.5 to 5.8 mg/L) and petunidin-3-O-glucoside (3.4 to 9.9 mg/L). These results align with data reported by Bautista Ortin et al. [1], who observed reduced concentrations of anthocyanidin monoglucosides in enzyme-treated wines compared to conventional wines, which is consistent with the results of the present study. However, their study indicates that thermovinification led to two-fold higher amounts of these compounds compared to conventional wine, in contrast with our findings, while Lukić et al. [35] detected lower amounts in thermovinified samples compared to the control. Conventional wine had the highest concentration of malvidin-3-O-acetylglucoside (6.8 mg/L), while all four other 3-O-acetylglucosides were present at the highest concentration in thermo-treated wine. The highest concentration of all (6-O-coumaroyl) glucosides was in conventionally produced wines, with the predominance of malvidin-3-(6-O-coumaroyl) glucoside (16.5 mg/L).

3.4. Wine Volatile Components

The volatile composition of Babica wines, analyzed using the SPME-GC/MS technique, is presented in

Table 3. Volatile profiles (%) of Babica wines produced by conventional, enzyme, and thermovinification.

Compound	RI	Conventional	Enzyme	Thermovinification
Alcohols
Isoamyl alcohol	767	41.18 ± 1.79 a	37.90 ± 2.65 a	43.73 ± 1.00 a
2-Methyl-1-butanol	769	7.11 ± 0.77 a	8.23 ± 1.72 a	8.00 ± 0.61 a
(R,R)-Butane-2,3-diol	796	0.89 ± 0.16 a	0.19 ± 0.00 b	0.30 ± 0.29 b
2,3-Butanediol	813	0.42 ± 0.03 a	0.31 ± 0.03 b	0.24 ± 0.02 b
(S)-(+)-3-Methyl-1-pentanol	851	0.20 ± 0.06 a	0.10 ± 0.00 a	0.14 ± 0.06 a
1-Hexanol	871	2.13 ± 0.17 a	1.96 ± 0.03 a	2.49 ± 0.04 b
1-Heptanol	971	0.18 ± 0.01 a	0.29 ± 0.34 a	0.09 ± 0.06 a
2-Ethyl-1-hexanol	1031	0.09 ± 0.02 a	0.04 ± 0.00 b	0.01 ± 0.01 b
2-Phenylethanol	1119	15.12 ± 0.44 a	15.04 ± 0.95 a	15.29 ± 0.16 a
1-Decanol	1275	0.02 ± 0.01 a	0.00 ± 0.00 a	0.01 ± 0.00 a
Esters
Ethyl butanoate	782	0.76 ± 0.04 a	0.46 ± 0.01 b	1.41 ± 0.05 c
Ethyl 2-hydroxypropanoate	824	0.24 ± 0.00 a	2.63 ± 0.05 b	0.28 ± 0.03 a
Ethyl α-methylbutyrate	855	0.59 ± 0.02 a	0.36 ± 0.00 b	1.05 ± 0.06 c
Ethyl isovalerate	858	0.86 ± 0.03 a	0.47 ± 0.01 b	1.34 ± 0.07 c
Isoamyl acetate	879	1.20 ± 0.14 a	3.14 ± 0.06 b	2.52 ± 0.03 c
Ethyl hexanoate	1000	6.24 ± 1.32 a	4.16 ± 1.21 a	3.56 ± 0.17 a
Ethyl 2-hydroxycaproate	1060	0.35 ± 0.04 a	0.26 ± 0.01 a	0.30 ± 0.03 a
Ethyl succinate	1182	8.59 ± 0.14 a	7.43 ± 0.08 b	7.17 ± 0.08 b
Ethyl octanoate	1199	2.92 ± 0.13 a	3.20 ± 0.10 a	1.15 ± 0.12 b
Diethyl malate	1271	0.10 ± 0.03 a	0.02 ± 0.04 a	0.03 ± 0.01 a
Ethyl decanoate	1398	0.35 ± 0.04 a	0.37 ± 0.04 a	0.07 ± 0.02 b
Ethyl 3-methylbutyl succinate	1432	0.61 ± 0.06 a	0.46 ± 0.04 a	0.38 ± 0.07 b
Ethyl phenyllactate	1452	0.07 ± 0.04 a	0.04 ± 0.02 a	0.02 ± 0.00 a
Ethyl dodecanoate	1598	0.25 ± 0.04 a	0.17 ± 0.02 a	0.06 ± 0.00 b
Dodecanoic acid, 1-methylethyl ester	1631	0.01 ± 0.01 a	0.02 ± 0.00 a	0.03 ± 0.00 a
Ethyl tridecanoate	1698	0.03 ± 0.00 a	0.01 ± 0.00 a	0.00 ± 0.00 a
Tetradecanoic acid, ethyl ester	1797	0.14 ± 0.05 a	0.07 ± 0.01 b	0.05 ± 0.01 b
Hexadecanoic acid, ethyl ester	1997	0.71 ± 0.06 a	0.15 ± 0.02 b	0.25 ± 0.03 b
Octadecanoic acid, ethyl ester	2168	0.03 ± 0.00 a	0.02 ± 0.01 a	0.02 ± 0.00 a
Glycoside
Methyl-ß-D-glucopyranoside	981	0.39 ± 0.25 a	0.60 ± 0.53 a	0.25 ± 0.11 a
Monoterpenes
Limonene	1033	0.34 ± 0.08 a	0.28 ± 0.02 a	0.32 ± 0.03 a
Eucalyptol	1037	0.10 ± 0.06 a	0.12 ± 0.05 a	0.06 ± 0.02 a
Linalool	1102	0.18 ± 0.01 a	0.26 ± 0.00 b	0.26 ± 0.03 b
Fatty acid
Octanoic acid	1173	0.35 ± 0.27 a	0.41 ± 0.23 a	0.55 ± 0.11 a
Aldehydes
Nonanal	1106	0.07 ± 0.02 a	0.11 ± 0.08 a	0.13 ± 0.04 a
Decanal	1209	0.18 ± 0.08 a	0.32 ± 0.04 a	0.10 ± 0.04 b
Dodecanal	1413	0.27 ± 0.07 a	0.94 ± 0.09 b	0.24 ± 0.04 a
Total identified compounds (%)		93.26	90.56	91.89

