The winemaking industry in the Republic of Moldova has a rich historical and cultural heritage, with white wines constituting a significant part of the country’s production. Traditional winemaking mainly relies on the yeast Saccharomycescerevisiae, which ensures a complete and effective alcoholic fermentation. However, this yeast alone often produces wines with similar aromatic profiles and less complexity [1]. In recent decades, commercial Saccharomycescerevisiae strains have been used as starter cultures, whereas non-Saccharomyces yeasts have largely been overlooked and considered of little technological importance.

In recent years, the study and use of non-Saccharomyces yeasts have gained significant attention as tools for diversifying and improving wine sensory qualities. These yeasts, naturally found on grape skins and in winery environments, possess unique metabolic activities that can enhance the aromatic and flavor profiles of white wines [2]. Studies conducted by Taran, N., on the native Codrinschii grape variety selected nine yeast strains with high biotechnological potential for producing dry red wines. These local yeast strains can adapt to specific environmental conditions, ferment carbohydrates from the must, and contribute to wines with characteristic qualities and high organoleptic standards typical of the wine-growing region [3]. Researchers Roudil L. and Russo P. (2005) noted that non-Saccharomyces yeasts can enhance wine aroma, quality, and food safety by producing various metabolites during the alcoholic fermentation of must samples [4].

As is known, the definition of vitivinicultural “terroir” according to resolution OIV/VITI 333/2010 refers to an area where collective knowledge of the interactions between the physical and biological environment and applied vitivinicultural practices develops, providing distinctive characteristics for products originating from the PGI area (PGI—noted protected geographical indication). The concept of “terroir” includes specific soil, topography, climate, landscape features, and biodiversity [5]. Based on the principles of sustainable vitiviniculture adopted by the OIV-CST 518-2016 RESOLUTION (OIV, 2016) [6], and the guidelines for their implementation adopted by the OIV-VITI 641-2020 RESOLUTION (OIV, 2020) [7], protecting soils, water, air, biodiversity, and landscapes is especially important in the vitivinicultural sector. Therefore, careful planning is essential before establishing new vineyards or other vitivinicultural facilities, using proven ecological principles and optimal management of both existing and new assets.

Biodiversity of living organisms is essential to implementing the principles of sustainable vitiviniculture. In this context, the OIV-VITI 655-2021 RESOLUTION (OIV, 2021) presents recommendations regarding the valuation and significance of microbial biodiversity in sustainable vitiviniculture [8].

In this context, research over the past 5 - 10 years in microbiology and biotechnology related to wine products has begun to focus on isolating and utilizing local yeast strains or species to produce natural, organic, sustainable wines with organoleptic qualities typical of the vineyards where the grapes are grown [9]. The use of microbial resources in wine production is essential for driving innovation and improving wine quality. Ongoing research on Saccharomyces cerevisiae and non-Saccharomyces species to enhance wine characteristics and accommodate changing consumer preferences is promoting a competitive, sustainable wine industry [10].

In this context, non-Saccharomyces yeast species such as Torulasporadelbrueckii, Lachanceathermotolerans, Metschnikowiapulcherrima, Schizosaccharomycespombe, and Pichiakluyveri are already commercially available as cultures suitable for various winemaking applications. However, they do not preserve the authenticity of the “terroir” microbiome, which imparts aromatic complexity and a unique regional expression to wines produced using them. Another benefit of species from the indigenous grape microbiome is that they can limit the growth of unwanted microorganisms during the early stages of alcoholic fermentation through competitive inhibition mechanisms and the secretion of antimicrobial substances. Recent studies by Rubio-Breton, P., have shown that yeasts such as Metschnikowiapulcherrima, Torulasporadelbrueckii, and Lachanceathermotolerans contribute to ensuring the microbiological stability of wine and contribute to aromatic complexity by producing unique metabolites, including aromatic esters and organic acids [11]. Non-Saccharomycesyeasts include a diverse range of genera and species that were once considered spoilage microorganisms. However, recent research has highlighted their beneficial role in controlled alcoholic fermentation. Some of the most important species are listed in Table 1.

Table 1.The primary impact of non-Saccharomyces on the sensory attributes of wines.

Each species has specific enzymatic and metabolic abilities that can be strategically combined with Saccharomycescerevisiae to produce more balanced, aromatic wines unique to the wine-growing Protected Geographical Indication (PGI) region.

