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Article

The Effects of Pre-Fermentative Treatments on the Aroma of Krstač and Žižak Wines

by
Valerija Madžgalj
1,*,
Iris Đorđević
2,
Ivana Sofrenić
3 and
Aleksandar Petrović
4
1
Faculty for Food Technology, Food Safety and Ecology, University of Donja Gorica, Oktoih 1, 81000 Podgorica, Montenegro
2
Faculty of Veterinary Medicine, University of Belgrade, Radova 43, 11000 Belgrade, Serbia
3
Faculty of Chemistry, University of Belgrade, Studentski trg 12-16, 11000 Belgrade, Serbia
4
Faculty of Agriculture, Institute of Food Technology and Biochemistry, University of Belgrade, Nemanjina 6, 11080 Belgrade, Serbia
*
Author to whom correspondence should be addressed.
Fermentation 2025, 11(10), 577; https://doi.org/10.3390/fermentation11100577
Submission received: 20 August 2025 / Revised: 21 September 2025 / Accepted: 4 October 2025 / Published: 7 October 2025
(This article belongs to the Special Issue Wine and Beer Fermentation, 2nd Edition)
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Abstract

Pre-fermentative treatments are essential in winemaking, as they significantly influence the quality and stability of white wines in particular. The synthesis of many compounds obtained from yeast, such as higher alcohols and esters, is influenced by the type and concentration of aromatic precursors present in the must, especially amino acids. Clarification has a positive effect on wine quality, mainly by improving organoleptic properties, with flavour being the most affected. In this study, the influences of different static settling times, different pressures during must extraction and the addition of different bentonite concentrations to the must on the aroma of wines from the autochthonous grape varieties Krstač and Žižak were investigated. The identification of aromatic compounds in the wine was performed using GC/FID-MS analysis. Wine subjected to the longest static settling time (30 h) showed the highest concentration of esters. Krstač wine, which underwent a 30 h of settling, was characterised by an increased concentration of esters, such as isoamyl acetate, ethyl decanoate and ethyl hexanoate, while Žižak wine was characterised by the presence of 2-phenylethyl acetate and isoamyl acetate. The total fatty acid content in Krstač wine obtained by pressing was higher (14.90 mg/L) than in wine produced from free-run juice (8.04 mg/L).

Graphical Abstract

1. Introduction

Aroma is a key component of white wine quality and plays an important role in sensory characteristics [1,2]. Early processes in white wine production are of particular importance, as they influence the extraction of aromatic compounds and their precursors from the grape into the must, which in turn affects the concentration of these compounds in the final wine [3,4]. Pre-fermentative processes in white wine production include skin maceration, the application of pressure when pressing the grapes, static settling of the must, the addition of bentonite and other treatments [5,6,7,8,9].
Solids in white grape juice can be removed by three main methods: static settling, filtration and flotation [10]. The clarification process removes excess proteins, oxidisable polyphenols and suspended solids that contribute to the turbidity of the must [8]. Solid particles are known to be a source of nutrients that are important for fermentation. These include compounds such as lipids, phospholipids, sterols, glycerolipids, polysaccharides and other molecules [5,10]. Due to their pronounced hydrophobic properties, the lipids in grape must are predominantly associated with solid particles [10]. The sedimentation rate of particles in the juice depends on the particle size and density, the viscosity of the juice and the colloidal content of the juice [11]. The physicochemical properties of these particles, such as size, composition and nature, were influenced by the grape variety and juice extraction [8].
The simplest and most natural form of clarification is static settling of the grape must at low temperatures. This is often used in oenological practice to remove solid particles and improve the overall quality of the wine [5,11,12]. Clarification is performed because solid particles have polyphenol oxidase activity, which can lead to browning of the juice, and esterase activity, which reduces the accumulation of esters synthesised by the yeast [13].
Apart from the static settling time, the grape variety used for winemaking has a significant influence on the amino acid content in the must, which in turn affects the aroma profile of the wine. Amino acids are the primary source of nitrogen for yeast metabolism and serve as precursors for the synthesis of aroma-active compounds [5]. Therefore, changes in the amino acid composition of the must can significantly influence the sensory characteristics of the resulting wine [5]. The balance between solids content and nitrogen availability is a critical parameter for regulating the fermentation process [10].
In addition, clarification of the must leads to the removal of suspended solids and colloidal particles, which can lead to instability of the wine or potential health problems [8]. High turbidity in musts is often associated with the emergence of herbaceous (vegetal) aroma compounds, as well as a tendency for the wine to develop darker hues (browning) in the finished wine [12,14]. However, excessive clarification of the must can suppress alcoholic fermentation and reduce the concentration of primary aroma compounds. In juices with low turbidity, yeast generally produces higher amounts of extracellular and cell wall polysaccharides during alcoholic fermentation. These compounds influence the wine’s taste and perceived sweetness, though their impact on nutritional value remains minor [15]. Therefore, determining the optimal settling time of the grape must is essential for achieving a desirable aroma profile in the finished wine [8,15,16].
The aromatic compounds in wine can be divided into two groups: volatile and non-volatile compounds. Volatile compounds, which are present in their free form, contribute directly to the olfactory properties of the wine. In contrast, non-volatile compounds are bound precursors that can be enzymatically or chemically converted into aroma compounds during fermentation [1,9,17]. The settling duration of the must influences the extraction of these aroma precursors, which are later converted into important aroma compounds such as esters, higher alcohols and other volatile substances during alcoholic fermentation.
The presence of intense aromatic compounds and the stability of proteins are two essential prerequisites for white wines [18]. The addition of bentonite to grape must promotes the sedimentation of suspended solid particles and clarification [19,20]. To avoid the undesirable effects of fining agents, the selection of the agent, the timing of its addition and the dosage are of critical importance [12]. The removal of oxidisable phenols is important to preserve the wine’s aroma and prevent oxidation and browning [6]. Some authors suggested that the addition of bentonite to the must leads to a reduction in pre-fermentative and varietal aromas [21]. The impact of pressure during pressing has not yet been sufficiently researched. In general, wines made from free-run juice exhibit greater vibrancy and freshness in their aromas [21].
The aim of this study was to investigate the effects of different static settling times, pressing pressures and the addition of different concentrations of bentonite to the must on the aroma profile of wines. For the first time, the influences of these pre-fermentative treatments on the aromatic profile of wines from the Montenegrin autochthonous varieties Krstač and Žižak were evaluated.

