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Article

Characterization of Microbial Population of Organic Grapes, Must and Natural Wine During Spontaneous Vinification of Limniona Red Grape Variety

by
Paraskevi Bouki
1,
Chrysanthi Mitsagga
1,
Aphrodite Tousia Becker
2 and
Ioannis Giavasis
1,*
1
Laboratory of Microbiology and Biotechnology, Department of Food Science and Nutrition, University of Thessaly, 43100 Karditsa, Greece
2
Kontozisis Wineries, Kanalia, 43060 Karditsa, Greece
*
Author to whom correspondence should be addressed.
Beverages 2025, 11(6), 160; https://doi.org/10.3390/beverages11060160
Submission received: 16 August 2025 / Revised: 26 October 2025 / Accepted: 4 November 2025 / Published: 17 November 2025
(This article belongs to the Section Beverage Technology Fermentation and Microbiology)
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Abstract

Natural wines represent a new trend in winemaking without the use of preservatives and starter cultures, revealing the unique quality traits of grapes, wine, and terroir, but are susceptible to spoilage or undesirable fermentations. This study investigated the microbial populations associated with organic grapes, must, and natural wines of the Limniona red grape variety, focusing on different production stages and fermentation vessels. Samples included immature and ripe grapes, initial and fermenting must, filtered and unfiltered wines, and final bottled and filtered wines. These were analyzed in order to enumerate key groups of microorganisms and identify beneficial yeasts and bacteria of alcoholic and malolactic fermentation, respectively, as well as potential markers of off-flavors. Culture-dependent methods were used to enumerate yeasts and bacteria, while Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) and 16S rRNA sequencing provided taxonomic resolution. Beneficial fermentation microorganisms (especially Saccharomyces yeasts) were scarce in initial grapes, where other contaminants or wild yeasts were present. Gradually, as fermentation progressed, there was a prevalence of Saccharomyces cerevisiae strains of increased diversity in matured wine, as well as several lactic acid bacteria (LAB) of malolactic fermentation. Most LAB were identified as Lactobacillus and Oenococcus species. Other bacteria from environmental sources, irrelevant to alcoholic/malolactic fermentation or spoilage, like Burkholderia, were also present during the vinification process. The type of vessel affected the type of LAB that prevail, with an abundance of Oenococcus in clay vessels versus Lactobacillus species in stainless-steel vessels. Notably, Lentilactobacillus parafarraginis can be linked to off-flavors if they represent a high percentage of the wine microbiota. These findings highlight the importance of understanding, monitoring and controlling microbial succession during production stages in order to prevent sensory faults and ensure the stable quality of natural wines.