The purpose of the study was to use microbiological methods and techniques to observe, isolate, and identify microorganisms in the examined grapes from the Stefan Voda PGI region. The aim was to determine whether harmful microorganisms were present or absent, with a particular focus on the native microflora that has technological importance in white wine production.

The practical study methods focused on identifying, isolating, and multiplying native flora, including both non-Saccharomyces yeast species and Saccharomyces yeast species of Muscat grape varieties from Javgur, Cimislia district (Stefan Voda PGI viticultural region), to select indigenous yeast species with technological interest.

The presence of Saccharomyces yeasts, acetic bacteria, Torulopsis, Metschnikowia, Hanseniaspora, and Bretanomyces species was detected in experimental samples during microbiological examination and is shown in Figure 2.

Figure 2.Photographs of Petri dishes with thermostatic culture media of experimental samples.

Table 2. The dynamics of microflora during white wine production.

Based on morphological classification of the indigenous microbiome, fermentation yeasts of the genus Saccharomyces have round or ellipsoidal, white-colored cell morphology. In contrast, microorganisms of the genus Torulopsis have beige, spherical morphology. The white, lemon-shaped, or cylindrical morphology is characteristic of microorganisms in the genera Hanseniaspora and Kloeckera. Metschnikowia sp. is ovoid to ellipsoidal in shape, reproduces by budding, with cell colonies of pink color and lactic bacteria of Lactobacillus forming large colonies of gray bacilli [22].

The results showed that the studied yeasts do not form true mycelium and reproduce vegetatively through multilateral budding and sexually via spores, confirming that these strains belong to the genus Saccharomyces. Based on assessments of morphological, cultural, and reproductive features, isolated yeast cultures from the indigenous microflora of grape PGI Stefan Voda were found to comprise uniform, viable cells, as shown in Table 2.

Out of the 145 identified strains, 65 were microbiologically characterized. Based on the evaluation of morphological, cultural, and reproductive traits observed in the developed Petri dish cultures, it was determined that yeast cultures isolated from the indigenous microflora are uniform and viable cell strains, with potential for use in winemaking.

The practical analysis examined 145 individual colonies of dominant fungi, with Ascomycotina accounting for 75.86% ± 2.08% and the Deuteromycotina genus representing 24.14% ± 2.68%, according to Figure 3. Over time, the microbiota population in the alcoholic fermentation medium decreases from 52 colonies during the must stage to 14 colonies in the raw material wine.

Figure 3.The structure of the microbial community during the wine production process.

In the alcoholic fermentation process of the studied Muscat must, species of Saccharomyces, Kloeckera, and Torulopsis are present at moderate levels. However, as non-Saccharomyces species are converted into alcohol, they become inactive, and in the raw material wine, these species account for 9.7% - 13.1% of the initial microbiota population.

Experimental grape samples and dry white wines from the study were subjected to physicochemical analysis, and the results are presented in Table 3.

Table 3.Physicochemical indices of experimental must samples, harvest 2024.

Comparing the physicochemical indices, the Traminer Rose must sample shows a higher total titratable acid concentration (6.62 g/L compared to 5.81 g/L for Muscat) and a significantly higher pH (0.34 pH units higher). The higher acidity in the Traminer Rose must does not indicate a sourer taste, as the must’s buffer system maintains a high pH. Muscat must have a more balanced acidity profile and a safer pH from a microbiological perspective.

The Traminer Rose must sample stands out for its amino acid concentration of over 100 mg/L, which is higher than that of Muscat and is referred to as technological Yeast Assimilable Nitrogen (YAN). This provides an excellent nutrient medium for alcoholic fermentation, reducing the risk of slow or stalled fermentation. Both must show normal turbidity levels for freshly pressed must. The slightly higher level in Traminer Rose (at 28.68 NTU) indicates a greater concentration of suspended solids (e.g., pulp particles and pectin), which require an effective decantation or clarification step before fermentation to produce higher-quality wine. Both must demonstrate good quality, with high phenological and technological maturity.

As an experimental observation, the kinetics of alcoholic fermentation in the two technological wine study options depended on the inoculated yeast strains. During alcoholic fermentation of the control samples (Muscat and Traminer Rose) using S. cerevisiae industrial oenological dry yeast, the total sugar concentration was depleted within the first 5 days of fermentation. In comparison, the duration of alcoholic fermentation with non-Saccharomyces strains (samples II) ranged from 8 to 10 days for the sequential fermentation with yeasts of Hanseniaspora, Kloeckera, Torulaspora, and indigenous species Saccharomycescerevisiae, which aligns with the literature data [23].