Significant differences (at 95% confidence level, p < 0.05) in compound concentrations among samples (n = 3) are marked using different letters (a–c).

. In total, 37 compounds were identified, belonging to various chemical classes, including alcohols, esters, diols, glycosides, monoterpenes, fatty acids, and aldehydes, providing a comprehensive insight into their composition and potential contribution to aroma and flavor. The percentage of total identified compounds varies among the different maceration techniques: 93.26% in conventional, 90.56% enzyme-treated, and 91.89% in thermovinified wines. Although previous studies [11,45] suggest that heat treatments can cause a significant loss of key grape-derived aroma compounds and an increase in compounds suggesting thermal degradation of volatiles, the differences in the aroma profiles of wines in the current study were not significant to confirm previous conclusions of pronounced degradation of volatile compounds. The highest percentages of identified volatile alcohols, mostly responsible for the herbaceous notes [46], were detected in thermovinified wine, followed by the conventional and enzymatically assisted vinification. The most prevalent alcohol among the identified compounds was isoamyl alcohol, known for its characteristic fruity and banana-like aroma, comprising 43.73% in thermovinified and 37.9% in enzyme-treated wines. These findings align with those of Geffroy et al. [11], who reported elevated isoamyl alcohol content in thermovinified wines from Carignan, Grenache, and Fer. Similar percentages of phenylethyl alcohol, with its distinctive floral rose aroma contributing sweet and slightly fragrant notes [47], have been detected in enzyme-treated (15.04%) and thermovinified (15.29%) wines. Additionally, active amyl alcohol (2-methyl-1-butanol), a compound with mild alcoholic and chemical aroma, was detected in amounts ranging from 7.11% in the conventional to 8.23% in enzyme-treated wines. These results can be partly explained by the fact that in the thermovinification, a higher percentage of alcohol is likely to develop due to more intense extraction from the grape skins, accelerated fatty acid oxidation, and enhanced enzyme activity at higher temperatures [35].

The volatile composition of wines is more influenced by the heating temperature than by its duration. Surprisingly, an increase in temperature did not uniformly enhance fermentation-derived volatiles. Several aromatic compounds, especially grape-derived molecules, were affected by the increase in temperature, likely through mechanisms of enzyme degradation and denaturation or as a consequence of modification of fermentation conditions. Wines processed at 50 °C may show increased concentrations of grape-derived volatiles. This indicates that heating temperature can be modulated to enhance aroma complexity in thermovinified wines. Looking from an olfactory point of view, wines obtained in this way are recognized by their sensory profile, the so-called “banana-yogurt”, largely driven by fatty acids and acetates [11,45].