Graphical Abstract

1. Introduction

Wine, derived from grapes (Vitis spp. L.), is one of the most significant horticultural products worldwide, renowned for its rich polyphenol composition and associated health benefits [1]. Growing health awareness and increasing demand for natural products have boosted the production of natural wines and vinegars. In 2021, Greece ranked sixth in global grape production, with 193,252 grape farms recorded in 2020 [2]. Indigenous Greek grape varieties have demonstrated superior adaptation to warmer climates compared to international cultivars, highlighting their value in the context of global climate change [3]. Natural wine, characterized by minimal intervention and lacking formal regulation, is increasingly promoted by wine professionals. France’s AVN (Association des Vins Naturels) defines it as wine made from organic or biodynamic grapes without additives, using native yeasts. S.A.I.N.S. (“Sans Aucun Intrant Ni Sulfite ajouté”) further prohibits any inputs of sulfites. Vin Méthode Nature, established in 2020 and recognized by the DGCCRF (Direction générale de la concurrence, de la consommation et de la répression des fraudes), offers two certified versions: one without added sulfites and another allowing up to 30 mg/L at bottling, both without addition of any starter cultures [4].
Natural wine can be conceptualized as a socially driven movement involving both consumers and producers, rather than as a strictly regulated form of agricultural production. This phenomenon reflects a broader evolution in consumer preferences, increasingly shaped by concerns related to personal health and environmental sustainability—factors that significantly influence firms’ strategies for quality differentiation [5].
A notable aspect of natural winemaking is the use of indigenous microbial cultures—native yeasts and bacteria associated with alcoholic and malolactic fermentation [6,7,8]. While this practice is often emphasized within the natural wine community, it remains largely peripheral in conventional winemaking and is of limited relevance to most industrial producers. Instead, it constitutes a niche approach primarily adopted by small-scale wineries seeking to differentiate themselves through the unalloyed expression of the terroir and the winery environment into the sensory properties of a natural wine. These native microorganisms of the vineyard ecosystem drive both alcoholic and malolactic fermentations, playing a critical role in shaping natural wine’s aroma and flavor, leading to wines with distinct sensory characteristics [9,10,11].
Advancements in biochemistry and molecular microbiology have significantly enhanced our understanding of fermentation, emphasizing the diversity and functional roles of native microbial populations. While a handful of commercial yeast strains dominate industrial production, indigenous strains naturally thrive in grape-rich environments, offering unique aromatic profiles and stability, particularly under oxygen-limited conditions common in natural winemaking [7,12,13,14].
Alcoholic fermentation transforms natural grape sugars into ethanol and CO2, defining the wine’s basic flavor profile, whereas malolactic fermentation softens acidity and enhances complexity by converting malic acid into lactic acid [4,15,16]. Although commercial yeast and bacterial starter cultures are widely employed to expedite fermentation and minimize the risk of excessive malolactic fermentation, spontaneous fermentation—driven by naturally occurring microorganisms—remains central to traditional artisanal winemaking and natural wine practices. This sequential process typically begins with the dominance of yeasts during the early fermentation stages, followed by a progressive increase in lactic acid bacteria (LAB) as fermentation advances [17,18,19,20].
Spontaneous vinification in natural wines initiates with the metabolism of sugars into alcohol and CO2 by different yeasts and the gradual transition to LAB dominance after sugar depletion. In contrast, commercial yeast starter cultures are usually used in conventional winemaking to accelerate and stabilize fermentation by ensuring the dominance of a specific strain of S. cerevisiae throughout the fermentation process. Indeed, the prevalence of beneficial yeasts at an adequate population cannot be guaranteed in natural wine fermentation. Thus, the molecular characterization of the types of yeast and bacteria, as well as their populations, during natural wine fermentation is critical for understanding the dynamic changes that occur during vinification and monitoring both beneficial as well spoilage bacteria and yeasts, especially under sulfite-free conditions where stability may be challenged [10,21].
In this study, we combined the enumeration, isolation and identification of indigenous microbial cultures from different varieties of organic grapes, as well as must and naturally fermented wine of the local red grape variety “Limniona” of Thessaly, Greece. Plate count methods were applied to quantify yeast and bacterial populations across various fermentation stages. For microbial identification, Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) was employed, offering rapid and cost-effective species-level identification with potential strain-level differentiation through Main Spectra Projection (MSP) dendrogram clustering. Complementary 16S rRNA gene amplicon sequencing via next-generation sequencing (NGS) enabled the comprehensive profiling of bacterial communities, allowing for the tracking of microbial succession during fermentation, detection of spoilage organisms, and identification of beneficial taxa.
Together, these approaches provide deeper insights into the microbial ecology of natural wine fermentation, supporting targeted strain selection, improved fermentation management, and the optimization of wine quality, particularly under sulfite-free enological practices.

2. Materials and Methods

2.1. Sample Collection

Samples were collected from Kontozisis Winery, located in the Karditsa region of Central Greece and transported at <5 °C, and stored at −80 °C until processing. Grape, must, and wine samples of the Limniona variety were split into two portions: one was analyzed immediately by plating on selective media, and another was stored at −80 °C for subsequent DNA extraction. This ensured that microbial enumeration was not influenced by freezing, while DNA integrity was preserved for sequencing. The sampling process encompassed key stages of the winemaking process to capture the microbial dynamics and diversity from the vineyard throughout wine fermentation. Specifically, the sampling included organic grape material collected at two ripening stages: (i) immature grapes (collected at the pre-veraison stage, approximately 4 weeks before harvest) and (ii) mature grapes at harvest. Limniona red grapes were obtained from two local regions: Zisi and Polia.
In addition, sequential samples were collected from Limniona winemaking at different fermentation stages: initial must at the beginning of alcoholic fermentation, fermenting must at mid-fermentation, and red wine at the end of fermentation. Final Limniona wine samples were obtained from either stainless-steel (inox) or traditional clay fermentation vessels (“pythari”), both before and after filtration and bottling. Sediment from the clay vessel was also sampled.
Spontaneous wine fermentations were carried out without starter cultures, and no sulfur dioxide (SO2) was added throughout the fermentation process. This comprehensive sampling allowed for an in-depth analysis of microbial succession and its potential influence on the sensory characteristics of the finished product.