The physicochemical indices of the study samples show significant differences for both Muscat and Traminer rose varieties, as shown in Table 4. Muscat wines are slightly more acidic (average pH ~3.28) than Traminer Rose wines (average pH ~3.44). A lower pH enhances the sensation of freshness. Variations among samples are minimal, indicating consistent winemaking. In terms of titratable acidity, Traminer Rose has a slightly higher concentration (~5.89 g/L) than Muscat (~5.30 g/L) due to higher acidity in the processed grapes. Traminer Rose samples have significantly higher alcohol content (12.66% v/v in both samples) than Muscat (~12.07% v/v). This is explained by the higher initial sugar content in Traminer Rose grapes, as shown in Table 3. Regarding residual sugar content, the wine samples are dry, with less than 4 g/L.

Table 4. Physicochemical indices of dry white wine samples.

One of the main advantages attributed to T. delbrueckii was its ability to lower volatile acidity in experimental wines. For the Muscat sample II wine, the reduction in volatile acidity was 0.16 g/L compared to Sample I, and for the Traminer Rose variety, it was 0.18 g/L. These technological results match those reported by other researchers, who observed decreases in the final volatile acidity concentration to 0.14 - 0.28 g/L compared to S. cerevisiae, as noted in Mas’s studies [24].

There may be a metabolic conflict between the inoculated species T. delbrueckii and Hanseniaspora, as evidenced by an increase in volatile acidity. In the present study, during the early stages of fermentation (the first days after inoculation), varietal aromatic complexity, mediated by fruit and flower esters, is observed to be dynamic in response to the selected Hanseniaspora species. Technically, slowing alcoholic fermentation with Hanseniaspora species can increase volatile acidity; however, in the practical study, the Saccharomyces inoculum was added on the third day of fermentation, thereby avoiding the side effect of increased volatile acidity. Additionally, applying T. delbrueckii can reduce the final ethanol concentration in wines by up to 1%, while increasing glycerol levels from 0.2 to 0.9 g/L, as reported by Yao [2]. In wines made with the technological variant II, glycerol content was higher by 0.35 g/L (Muscat) and 0.38 g/L (Traminer Rose) compared to the control method (Sample I). These glycerol levels stay within the range of 0.2 - 0.9 g/L, as shown by Van Leeuwen’s [25] and Martin’s [26] studies.

Several authors (Di Canito, 2021, and Morata, 2020) report that T. delbrueckii releases more mannoproteins than Saccharomyces and other non-Saccharomyces species [1][27]. In this study, however, this mannoprotein content was not measured.

The levels of secondary fermentation compounds, glycerol and 2,3-butylene glycol, indicate proper alcoholic fermentation, with concentrations ranging from 5.45 to 6.42 g/L in the samples. Traminer wines had an average 2,3-butylene glycol level of 238.62 mg/L, which is notably higher than that of Muscat wines (147.03 mg/L). These elevated levels suggest increased body, texture, and naturalness in the wine, aligning with Morata’s research, as noted in the specialized literature [28].

The total phenolic compounds in wine samples are higher, ranging from 146.5 to 168.5 mg/L. These compounds contribute to the structure, taste, and stability of the wines. Both wine varieties, in terms of organoleptic characteristics and quality, are described as clear, dry, and free from defects.

In general, in both cases, Sample II was evaluated as having higher overall quality than Sample I in the study samples.

Among the many researchers mentioned in section 1 of the article, non-Saccharomyces yeasts that enhance the complexity and fruity characters of experimental Muscat and Traminer rose dry white wines are discussed in Figure 4 below.

The aroma impact of Sample II’s technological method influenced the wine’s aromatic quality. The aroma of T. delbrueckii is often described as “fruitiness,” which aligns well with the sensory analysis [29]. Muscat Sample II (orange) generally scores slightly higher than control Sample I (blue) across most attributes, including Persistence, Body, and Sweetness. Traminer Rose Sample II (yellow) stands out with the highest Sweetness score of all four samples (nearly 3.5). In contrast, the control sample (gray) exhibits more pronounced Astringency and Structure, as well as aroma characteristics detected by the panel, especially Field flowers.