2. Materials and Methods

2.1. Chemicals

The following chemicals were used in this study: methyl alcohol, anhydrous sodium sulfate, methylene chloride and 4-methyl-1-pentanol. All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA), with the exception of methylene chloride, which was purchased from Merck (Darmstadt, Germany). Analytical grade solvents (methylene chloride and methanol) were used, which were additionally purified by distillation and then dried with anhydrous calcium chloride or sodium sulphate [22].

2.2. Winemaking

Experiment 1: Krstač (K) and Žižak (Z) are autochthonous grape varieties grown in a vineyard in Ćemovsko polje, Montenegro. The grapes were harvested by hand when fully ripe and under good phytosanitary state, as determined by visual inspection. After harvesting, the grapes were destemmed, crushed and treated with potassium metabisulphite (K2S2O5) at a dosage of 10 g per 100 kg of crushed grapes. Vinification of the white wine involved crushing the grapes and separating the stems with a destemmer (capacity: 3–5 t/ha; Nuova Enopieve, Italy), followed by soft pressing with a 300 L hydraulic press (Nuova Enopieve, Italy). The resulting must was clarified by static settling at 5 °C for 0, 10, 20 and 30 h (Table 1) in 15 L glass containers. After settling, the clarified juice was racked off the sediment.
All musts were inoculated with the yeast Saccharomyces cerevisiae (ICVD47, Lallemand, Montreal, QC, Canada) at a concentration of 20 g/hL. Each treatment was carried out in triplicate. For each treatment, vinification was carried out in three glass flask (15 L) with an identical sample volume. The balloons were closed with hydraulic crow-bars. All vinifications were carried out at 15 ± 1 °C. After alcoholic fermentation, the first racking was carried out after one month, and the free SO2 content was adjusted to 30 mg/L by adding 3 g K2S2O5/hL. A second racking was carried out after one and a half months with the same SO2 setting. No nutrients (e.g., ammonium or amino acids), bentonite, pH adjustment, or malolactic fermentation were applied in this experiment.
Experiment 2: The grapes of both varieties were processed with a destemmer (capacity: 3–5 t/ha; Nuaova Enopieve, Italy) and sulphited with 10 g K2S2O5 per 100 kg of crushed grapes. The juice was fractionated into two components using a pneumatic press (model PST 5, Škrlj, Slovenia): free-run juice (0 bar) and pressed juice (approximately 1.8 bar) (Table 1). Both juice fractions were settled by gravity at 5 °C for 48 h. After clarification, the juice was racked and inoculated with S. cerevisiae (ICV D47, Lallemand, Canada). Each treatment (Krstač and Žižak) was carried out in triplicate. For each treatment, vinification was carried out in three glass flasks (15 L) with an identical sample volume. The balloons were closed with hydraulic crow-bars. All vinifications were carried out at 15 ± 1 °C. Post-fermentation wine care was carried out in the same way as in experiment 1.
Experiment 3: After observing the ripening dynamics of the grapes, the Krstač and Žižak grapes were harvested by hand. During white vinification, the grapes were destemmed, crushed, sulphited (10 g K2S2O5/100 kg) pressed with a hydraulic press. The must was allowed to settle statically for 48 h at 5 °C, after which the clarified juice was racked. Subsequently, the must was divided into 15 L glass flasks to which increasing concentrations of bentonite were added (Table 1). Each treatment (Krstač and Žižak) was carried out in triplicate. Fermentation was initiated with 20 g/hL of S. cerevisiae (ICV D 47) at 15 °C for both varieties. Subsequent treatment of the wines was identical to the previous trials.

2.3. Liquid–Liquid Extraction

Samples were prepared according to the liquid–liquid extraction method described by Avram et al. [23]. Briefly, 25 mL of wine and 5 mL of methylene chloride were added to an Erlenmeyer flask. The mixture was stirred magnetically for 1 h at 0 °C in an ice bath to facilitate extraction. The resulting mixture was then placed in an ultrasonic bath for 5 min to “break” the emulsion formed during the extraction. The organic phase was then separated, dried with anhydrous sodium sulphate and then filtered. Subsequently, 0.6 mL of the extracted wine was analysed using the GC/FID-MS method [22].

2.4. GC/FID-MS Analysis

The volatile compounds were analysed using the GC/FID-MS system, based on a previously published method with minor modifications [24]. The analysis was performed using the Agilent 7890A gas chromatograph (GC) (Santa Clara, CA, USA) equipped with an Agilent 19091N-113 HP-INNOWax fused silica capillary column (30 m × 0.32 mm i.d., 0.25 μm film thickness). Sample injections were carried out in split mode (3:1) using helium as the carrier gas with a flow rate of 1.46 mL/min and an injection volume of 1 μL. The temperature of the GC oven was first held at 40 °C for 5 min, then programmed to 220 °C at 10 °C min−1 and held at this final temperature for 4 min.
The device was equipped with dual detectors: an inert mass selective detector (MSD) 5975C XL EI/CI MSD and a flame ionisation detector (FID), which was connected to the make-up gas via a 2-way capillary splitter. The ion source of the MSD and the temperature of the transfer line of the MSD were kept at 230 °C and 280 °C, respectively. The MSD operated in positive ion electron impact (EI) mode. Electron impact spectra were collected in scan mode at 70 eV over a mass range from 35 to 500 m/z. The FID detector was operated at a temperature of 300 °C [25]. Quantitative analysis was performed using the internal standard (IS) with a known amount of 4-methyl-1-pentanol. The (relative) proportions of each volatile compound were calculated from the peak areas in the gas chromatograms. The concentration of each volatile compound was determined from the peak area of the internal standard and expressed as the relative concentration of each component in the analysed sample. The compounds were identified by comparison with reference spectra (Wiley and NIST databases) [9].