The results of the current research showed differences in the amount of identified volatile esters depending on the applied maceration technique. Volatile esters play a crucial role in wine aroma by imparting desirable fruit, floral, and confectionery characters [48]. The highest share of volatile esters was found in samples obtained using the conventional method (24.12%), followed by the enzyme-treated (23.5%) and thermovinified wines (19.61%). These findings underscore the influence of fermentation temperature on the presence of volatiles in wines. Ethyl esters, particularly ethyl succinate, hexanoate, and octanoate, were the most prevalent within that group. This is in accordance with Beltran et al. [49] and Molina et al. [50], who noted that ester production can be influenced by yeast detoxification stress response, particularly in fermentations with fewer suspended solids. Gene regulation and membrane fatty acid composition also impact ethyl ester formation at lower fermentation temperatures, while higher temperatures may induce ester evaporation, contributing to their overall decline in thermovinified wines. In the enzyme-treated wine, isoamyl alcohol stands out as a compound contributing to the fruity aroma of wine (3.14%), the lowest amount of which was detected in conventional wine (1.20%). Monoterpenes, grape-derived secondary metabolites, are key factors in the typicality of the variety and largely contribute to its unique aroma [51]. In all samples, limonene was the most abundant, from 0.28% in the enzyme-treated to 0.34% in the conventional wine. These differences in volatile profiles among samples highlight the significant impact of maceration technique on it and provide valuable information for further research and optimization of the vinification process, with the aim of achieving the desired wine characteristics. Further research should focus on the stability and sensory perception of these volatiles during wine aging.

3.5. Wine Sensory Descriptive Analysis

Descriptive sensory analysis was used to describe the sensory attributes of cv. Babica wines produced with three different maceration techniques (conventional, enzyme-treated with a β-glucanases (β1-3, β1-6), and short-term vinification of the pomace (Figure 3). A trained panel selected ten key sensory aroma descriptors (blueberry, raspberry, black currant, cherry, raisin, dried prune, dried fig, hay, tobacco, and smoky) describing the aroma of the produced wines (Table S1). The olfactory attributes (body, sourness, sweetness, bitterness, and astringency) were also evaluated. The sensory profile of the wines studied was characterized by aromas of dark berry fruits (blueberry, blackcurrant), red berry fruits (raspberry, cherry), dried fruits (raisins, dried prune, dried fig), and dried herbal (hay and tobacco), as well as woody, burnt aromas (smoky). Sensory attribute intensity scores were quite low, ranging from 1.9 for tobacco (thermovinification) to 4.9 for blueberry (thermovinification).

Babica wine produced by thermovinification had a significantly higher intensity of blueberry aroma (score 4.9) compared to wines produced by conventional (score 3.9) and enzymatic treatment (score 2.8). Higher perception of fruity aroma in thermovinified wines could be attributed to increased extraction of aroma precursors and the transformation of glycosidically bond compounds under higher temperature [52]. The intensity of astringency was also significantly higher with thermovinification (score 4.4) compared to conventional (score 2.9) and enzyme-treated wines (score 3.5). Previous studies have shown that the extraction of phenolic content increases with the temperature of thermovinification [10]. Treatment of the wine with the enzyme increased the intensity of the hay and decreased the intensity of the sour taste compared to the other two wine samples. No significant differences were observed in other sensory attributes among the three wines studied.

Considering overall sensory balance, thermovinification appears beneficial for enhancing fruity aromas characteristic of high-quality Babica wines, but its accompanying rise in astringency should be carefully managed. Enzyme treatment may be advantageous for softening acidity, though its impact on aroma profile warrants consideration to avoid diminishing fruit-forward character.

4. Conclusions

Recent trends in winemaking have significantly influenced the impact of consumer preferences, which are partially focused on the moderate consumption of red wines with a good structure, taste, and roundness. According to the kinetic data, the critical extraction of the anthocyanins and flavonoids in Babica wine occurs within the first few days of maceration, highlighting the importance of pre-fermentative and early fermentative handling of pomace. Thermovinification yielded the highest color intensity and increased the extraction of total phenols and flavonoids. Despite initially higher anthocyanin levels during early maceration, final anthocyanin and tannin concentrations were lowest in thermovinified wine, probably due to thermal degradation and limited polymerization. Thermovinified wine had the highest content of volatile alcohols, among which isoamyl alcohol was the most abundant and contributed to a stronger fruity aroma, as confirmed by sensory analysis. In enzyme-treated wine, hay aroma notes predominate, and on the palate, the sourness was reduced, while conventional wine had the best-preserved anthocyanin and tannin contents through maceration and a more balanced phenolic profile later in wine, indicating their better suitability for aging. Those also had the highest concentrations of esters, which contribute to fruity and floral sensory expression. The obtained results can serve as guidelines for wine producers in selecting the optimal winemaking technique depending on the desired wine style—whether for early consumption with intense color and pronounced dark fruit aroma notes (thermovinification) or for wines with aging potential and a more complex sensory profile (conventional vinification). Further research should focus on investigating the long-term stability of wines produced using different maceration techniques, particularly in relation to anthocyanin and tannin degradation during aging.