Figure 4. Radar chart of the sensory evaluation for the experimental dry white wine.

Another aspect to consider is the dominant attributes of each sample. Traminer Rose II (yellow) shows the highest sweetness and color intensity. In contrast, Muscat Sample I (blue) has the least structure and bitterness, indicating a light structure and a slight perception of bitterness. Balance scores are the highest for all samples except the control Muscat samples. These organoleptic analysis results align with those in Table 4, which explain why the Traminer Rose wines are, from both a chemical and sensory perspective, more intense and complex: they have higher alcohol content, titratable acidity, phenolic compounds, color, and 2,3-butylene glycol. These attributes contribute to their higher quality scores. Muscat wines are characterized by a fresher, smoother profile, lower pH, lower alcohol levels, and more delicate citrus and floral aromas.

The sensory analysis results of the wines (tasting sheets) were analyzed using principal component analysis (PCA). The PCA method helps visualize differences in organoleptic properties and panel preferences for wine samples produced by two methods (classic with selected industrial yeasts and sequential fermentation).

Figure 5 shows the compounds responsible for the most significant differences between the two samples. The first principal component (Factor 1) explained 71.63% of the total variation, while the second principal component (Factor 3) explained an additional 8.84% (totaling 80.47%).

Based on the PCA results, it was possible to distinguish the samples produced through both sequential fermentation and control studies [30].

In summary, the most organoleptically appreciated experimental samples were those produced by a mixture of non-Saccharomyces and Saccharomyces indigenous yeast species. They featured a complex aroma profile specific to the grape variety, a harmonious balance, and a blend of fruit and floral notes, with slight freshness from the acidity level.

Figure 5. Principal component analysis (PCA) of wine aroma features evaluated by panel tasters.

The evaluation of the correlation level of indigenous grape yeasts, included in Table 5, likely involves coexistence, competition, or succession as a final step, which allowed us to establish the following aspects:

Positive values close to “1” indicate a strong positive correlation, such as 0.974 between Kloeckeraapiculata and Saccharomycescerevisiae. This suggests that the two species tend to occur together or thrive under similar conditions.Negative values close to “−1” indicate a strong negative correlation, such as -0.652 between Saccharomycescerevisiae and Saccharomycesoviformis. This could be due to a mutually exclusive or highly competitive relationship, where the presence of one species inhibits the other.Values close to “0” indicate a weak or no correlation (e.g., 0.085 between Saccharomycesbailii and Saccharomycescerevisiae).Strong positive correlations (1.000) between species Candidamycoderma, Saccharomycesbayanus, and Pichiamembranefaciens, indicating a close association.Kloeckeraapiculata, a common wild yeast early in fermentation, shows strong correlations with most species of the genus Saccharomyces (the primary yeasts responsible for efficient alcoholic fermentation).

Table 5. Correlation levels of the microbial community at different stages of wine technology.

Dekkerabruxellensis (also known as Brettanomyces) and the genus Saccharomyces exhibit negative correlations with most other species, suggesting competitive interactions or different stages of development.The population dynamics show that Kloeckeraapiculata (a “non-Saccharomyces” yeast) is the dominant species at the start of alcoholic fermentation. It multiplies rapidly in fresh must, consuming sugars and producing various aroma compounds, except alcohol.As fermentation progresses, Saccharomyces cerevisiae (the primary winemaking yeast) becomes dominant because of its traits, which allow it to tolerate higher sulfur dioxide (SO₂) and produce high levels of ethanol. These conditions quickly suppress Kloeckeraapiculata and other non-Saccharomyces yeasts [31].

In terms of oenological impact, the Kloeckeraapiculata species and other non-Saccharomyces yeasts initially contribute positive aromas and flavor precursors, as detected by the panelists, as illustrated in Figure 4.

Based on the experimental wine samples, various microflora and fermentation processes can significantly influence the chemical and sensory qualities of wines. Indigenous microflora contributes to a more balanced aroma, underscoring the importance of carefully selecting both viticultural and fermentation practices to shape the wine’s local characteristics.

As noted above, plant-associated microbiomes are essential to viticulture and winemaking, where various fungi and bacteria can have positive, negative, or neutral effects on vine health and wine quality. Therefore, the sources and persistence of wine-related microbiota in vineyards are critical for the final product quality. Additionally, it is well established that human intervention can influence the vineyard microbiome through multiple direct and indirect pathways [32], with potential impacts on microbial terroirs (OIV, resolution 2010) [33].