2.5. Statistical Analysis

One-way analysis of variance (ANOVA) was used to compare the effects of different static settling times, pressing pressures and bentonite concentrations on the content of individual aromatic compounds. Tukey’s post hoc test with a significance level of p < 0.05 was performed to compare the mean values. One-way ANOVA and Tukey’s post hoc test were performed with the R package ‘agricolae v1.3-7’ [26].
Principal component analysis (PCA) was performed to analyse the differences between the wine samples based on the concentration of their volatile components. PCA was performed using the function ‘prcomp’ from the R package ‘stats’ [27]. We used supervised Sparse Partial Least-Squares Discriminant Analysis (sPLS-DA) to identify the key variables that discriminate between the wine groups. Each group consisted of three wines belonging to the same treatment. sPLS-DA was performed using the ‘splsda’ function from the MixOmics package v6:30.0 [28].

2.6. Sensory Analysis

The sensory evaluation of the wine samples was carried out according to the Buxbaum method [29]. The judging panel consisted of three experienced evaluators, aged between 35 and 45, who are members of the State Commission for Wine Evaluation. Each evaluator tasted each treatment in triplicate, and the overall score for each wine characteristic was averaged. The wine samples were evaluated according to colour, clarity, odour and taste. Each attribute was assessed individually, with the highest possible total score being 20 points [9].

3. Results and Discussion

3.1. Effect of Pre-Fermentative Static Settling on the Aroma of Wine Produced from Krstač and Žižak Variety

Higher alcohols, also known as fusel oils, are aliphatic or aromatic compounds containing more than two carbon atoms [30]. There are two primary pathways for alcoholic fermentation: the anabolic (biosynthetic) pathway, which is derived from glucose, and the Ehrlich pathway, which involves the catabolism of amino acids [9,31]. In this study, the highest concentrations were found for isoamyl alcohol, 2-phenylethyl alcohol and isobutyl alcohol (Supplementary Material Tables S1 and S2). While higher alcohols in elevated concentrations generally contribute negatively to the flavour, 2-phenylethyl alcohol is an exception as it produces a pleasant, rose-like odour and is synthesised from the amino acid phenylalanine [32]. The ANOVA test revealed no statistically significant differences in total higher alcohol content between the different treatments of Krstač wines. In contrast, a statistically significant difference was found between the control (Z0) and the Z10 and Z30 treatments in the Žižak wines (Supplementary Material Tables S1 and S2).
Among the fatty acids, octanoic acid had the highest concentration in the Krstač wines (between 4.16 and 11.41 mg/L), while its content was significantly lower in the Žižak wines (between 3.50 and 6.27 mg/L) (Supplementary Material Tables S1 and S2). This indicates that the total fatty acid content is influenced by the grape variety used. A statistically significant difference in total acidity was found between K0, K10, K20 and K30 Krstač wine, while in Žižak wine, the control was significantly different from all wines produced with different static settling times (Supplementary Material Tables S1 and S2). The synthesis of medium-chain fatty acids and their corresponding esters was influenced by the content of assimilable nitrogen [30]. They are formed during alcoholic fermentation and contribute to undesirable flavours, such as fatty, rancid and cheesy odours [30]. Ethyl esters of fatty acids (ethyl hexanoate, ethyl decanoate, ethyl octanoate), on the other hand, contribute to the desirable aroma of wines [30].
Esters are compounds that typically have pleasant flavours. Their concentrations were generally well above the sensory thresholds [31,33]. In our study, the following compounds contributed to a pleasant, fruity and floral flavour: ethyl butyrate (apple, banana), ethyl hexanoate (ripe banana, green apple), ethyl lactate (milky), ethyl octanoate (fruity, pear), isoamyl acetate (banana) and 2-phenylethyl acetate (fruity, rose-like) [3,9]. The concentration of esters ranged from 6.88 to 11.42 mg/L in Krstač wines and from 5.34 to 8.37 mg/L in Žižak wines. The highest ester concentration was observed in wine samples subjected to prolonged static settling (K30, Z30), which is consistent with the results of Burin et al. [34]. Wines produced with an extended static settling time (36 h) had a higher ester content than those subjected to a static settling time of 24 h [35]. A statistically significant difference in total ester content was found between the control and K20 and K30 Krstač wines and between the control and K10 and K30 Žižak wines.