2.2. Sample Analysis

2.2.1. Microbiological Analysis

Bacterial and yeasts counts were performed on each sample in triplicate using the standard plate count method with selective media under specific incubation conditions. The results were expressed as colony-forming units (CFU) per gram of sample. To prepare samples, 10 g of sample was aseptically homogenized in 90 mL of sterile Maximum Recovery Diluent (MRD, Oxoid, Basingstoke, UK). This was followed by tenfold serial dilutions in 9 mL of sterile MRD.
The following groups of microorganisms were analyzed in triplicate for each sample:
(i)
(Presumptive Lactococci [Gram-positive, catalase-negative] were enumerated on M17 agar (Oxoid, Basingstoke, UK) at 37 °C, for 48 h.
(ii)
Presumptive Lactobacilli [Gram-positive, catalase-negative rods] were cultured on acidified MRS agar (pH 5.4; Oxoid, Basingstoke, UK) under a microaerophilic atmosphere (<1% O2, 10% CO2) at 37 °C, for 48 h in a Bactron 300-2 (Sheldon Manfacturing Inc. Cornelius, OR, USA).
(iii)
Yeasts and molds were cultured on Potato Dextrose Agar (PDA) (Oxoid, Basingstoke, UK) at 21 °C for five days and on Wort Agar (Oxoid, Basingstoke, UK) at 25 °C for five days.
To prevent fungal contamination, 100 μg/mL of cycloheximide (Sigma-Aldrich, Darmstadt, Germany) was added to both MRS and M17 agar media [22]. After incubation, colonies were counted, and results were reported as the logarithm of mean CFU per gram.

2.2.2. Bacterial Isolation from Selective Media

Representative colonies from MRS, M17, PDA and Wort agar plates were randomly selected, transferred to Nutrient broth (Oxoid, Basingstoke, UK), and incubated at 30 °C for 24 h. These colonies were purified by streaking onto Nutrient agar plates (Oxoid, Basingstoke, UK), incubating at 30 °C for 24 h, and then transferring individual colonies back to MRS broth for an additional 24 h of incubation. Purified colonies were preserved in a mixture of Nutrient broth and 5% (v/v) glycerol (Sigma-Aldrich, Darmstadt, Germany) at a ratio of 2:1 and stored at −80 °C. Before MALDI-TOF MS analysis, each isolate was sub-cultured on Tryptone Soy Agar (TSA) (Oxoid, Basingstoke, UK) for 24 h at 37 °C.

2.3. MALDI—TOF MS Analysis

Culturable isolates were identified through MALDI-TOF MS using the MALDI Microflex LT system (Bruker Daltonics, Bremen, Germany) [22,23]. A modified mild protein extraction protocol was employed to enhance spectrum quality. Specifically, a single colony from freshly grown isolates was directly spotted onto a 96-spot steel MALDI target plate. Subsequently, 1 μL of 70% formic acid (PENTA) was applied to each target and allowed to air-dry at room temperature. This was followed by the application of 1 μL of a saturated solution of α-cyano-4-hydroxycinnamic acid (HCCA) (Bruker Daltonics, Bremen, Germany), which was co-crystallized at room temperature.
Protein profiles were acquired in linear positive mode with a laser frequency of 20 Hz. Raw spectra were automatically captured using the AutoXecute control software (FlexControl 3.4; Bruker Daltonics, Bremen, Germany) and recorded within the range of 2000–20,000 Da. Identification was performed using the MALDI Biotyper Software, version 4.0, with default settings. Acquired spectra were compared against the reference mass spectral library (6093 MSPs). External calibration was conducted using the Bruker Bacterial Test Standard (BTS), an extract of E. coli DH5α spiked with RNAase A and myoglobin to extend the upper mass range.
Results were classified according to the modified score values recommended by the manufacturer. A score between 0.000 and 1.699 indicated unreliable identification; a score from 1.700 to 1.999 indicated probable genus-level identification; a score from 2.000 to 2.299 indicated secure genus and probable species-level identification; and scores from 2.300 to 3.000 indicated highly probable species-level identification.
To cluster the most frequently isolated strains, an MSP dendrogram was constructed based on their protein profiles. Each acquired spectrum was subjected to baseline subtraction and smoothing procedures and subsequently processed using the MALDI Biotyper Offline Classification 4.0 software under default settings for MSP creation.