Fermentative yeasts are used industrially in wine production, primarily for their ability to ferment simple carbohydrates anaerobically, producing ethanol and carbon dioxide. Many microorganisms are present, especially during grape ripening. After harvest in autumn, yeasts on the leaves fall into the soil with their fallen leaves, where they remain until spring. This process allows natural selection to occur, resulting in the survival of the most resistant species.

The practical analysis reveals the presence of 145 individual colonies of dominant fungi, with Ascomycotina (75.86% ± 2.08%) and Deuromycotina genus (24.14% ± 2.68%) being predominant. During the alcoholic fermentation of the studied Muscat must varieties, Saccharomyces, Kloeckera, and Torulopsis species are present in moderate amounts. However, as monosaccharides are converted into alcohol, non-Saccharomyces species become inactivated, lowering their initial population in the raw material wine to a range of 9.7% - 13.1%.

One of the initial benefits linked to the T. delbrueckii species was a reduction in volatile acidity in the experimental wines: the volatile acidity decreased by 0.16 g/L in Muscat sample II compared to Sample I, and by 0.18 g/L in Traminer Rose wine. Consequently, the glycerol content was higher in samples II by 0.35 g/L in Muscat and 0.38 g/L in Traminer Rose than in the control (Sample I).

Another aspect was the organoleptic aroma-dominant attributes per sample: the Traminer Rose II sample had the most pronounced sweetness and color-intensity profile. At the same time, the Muscat Sample I presented a minor content in structure and bitterness-free (lack of bitterness), indicating a light structure and a slight perception of bitterness. The Traminer Rose wines analyzed, from a chemical and sensory perspective, are more intense and complex due to their higher levels of alcohol, titratable acidity, phenolic compounds, color, 2,3-butylene glycol, and higher organoleptic quality scores.

In terms of oenological impact, Kloeckeraapiculata and other non-Saccharomyces yeasts initially contribute positive aromas and flavor precursors; however, if they remain dominant for too long, they can lead to the formation of undesirable volatile acidity (e.g., ethyl acetate) and reduced alcohol yield. The modern use of selected Saccharomyces yeasts helps control this transition, ensuring efficient fermentation and a balanced flavor profile [34].

This study shows that using starter cultures results in faster complete fermentation and produces more alcohol than spontaneous fermentation. The sensory characteristics are specific to the grape variety and terroir, and the grapes demonstrate high resistance to microbial changes. T. delbrueckii and Kloeckera enhance the intensity and quality of wine aroma, boosting the overall impression and highlighting the varietal and fruity qualities.

The results showed that using selected starter cultures can produce balanced wine and may also help develop wines that reflect their geographical origin. Kloeckera, T. delbrueckii, and Torulaspora species in the Republic of Moldova can naturally occur on grapes, making them potential fermentation promoters, particularly for local wine-industry applications. However, their abilities need to be verified later, considering that they do not tolerate ethanol concentrations higher than 4% - 6% v/v.

This study demonstrated that by selecting and multiplying indigenous starter cultures and using them for fermentation, it is possible to preserve the wine’s authenticity linked to a specific geographical area. A sustainable use of the grape microbiome involves this method, aligning with recent trends in microbiology and biotechnology, and results in natural, organic, sustainable wines with organoleptic qualities typical of the vineyards where the grapes are cultivated.

For a more detailed study of the influence of indigenous yeast species, we plan to investigate the mannoprotein content (a technological byproduct of T. delbrueckii) in the wine samples prepared, as well as to perform HPLS-DAD-MS analysis to quantify the aromatic compounds involved. This would complement the organoleptic analysis conducted in this article.

The Institutional Project supported the research, under subprogram 02.04.05 “Optimizing food processing technologies in the context of the circular bioeconomy and climate change”, Bio-OpTehPAS, which is being implemented at the Technical University of Moldova, Department of Oenology and Chemistry.

This research was funded by the Young Researchers Project 23.70105.5107.04T, titled “Valorization of the indigenous flora of the Ștefan Vodă wine-growing region to increase the authenticity and competitiveness of Moldovan wines,” carried out within the Oenological Research Center, Department of Oenology and Chemistry, Faculty of Food Technology, TUM.