PCA and Multivariate sPLS-DA

The results of the principal component analysis (PCA) for the Krstač wines showed that the first component (PCA 1) explained 63.3% of the variability, while the second component (PCA 2) accounted for 26.66%, with a cumulative total of 89.96% of the variance (Figure 1). PCA 1 separated the Krstač K30 wines from the K0, K10 and K20 wines. PCA 2 further separated the K0 and K30 wines from the K10 and K20 wines.
Component PCA 1 distinguished 8 aromatic compounds (3-(methylthio)-1-propanol (3M1P), 2-phenylethyl alcohol (2Pa), ethyl butyrate (Eb), diethyl succinate (Ds), ethyl hydrogen succinate (Ehs), ethyl lactate (El), 9-decenoic acid (9Da) and γ-butyrolactone (Bl)) from the remaining 17 aromatic compounds. PCA 2 contrasted 8 compounds (1-hexanol (1H), isobutyl alcohol (Iba), 3-ethoxy-1-propanol (3E1P), ethyl butyrate (Eb), diethyl succinate (Ds), 1,3-propanediol diacetate (1.3Pd), hexanoic acid (Ha) and isobutyric acid (Ia)), which correlated positively with PCA 2, while the remaining 17 compounds showed a negative correlation (Figure 1). The abbreviations of all compounds can be found in Supplementary Material Table S3.
To identify the aromatic compounds most responsible for the differences between the different pre-fermentation treatments, sPLS-DA was performed (Figure 2). In Figure 2A, samples K0, K10, K20 and K30 are clearly separated along components 1 and 2. The sPLS-DA identified 16 compounds that significantly contribute to the differentiation of wines subjected to different treatments (Figure 2B,C).
Figure 2B shows that the Krstač wines subjected to 30 h of static settling of the must (K30) were characterised by volatile compounds such as higher alcohols: 3-ethoxy-1-propanol (3E1P), isobutyl alcohol (Iba), esters: 1,3-propanediol diacetate (1.3Pd), isoamyl acetate (Iac), ethyl decanoate (Ed), ethyl hexanoate (Eh) and fatty acid: isobutyric acid (Ia), all of which correlate positively with component 1. Burin et al. [34] reported that the wines obtained by 30 h of sedimentation showed a correlation with the ethyl esters of the fatty acids.
The settling time of the must is an important factor that influences the concentration of aroma precursors, thus playing a key role in the development of wine aroma [34]. Kechagia et al. [36] reported higher concentrations of isoamyl acetate in wines where longer static settling times were applied, while other studies showed opposite results [34]. It was also found that wines produced from must with lower nitrogen content contained a higher concentration of fatty acid ethyl esters [34].
The Krstač control wines (K0) were characterised by ethyl lactate (El), 9-decenoic acid (9Da) and γ-butyrolactone (Bl), which correlate negatively with component 2. The wines K30 and K0 had the highest concentrations of the aforementioned compounds compared to those subjected to other static settling times. The concentrations of compounds most responsible for these differences are presented in Supplementary Material Table S4. Figure 2D shows a cluster image map (CIM) of 16 aromatic compounds in Krstač wines, selected by sPLS-DA.
The PCA results for Žižak wines showed that PCA 1 explained 35.85% of the variability and PCA 2 explained 31.67%, totalling 67.52% of the variance (Figure 3). PCA 1 distinguished the Z10 wines from the remaining Žižak wines (Z0, Z20 and Z30), while the PCA 2 component separated the Z0, Z20c, Z10c and Z10b wines from the remaining samples (Z10a, Z20a, Z20b and Z30).
The PCA 1 component compared 11 compounds (1-hexanol (1H), 2-phenylethyl ethanol (2Pa), isobutyl alcohol (Iba), 2,3-butanediol (2.3B), 3-(methylthio)-1-propanol (3M1P), isoamyl alcohol (Iaa), ethyl octanoate (Eo), diethyl hydroxybutanedioate (Dhb), ethyl decanoate (Ed), ethyl 4-hydroxybutanoate (E4h) and decanoic acid (Da)). PCA 2 contrasted 4 volatile compounds, namely 1-hexanol (1H), 1,3-propanediol diacetate (1.3Pd), ethyl butyrate (Eb) and ethyl octanoate (Eo), which were positively correlated with PCA 2, while the remaining 20 compounds showed a negative correlation.
The application of sPLS-DA (Figure 4A) clearly separated the Žižak wines by treatment. The first component distinguished Z10 wines from Z0, Z20 and Z30, while the second component separated Z0 from Z10, Z20 and Z30. The analysis identified 16 compounds that contributed most to the differences between the wines.
As shown in Figure 4B, the Z30 wines were characterised by esters (ethyl hydrogen succinate (Ehs), diethyl succinate (Ds), 2-phenylethyl acetate (2Pac), isoamyl acetate (Iac)) and isobutyric acid (Ia)), which were positively correlated with component 1, while octanoic acid (Oa), 9-decenoic acid (9Da) and hexanoic acid (Ha) were positively correlated with component 2. It has been shown that the settling of must stimulates the formation of medium-chain fatty acids [35]. The formation of fatty acids increased significantly with a longer static settling time of the must, which subsequently led to an increased synthesis of fatty acid esters in the wine. The wines from the Z10 treatment were characterised by compounds 1-hexanol (1H), ethyl octanoate (Eo) and diethyl hydroxybutanoate (Dhb), which were positively correlated with component 1, while 2,3-butanediol (2.3B), isoamyl alcohol (Iaa), ethyl decanoate (Ed) and decanoic acid (Da) were positively correlated with component 2.
A significant presence of fatty acid precursors was generally associated with a higher concentration of 1-hexanol [37,38]. However, a significant loss of C6 compound precursors typically occurs in the early stages of must settling [34,35], which explains the minimal differences in 1-hexanol concentrations between the static settling treatments of 20 and 30 h (Supplementary Material Table S2).
The influence of static settling time on the concentration of higher alcohols, including 1-hexanol, was highly dependent on the content of insoluble solids in the must. According to the literature, musts with a higher solids content tend to have a higher concentration of isoamyl alcohol [39], although other studies have reported contrasting results [34]. The concentrations of the compounds that contributed most to the differences between the Žižak wines with different static settling times are listed in Supplementary Material Table S5. The clustered image map (CIM) of 16 aromatic compounds in Žižak wines selected by sPLS-DA is presented in Figure 4D.

3.2. Effect of Pressing Pressure on the Aroma of Wine Produced from Krstač and Žižak Varieties

According to the GC/FID-MS analysis, the concentrations of total aromatic compounds detected in Kfr and Zfr were 224.31 mg/L and 211.12 mg/L, while in Kp and Zp, they were 146.53 mg/L and 199.59 mg/L, respectively (Supplementary Material Table S6). The total flavour content was higher in wines produced by free-run juice fermentation (Kfr, Zfr) than in wines produced by pressurised pressing of grape must (Kp, Zp). Based on Tukey’s post hoc test, a statistically significant difference in total aromatic compound content was found between Kfr and Kp wines (Supplementary Material Table S6). An important parameter that determines must composition for winemaking is the amount of pressure exerted during pressing [3]. According to the literature, pressing reduces the quality of the must intended for winemaking, leading to an increase in total phenolic compounds, browning and an increased content of C6 compounds [40].
The total alcohol concentration was higher in the wines from Krstač and Žižak, which were produced by fermentation of free-run juice. In Krstač wines, the content of 2-phenylethyl alcohol was 4.1 times higher and in Žižak wines 1.6 times higher than in Kp and Zp wines. 2-Phenylethyl alcohol is a derivative of the phenylpropanoid metabolism in grapes but is mainly synthesised by yeasts during fermentation from various grape-derived precursors [40,41].
When pressing (p~1.8 bar) was applied, the concentrations of 1-hexanol were higher in both Krstač and Žižak wines than in Kfr and Zfr (Supplementary Material Table S6), which is consistent with the results of Selli et al. [40] regarding C6 alcohols in Emir juice. C6 compounds are formed during the pre-fermentative stages, such as harvesting, pressing and maceration [42,43]. In Kp wines, where a pressure of about 1.8 bar was applied, the content of 1-hexanol was 5.5 times higher and in Zp wines 7 times higher than in Kfr and Zfr wines. These results are consistent with the studies by Selli et al. [40]. Parish-Virtue et al. [7] reported a notable rise in hexanol content, which was consistently observed during the entire pressing process.
In Krstač and Žižak wines, fatty acids are the most important compounds after alcohols and esters. In the wines from Krstač and Žižak, the most abundant fatty acids were octanoic, hexanoic and decanoic acids, while isobutyric and 9-decenoic acids were present in lower concentrations (Supplementary Material Table S6). The total fatty acid content of Krstač wine obtained by pressing was higher (14.90 mg/L) than that of wine from free-run juice (8.04 mg/L). Using Tukey’s post hoc test, statistically significant differences in total fatty acid content were found between the Kfr and Kp wines and between the Zfr and Zp wines (Supplementary Material Table S6).
The volatile esters are particularly important in white wines, as they contribute significantly to the floral and fruity characteristics. Esters are primarily present as acetate and ethyl esters, produced through yeast metabolism via fatty acyl-CoA and acetyl-CoA pathways. Acetate esters result from condensation reactions between acetyl-CoA and higher alcohols generated during yeast amino acid metabolism, whereas ethyl esters are formed by the esterification of ethanol with acyl-CoA intermediates, a reaction catalysed by esterases and transferases [44,45].
The total ester content was higher in wines produced from pressed must (Kp, Zp) and was 16.57 mg/L and 12.84 mg/L, respectively, compared to 8.81 mg/L and 8.69 mg/L in wines produced from free-run juice (Kfr, Zfr). Using Tukey’s post hoc test, a statistically significant difference in total ester content was found between Kfr and Kp wines as well as Zfr and Zp wines (Supplementary Material Table S6).