2.4. NGS-DNA Extraction and Microbial Community Profiling

Microbial community profiling was carried out for unfiltered and filtered natural Limniona wine from inox and clay vessels, as well as samples from the sediment of clay fermentation vessel at the end of fermentation. In addition, two bottled wines with atypical characteristics (i.e., wines with off-flavors or sensory deviations like oxidation, reduction, or other spoilage notes) were analyzed, namely a “Limniona” red wine, as well as a “Malagouzia” white wine from the previous vintage and from stainless-steel vessels. This was performed to identify potential spoilage indicators that can be present in red or white wines of the same producer, which are absent in natural (Limniona) wine with typical (normal) sensory characteristics. NGS was performed using the Ion 16S™ Metagenomics Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions, without prior DNA extraction. DNA concentration and purity were assessed using a Qubit™ 4 Fluorometer (Thermo Fisher Scientific). Sequencing targeted the hypervariable regions V2, V3, V4, V6–7, V8, and V9 of the 16S rRNA gene on the Ion GeneStudio™ S5 System (Thermo Fisher Scientific). Two separate PCR reactions per sample were performed with primer pools targeting V2, V4, V8 (Pool 1) and V3, V6–7, V9 (Pool 2), and equal volumes were combined. Libraries were prepared using the Ion Plus Fragment Library Kit™ and Ion Xpress™ Barcode Adapters, purified with AMPure XP beads (Beckman Coulter, Indianapolis, IN, USA), and assessed for quality on an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Libraries were normalized to 50 pM, pooled equimolarly, and sequenced on an Ion 316™ v2 chip using the Ion 400™ sequencing kit, with template preparation on the Ion Chef™ System (Thermo Fisher Scientific, Waltham, MA, USA). Base calling and demultiplexing were performed using Torrent Suite™ Software v4.4.2, and FASTQ files were generated with FileExporter v4.4.0.0. Taxonomic classification was performed using the Ion Reporter™ Metagenomics 16S workflow against the curated Greengenes database (version 13_8) and the MicroSEQ® ID 16S rRNA database. A minimum sequencing depth of 50,000 reads per sample was achieved.

2.5. Statistical Analysis

All experiments for microbial enumeration were performed in triplicate, and results are expressed as mean values ± standard error (SE). Statistical differences among groups were assessed using one-way ANOVA in Microsoft Excel (Office 365). Post hoc comparisons were conducted using Tukey’s Honestly Significant Difference (HSD) test, with significance set at p < 0.05.

3. Results

3.1. Microbial Populations

The microbiological populations cultured on MRS agar, M17 agar, Potato Dextrose agar, and Wort agar are presented in Table 1 and Table 2. These include data from two immature grape samples (Limniona Zisi, Limniona Polia), mature grapes at harvest, initial must before fermentation, must at different fermentation stages, and samples collected from the traditional clay fermentation vessel at the end of fermentation.
Notably, the populations of either LAB or yeasts were very low in all immature grapes that were tested (Table 1), probably due to their high acidity and lack of fermentable sugars at this stage. LAB counts on M17 and MRS agar ranged from 1.1 log CFU/g to 1.39 ± 0.36 log CFU/g. Similarly, yeasts counts on PDA and WORT agar ranged from 1.44 log CFU/g to 1.83 ± 1.44 log CFU/g.
The region seemed to play some role in the population levels, as in the case of Limniona grapes from Polia, which had measurable populations of Lactobacilli and yeasts, while in Limniona grapes from Zisi region, the corresponding counts in MRS and Wort agar were below 1 log CFU/g (Table 1).
The microbial communities cultured from mature grapes at harvest, initial must prior to fermentation, must during fermentation, and sample collected from the traditional clay fermentation vessel, exhibited significant variations across the different sample types and substrates, with statistical significance observed at both p < 0.05 and p < 0.01 levels (Table 2).
Lactococci on M17 agar ranged from 4.24 ± 0.06 log CFU/g to 7.49 ± 0.09 log CFU/g, while those on MRS agar varied 4.25 ± 0.07 to 7.68 ± 0.27 log CFU/g. Similarly, Yeasts on PDA agar ranged from 4.28 ± 0.09 log CFU/g to 7.50 ± 0.04 log CFU/g, and those on Wort agar ranged from 4.28 ± 0.07 log CFU/g to 7.65 ± 0.39 log CFU/g.
Statistically significant differences in microbial populations were observed among samples collected at different fermentation stages across all substrates used for enumeration. Microbial populations cultured on M17 agar displayed statistically significant differences (p < 0.01) across all sample comparisons, including grape samples, initial must, fermenting must, and unfiltered wine (with sediment). Similarly, populations grown on MRS agar exhibited significant differences (p < 0.01) across most comparisons; however, no statistically significant differences were observed between mature grape samples and initial must, or between mature grape samples and unfiltered wine.
Yeasts enumarated on PDA agar exhibited significant variations (p < 0.01) across all sample types, with the exception of the comparison between grape samples and unfiltered wine, which showed a weaker but still statistically significant difference (p < 0.05). Yeasts cultured on WORT agar also showed significant differences (p < 0.01) in most sample comparisons, although no significant differences were detected between mature grape samples and initial must, between mature grape samples and unfiltered wine, or between initial must and unfiltered wine.