PCA and Multivariate sPLS-DA

The results of the PCA for Krstač and Žižak wines show that the first principal component (PCA 1) explained 44.92% of the total variability, while the second component (PCA 2) accounted for 27.83%, corresponding to a cumulative variance of 72.75% (Figure 5).
PCA 1 clearly distinguished between Krstač and Žižak wines made from free- run grape juice (Kfr and Zfr) and Žižak and Krstač wines made from pressed juice (Zp and Kp). PCA 2 separated Krstač and Žižak wines made from pressed juice (Kp and Zp) from those made from running juice (Kfr and Zfr).
Using sPLS-DA, component 1 separated Kfr, Zfr and Zp wines from Kp wines. Component 2 distinguished between Krstač and Žižak wines made from free-run juice (Kfr and Zfr) and those made from pressed juice (Kp and Zp) (Figure 6A). Figure 6B shows that Kp wines are characterised by higher concentrations of higher alcohol, 3-ethoxy-1-pentanol (3E1P) and esters such as hexyl acetate (Hac), isoamyl acetate (Iac), ethyl butyrate (Eb) and decanoic acid (Da), all of which correlate positively with component 1. These compounds were more concentrated in the Kp wines than in the others. Zp wines were characterised by higher concentrations of the higher alcohols 3-(methylthio)-1-propanol (3M1P), isoamyl alcohol (Iaa) and ester diethyl succinate, all of which were negatively correlated with component 1. Wines characterised by these compounds showed higher concentrations compared to the other wine samples (Supplementary Material Table S7).
Figure 6C shows that Kfr wines are characterised by a higher alcohol content of phenylethyl alcohol (2Pa) and esters such as 2-phenylethyl acetate (2Pac), ethyl hydrogen succinate (Ehs) and 9-decenoic acid (9Da), all of which correlate positively with component 2. Žižak wines from pressed juices (Zp) showed the highest concentrations of ethyl lactate (El), ethyl octanoate (Eo), ethyl 4-hydroxybutanoate (E4h) and isobutyl alcohol (Iba) (Figure 6C, Figure 6D).

3.3. Effect of Bentonite Addition to the Must on the Aroma of Wines Produced from Krstač and Žižak Varieties

In this study, increasing concentrations of bentonite (0, 50, 100, 200 g/hL) were added to the must of two grape varieties, Krstač and Žižak. In the wines, the highest concentrations were found for higher alcohols, followed by esters and acids. Among the higher alcohols, isoamyl alcohol, 2-phenylethyl alcohol and isobutyl alcohol were the most common. The wines from Krstač had significantly higher concentrations of 2-phenylethyl alcohol, ranging from 48.62 to 60.61 mg/L, compared to the wines from Žižak, in which the concentrations ranged from 43.18 to 47.64 mg/L (Supplementary Material Tables S8 and S9). The presence of 2-phenylethyl alcohol is particularly important for wine aroma, as it imparts a pleasant, rose-like flavour [3,9].
In the wines produced from Krstač and Žižak grape varieties, the most common fatty acids were octanoic, hexanoic and decanoic acids. The concentration of octanoic acid was significantly higher in the wines from Žižak (7.21 to 7.60 mg/L) than in the wines from Krstač, where the concentration was between 4.55 and 5.60 mg/L. In addition, the concentrations of hexanoic and decanoic acids were also significantly higher in Žižak wines compared to Krstač wines. A reduction in octanoic acid was observed in Žižak wines with the addition of bentonite, and the concentration of decanoic acid was statistically significantly reduced in all wines compared to the control.
Several authors have reported a reduction in C6, C8 and C10 fatty acids in wine after the addition of bentonite to the must compared to control wines [12,46,47]. According to Tukey’s post hoc test, the total ester content was significantly lower in all Krstač wines (KB50, KB100, KB200) compared to the control (KB0). In the Žižak wines, a statistically significant reduction in total ester content was observed in the ZB200 wines compared to the ZB50 and ZB100 wines. In addition, Krstač wines made from the must treated with the highest dose of bentonite (KB200) had significantly lower concentrations of Eh, Eo, Dhb and Iac, while Žižak wines had significantly lower concentration of Eh, Eo, Ed and 2Pac compared to the control.
Some authors emphasise that clarification with bentonite can impair the removal of certain flavouring substances [12,48,49]. Bentonite can eliminate substances that affect yeast growth, reducing the synthesis of fermentative aromatic compounds [50]. In the ZB50 and ZB100 wine samples, the ester content increased, confirming that bentonite can influence the production of fermentative aromas [51]. Lambri et al. [52] and Kumar and Suhag [53] reported that the use of fining agents can have both positive and negative effects on the composition of the must.
Solid particles present in the must can inhibit ester production and their removal can be achieved by the addition of bentonite [54]. Piug Deu et al. [19] and Casalta et al. [10] emphasised the importance of removing solids from the must, as they promote the synthesis of certain ethyl esters while limiting the formation of fusel alcohols. In addition, the composition of the must and its nitrogen content have a significant influence on the development of the fermentation flavour [16,34].
A clear difference in ester concentrations was found between the wines from Krstač and Žižak. When comparing the two varieties, significant differences were found in the content of the individual esters. Krstač wines contained significantly higher concentrations of ethyl butyrate, ethyl lactate, diethyl succinate and ethyl hydrogen succinate, while Žižak wines had significantly higher concentration of ethyl hexanoate, ethyl octanoate, ethyl decanoate, ethyl 9-decenoate, ethyl 4-hydroxybutanoate and isoamyl acetate. These results indicate that the type and concentration of the precursor compounds present in the must of the individual grape varieties have a major influence on the ester content.

PCA and Multivariate sPLS-DA

PCA for Krstač wines revealed that the first principal component (PCA 1) explained 34.12% of the variability, while the second component (PCA 2) accounted for 21.47%, totalling 55.59% of the total variance. PCA 1 separated the KB0, KB50 and KB100 groups from KB200, while PCA 2 separated KB0 and KB200 from KB50 and KB100 wines. The PCA 1 distinguished 16 volatile compounds from the other 8, while PCA 2 distinguished 7 volatile compounds from the remaining 17 (Figure 7).
Figure 8A (sPLS-DA) demonstrated that component 1 separates the wines KB0, KB50 and KB100 from KB200, while component 2 separates KB50 and KB100 from KB0 and KB200. KB0 was characterised by higher concentrations of alcohols, such as 2,3B, esters, including Ehs, Iac, Eh and Eo, all of which correlated positively with component 1 (Figure 8B,D). In addition, KB0 was also characterised by higher levels of higher alcohols (Iaa and Iba), esters (Eb, Iac and 1, 3Pd) and acids (Da and Ia), all of which correlated with component 2. KB200 was associated with 3M1P and 9Da, which were correlated with component 1, and Ha was negatively correlated with component 2. The results show that wine KB0 contained most of the compounds in elevated concentrations compared to the wines produced with bentonite-treated must (KB50, KB100 and KB200) (Supplementary Material Table S10).
PCA 1 for Žižak wines explained 46.03% and PCA 2 explained 19.72%, resulting in a cumulative variance of 65.75%. PCA 1 abolished 10 compounds from the remaining 13, while PCA 2 abolished 8 compounds from the remaining (Figure 9).
Based on the sPLS-DA, component 1 distinguished ZB0 from ZB50, ZB100 and ZB200. ZB50 and ZB100 overlap in the lower part of the diagram. Component 2 distinguished ZB0 and ZB200 from ZB50 and ZB100 (Figure 10A). Figure 10B,C show the contributions of compounds correlated with components 1 and 2, respectively.
ZB0 was characterised by esters (Eo, Ed and 2Pac) and acid (Da) (Figure 10D) that were negatively correlated with component 1, while 2.3B, Dhb and Bl were negatively correlated with component 2. ZB50 wines were characterised by the presence of higher alcohol 3M1P, increased acidity Ia and Ehs, all of which are positively correlated with component 1, while Ia was negatively correlated with component 2.
ZB100 was characterised by Ds, while Iaa, 1.3Pd, Iba and E4h were negatively correlated with component 2. The concentrations of compounds most responsible for these differences are presented in Supplementary Material Table S11.

3.4. Sensory Evaluation

Wines produced from must subjected to longer settling times (K20 and K30) exhibited the most visually appealing and brightest colour. Among these, K30 displayed the cleanest and most elegant aroma, characterised by freshness and pronounced fruity notes. GC/MS-FID analyses showed that K30 contained markedly higher concentrations of total esters, particularly ethyl esters of fatty acids, including ethyl hexanoate (green, apple, banana), ethyl octanoate (fruity, floral, pear) and ethyl decanoate (fruity, grape), together with acetate esters of higher alcohols, such as isoamyl acetate (banana, fruity) and 2-phenylethyl acetate (rose, floral) (3,9). Given their low sensory thresholds, these compounds imparted the freshness and elegance of K30 wines. Consequently, K30 achieved the highest sensory score (18.0 out of 20.0) (Figure 11A).
Aroma quality improved proportionally with increasing must settling times, consistent with the greater synthesis of ethyl esters and acetate esters responsible for fruity and floral attributes. K30 wines were described as moderately full-bodied and elegant, exhibiting good aromatic balance, whereas wines derived from must with shorter settling times were judged as fuller-bodied but exhibited harsher, more astringent notes, likely associated with elevating phenolic concentrations. The control wines received the lowest score (16.6 out of 20.0) (Figure 11A).
The colour of the Žižak wines was straw yellow and showed approximately the same intensity in all samples. Z30 Žižak wines exhibited higher concentrations of total esters, acetates and 2-phenylethyl alcohol. Among the alcohols, 2-phenylethyl alcohol contributed significantly to sensory perception, imparting a floral, rose-like aroma. The control wine samples had a sharp and astringent flavour. Among the Žižak wines, Z30 and Z20 received the highest overall sensory scores (17.6 and 17.7 out of 20.0), while Z10 received the lowest (17.2 out of 20.0) (Figure 11A).
The wines produced from the Krstač grape variety whose musts were treated with bentonite at concentrations of 100 g/hL and 200 g/hL had a lighter yellow colour. The wines produced from must treated with 100 g/hL and 200 g/hL received the highest score (17.5 and 18.0 out of 20.0) (Figure 11B).
The colour of the Žižak wines was straw yellow, with no significant differences between the samples in terms of appearance. The wines produced with 100 g/hL bentonite (ZB100) had the most balanced flavour profile: they were soft, smooth and free of astringency. The addition of 200 g/hL bentonite (ZB200) significantly reduced the total ester concentration. Consequently, the flavour of ZB200 wines was lighter and of lower quality. The ZB100 Žižak wines received the highest sensory score (18.1), whereas ZB200 wines received the lowest (17.3 out of 20.0) (Figure 11B).
When pressed during white wine production, the Krstač pressed wines (Kp) exhibited an intense colour with brownish tones. Their aroma was intriguing but showed altered varietal character. The flavour was moderately astringent with a full mouthfeel. For Žižak, the pressed wines also showed brownish tones. The flavour was full-bodied and non-astringent.

4. Conclusions

The aim of this study was to analyse the influence of pre-fermentation treatments on the aromatic profile of wines from the indigenous grape varieties Krstač and Žižak. The Krstač wines (K30) were characterised by esters such as isoamyl acetate and ethyl esters of medium-chain fatty acids (ethyl hexanoate and ethyl decanoate). The wines of the Žižak Z30 variety are characterised by esters (2-phenylethyl acetate and isoamyl acetate) and fatty acids (octanoic, hexanoic and 9-decenoic acid). Prolonged static settling of the must significantly increased the formation of fatty acids, which in turn improved the synthesis of fatty acid esters in the wines. Sensory evaluation and GC/MS-FID analysis demonstrated that static settling time significantly influenced wine aroma. Krstač and Žižak wines settled for 30 h showed the most favourable aromatic characteristics.
Krstač and Žižak wines produced by free-run fermentation had significantly higher concentrations of 2-phenylethyl alcohol, which contributed to the sensory characteristics of the wines. In contrast, Krstač and Žižak wines made from pressed juice showed higher concentrations of fatty acids, especially the C6 alcohol 1-hexanol. Kfr wines were characterised by higher concentrations of 2-phenylethyl alcohol, 2-phenylethyl acetate and 9-decenoic acid. Krstač and Žižak wines produced from free-run juice exhibited better sensory characteristics than those obtained from pressed juice. The addition of bentonite led to a reduction in the concentrations of octanoic and decanoic acids in Žižak wines. This study shows that the use of bentonite can have both positive and negative effects on the content of aromatic compounds, depending on the concentration of bentonite used. In the experiment with bentonite, the best results regarding aroma were observed in Žižak wines, where 100 g/hL of bentonite was added during the pre-fermentative stage.
The results of this study can help oenologists determine the most effective pre-fermentative treatments to achieve the desired wine flavour profile. They shed light on the specific compounds that characterise each treatment and are responsible for the aromatic differences between them. This is the first study to investigate the influence of selected pre-fermentative treatments on the aroma profiles of Krstač and Žižak wines. Considering the limited number of studies on this topic, these results contribute to the expansion of current knowledge in this field. Future research could investigate different must clarification methods, the application of a greater number of different pressures when pressing the must and the use of different agents for must clarification.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/fermentation11100577/s1, Table S1. The content of aromatic compounds in wines of the Krstač variety, obtained by applying different static settling times (0 h, 10 h, 20 h, 30 h). Table S2. The content of aromatic compounds in wines of the Žižak variety, obtained by applying different static settling times (0 h, 10 h, 20 h, 30 h). Table S3. The abbreviations of compounds. Table S4. Concentrations of aromatic compounds (mg/L) responsible for the differences among Krstač wines produced with different static settling times. Table S5. Concentrations of aromatic compounds (mg/L) responsible for the differences among Žižak wines produced with different static settling times. Table S6. Content of aromatic compounds in Krstač and Žižak wines, obtained by applying pressure (0; ~1.8 bar) during pressing. Table S7. Concentrations of aromatic compounds (mg/L) responsible for the differences among Krstač and Žižak wines produced under different pressing pressures. Table S8. The content of aromatic compounds in the wines of Krstač, obtained by applying increasing concentrations of bentonite (0, 50, 100, 200 g/hL). Table S9. The content of aromatic compounds in wines of the Žižak varieties, obtained by applying increasing concentrations of bentonite (0, 50, 100, 200 g/hL). Table S10. Concentrations of aromatic compounds (mg/L) responsible for the differences among Krstač wines produced with increasing amounts of bentonite. Table S11. Concentrations of aromatic compounds (mg/L) responsible for the differences among Žižak wines produced with increasing amounts of bentonite.

Author Contributions

Conceptualisation, A.P. and V.M.; methodology, V.M., I.S., I.Đ. and A.P.; formal analysis, A.P. and V.M.; investigation, V.M. and I.S.; resources, I.S.; data curation, V.M. and I.S.; writing—original draft preparation, V.M.; writing—review and editing, A.P. and V.M.; visualisation, V.M.; supervision, A.P. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

In accordance with the national laws, ethical approval is not required for studies involving sensory evaluation. There are no formal documentation procedures from human ethics committees available for sensory evaluation. The sensory protocol employed in this research was conducted in compliance with the relevant operation specifications in Serbia.

Informed Consent Statement

Written informed consent for participation was obtained from all subjects in accordance with the General Data Protection Regulation (GDPR) 2016/679. The study was conducted following the principles of the Declaration of Helsinki.

Data Availability Statement

The original contributions presented in this study are included in the article and the Supplementary Materials (https://doi.org/10.5281/zenodo.17273655). Further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Principal component analysis (PCA): Krstač wines with different treatments: K0, K10, K20 and K30 (Table 1).
Figure 1. Principal component analysis (PCA): Krstač wines with different treatments: K0, K10, K20 and K30 (Table 1).
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Figure 2. (A) Sparse partial least-squares discriminant analysis (sPLS-DA) in different wines with different treatments: K0, K10, K20 and K30; Treatments are shown in different colours: blue, orange, grey, and green. (B) differences along component 1; (C) differences along component 2; the colour of each bar represents the group where corresponding volatile compounds show the highest medium abundance; (D) clustered image map (CIM) of 16 aromatic compounds in Krstač wines chosen by sPLS-DA.
Figure 2. (A) Sparse partial least-squares discriminant analysis (sPLS-DA) in different wines with different treatments: K0, K10, K20 and K30; Treatments are shown in different colours: blue, orange, grey, and green. (B) differences along component 1; (C) differences along component 2; the colour of each bar represents the group where corresponding volatile compounds show the highest medium abundance; (D) clustered image map (CIM) of 16 aromatic compounds in Krstač wines chosen by sPLS-DA.
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Figure 3. Principal component analysis (PCA): Žižak wines with different treatments: Z0, Z10, Z20 and Z30 (Table 1).
Figure 3. Principal component analysis (PCA): Žižak wines with different treatments: Z0, Z10, Z20 and Z30 (Table 1).
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Figure 4. (A) Sparse partial least-squares discriminant analysis (sPLS-DA) in different wines with different treatments: Z0, Z10, Z20 and Z30; Treatments are shown in different colours: blue, orange, grey, and green. (B) differences along component 1; (C) differences along component 2; the colour of each bar represents the group where corresponding volatile compounds show the highest medium abundance; (D) clustered image map (CIM) of 16 aromatic compounds in Žižak wines chosen by sPLS-DA.
Figure 4. (A) Sparse partial least-squares discriminant analysis (sPLS-DA) in different wines with different treatments: Z0, Z10, Z20 and Z30; Treatments are shown in different colours: blue, orange, grey, and green. (B) differences along component 1; (C) differences along component 2; the colour of each bar represents the group where corresponding volatile compounds show the highest medium abundance; (D) clustered image map (CIM) of 16 aromatic compounds in Žižak wines chosen by sPLS-DA.
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Figure 5. Principal component analysis (PCA): Krstač and Žižak wines with different treatments KFR, KP, ZFR, ZP (Table 1).
Figure 5. Principal component analysis (PCA): Krstač and Žižak wines with different treatments KFR, KP, ZFR, ZP (Table 1).
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Figure 6. (A) Sparse partial least-squares discriminant analysis (sPLS-DA) in different wines with different treatments: KFR, KP, ZFR and ZP (Table 1); Treatments are shown in different colours: blue, orange, grey, and green. (B) Differences along component 1. (C) Differences along component 2; the colour of each bar represents the group where corresponding volatile compounds show the highest medium abundance. (D) Clustered image map (CIM) of 16 aromatic compounds in Krstač and Žižak wines chosen by sPLS-DA.
Figure 6. (A) Sparse partial least-squares discriminant analysis (sPLS-DA) in different wines with different treatments: KFR, KP, ZFR and ZP (Table 1); Treatments are shown in different colours: blue, orange, grey, and green. (B) Differences along component 1. (C) Differences along component 2; the colour of each bar represents the group where corresponding volatile compounds show the highest medium abundance. (D) Clustered image map (CIM) of 16 aromatic compounds in Krstač and Žižak wines chosen by sPLS-DA.
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Figure 7. Principal component analysis (PCA): Krstač wines with different treatments (KB0, KB50, KB100 and KB200 (Table 1)).
Figure 7. Principal component analysis (PCA): Krstač wines with different treatments (KB0, KB50, KB100 and KB200 (Table 1)).
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Figure 8. (A) Sparse partial least-squares discriminant analysis (sPLS-DA) in different wines with different treatments: KB0, KB50, KB100 and KB200 (Table 1); Treatments are shown in different colours: blue, orange, grey, and green. (B) Differences along component 1. (C) Differences along component 2; the colour of each bar represents the group where corresponding volatile compounds show the highest medium abundance. (D) Clustered image map (CIM) of 16 aromatic compounds in Krstač wines chosen by sPLS-DA.
Figure 8. (A) Sparse partial least-squares discriminant analysis (sPLS-DA) in different wines with different treatments: KB0, KB50, KB100 and KB200 (Table 1); Treatments are shown in different colours: blue, orange, grey, and green. (B) Differences along component 1. (C) Differences along component 2; the colour of each bar represents the group where corresponding volatile compounds show the highest medium abundance. (D) Clustered image map (CIM) of 16 aromatic compounds in Krstač wines chosen by sPLS-DA.
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Figure 9. Principal component analysis (PCA): Žižak wines with different treatments (ZB0, ZB50, ZB100 and ZB200).
Figure 9. Principal component analysis (PCA): Žižak wines with different treatments (ZB0, ZB50, ZB100 and ZB200).
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Figure 10. (A) Sparse partial least-squares discriminant analysis (sPLS-DA) in different wines with different treatments: ZB0, ZB50, ZB100 and ZB200 (Table 1); Treatments are shown in different colours: blue, orange, grey, and green. (B) Differences along component 1. (C) Differences along component 2; the colour of each bar represents the group where corresponding volatile compound show the highest medium abundance. (D) Clustered image map (CIM) of 16 aromatic compounds in Žižak wines chosen by sPLS-DA.
Figure 10. (A) Sparse partial least-squares discriminant analysis (sPLS-DA) in different wines with different treatments: ZB0, ZB50, ZB100 and ZB200 (Table 1); Treatments are shown in different colours: blue, orange, grey, and green. (B) Differences along component 1. (C) Differences along component 2; the colour of each bar represents the group where corresponding volatile compound show the highest medium abundance. (D) Clustered image map (CIM) of 16 aromatic compounds in Žižak wines chosen by sPLS-DA.
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Figure 11. Sensory analysis of Krstač and Žižak wines: (A) static settling; (B) bentonite.
Figure 11. Sensory analysis of Krstač and Žižak wines: (A) static settling; (B) bentonite.
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Table 1. Different treatments in the production of Krstač and Žižak wines.
Table 1. Different treatments in the production of Krstač and Žižak wines.
TreatmentsAbbreviation
(Wine Samples)		Explanation of Treatments
Static settlingK0, Z0Without static settling
K10, Z10Static settling for 10 h
K20, Z20Static settling for 20 h
K30, Z30Static settling for 30 h
PressureKfr, ZfrFree-run juice
Kp, ZpPressure ~1.8 bar
BentoniteKB0, ZB0Without bentonite
KB50, ZB50Addition of 50 g/hL bentonite
KB100, ZB100Addition of 100 g/hL bentonite
KB200, ZB200Addition of 200 g/hL bentonite
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Madžgalj, V.; Đorđević, I.; Sofrenić, I.; Petrović, A. The Effects of Pre-Fermentative Treatments on the Aroma of Krstač and Žižak Wines. Fermentation 2025, 11, 577. https://doi.org/10.3390/fermentation11100577

AMA Style

Madžgalj V, Đorđević I, Sofrenić I, Petrović A. The Effects of Pre-Fermentative Treatments on the Aroma of Krstač and Žižak Wines. Fermentation. 2025; 11(10):577. https://doi.org/10.3390/fermentation11100577

Chicago/Turabian Style

Madžgalj, Valerija, Iris Đorđević, Ivana Sofrenić, and Aleksandar Petrović. 2025. "The Effects of Pre-Fermentative Treatments on the Aroma of Krstač and Žižak Wines" Fermentation 11, no. 10: 577. https://doi.org/10.3390/fermentation11100577

APA Style

Madžgalj, V., Đorđević, I., Sofrenić, I., & Petrović, A. (2025). The Effects of Pre-Fermentative Treatments on the Aroma of Krstač and Žižak Wines. Fermentation, 11(10), 577. https://doi.org/10.3390/fermentation11100577

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