3.2. MALDI—TOF MS Identification

In total, 72 isolates were submitted to MALDI-TOF MS for identification. The spectral profile of the unknown strains was compared to the spectra of reference strains 1.754 to 2.223, indicating probable to secure identification at the genus and species level, according to standard MALDI-TOF MS interpretation criteria. The isolated strains were identified as seven distinct species, including both yeasts—Candida lusitaniae, Candida krusei, and Saccharomyces cerevisiae—and bacteria—Bacillus amyloliquefaciens ssp. plantarum, Staphylococcus epidermidis, Serratia marcescens, and Klebsiella aerogenes. The results of MALDI-TOF identification concerning genus and species identification, the number of the isolates classified as belonging to each species, as well as the score values recorded are shown in detail in Table 3.
MALDI-TOF MS profiling revealed diverse microbial communities across distinct stages of the winemaking process, with identification scores ranging from 1.754 to 2.298, reflecting variable resolution accuracy. In ripe Limniona grapes, Candida krusei yielded high-confidence scores, while Candida lusitaniae, Staphylococcus epidermidis, and Bacillus amyloliquefaciens were detected with lower reliability. In the initial must, Saccharomyces cerevisiae was identified, although all scores remained below the species-level confidence threshold. In contrast, Serratia marcescens and Klebsiella aerogenes were identified with high-certainty scores. Throughout fermentation and in the unfiltered wine samples, S. cerevisiae dominated; however, it consistently exhibited lower identification scores, highlighting the method’s strength in bacterial discrimination and its limitations in yeast strain-level resolution.
The MSP dendrograms of Saccharomyces cerevisiae isolates across different fermentation stages demonstrate progressive shifts in strain-level diversity. In the initial must (Figure 1a), isolates formed three closely related clusters, indicative of low diversity and the early establishment of a limited number of dominant strains, likely originating from the vineyard environment or winery surfaces. During active fermentation (Figure 1b), diversity increased moderately, with most isolates forming a compact cluster suggestive of a predominant strain, while isolates 47 and 48 exhibited substantial proteomic divergence, indicating the presence of genetically distinct or potentially wild-type variants. In the unfiltered wine sample (Figure 1c), a higher degree of intraspecific variation was observed. Isolates 61 and 62 displayed marked dissimilarity compared to the rest, while the remaining isolates grouped into two well-defined subclusters, reflecting the persistence or emergence of multiple strains during the late stages of fermentation. These findings underscore the dynamic nature of S. cerevisiae populations under fermentative selection pressures.

3.3. Next-Generation Sequencing

The high-throughput sequencing of the 16S rRNA gene was employed to characterize the microbial communities associated with wine samples. This method enabled the detailed taxonomic profiling of bacterial populations in unfiltered wine from traditional clay fermentation vessels, filtered wine from traditional clay fermentation vessels, bottled wine fermented in traditional clay vessels, unfiltered wine from stainless-steel (inox) fermentation vessels, filtered bottled wine fermented in stainless-steel (inox) vessels, and filtered bottled wine derived from stainless-steel (inox) vessel from the previous vintage (2024). In addition to the above wine samples which had no signs of off-flavors, another two bottled wines (of “Limniona” and “Malagouzia” grape variety) from the previous vintage and stainless-steel (inox) vessels, with atypical characteristics (off-flavors), were also analyzed in order to identify potential consistent markers of spoilage or undesirable fermentation that may exist not only in Limniona wine, but also in natural wines from other grape varieties. These analyses could provide insights into microbial diversity and the relative abundance of taxa across multiple hierarchical levels, from the phylum to the genus.
Fermentation in traditional clay vessels results in a distinct microbial profile across different stages of wine production, as revealed by 16S rRNA gene sequencing (Table 4). In the sediment from clay vessel, Oenococcus oeni was the dominant species (30.6%), indicating early malolactic activity. This was accompanied by several Arcobacter species, including A. venerupis (8.78%), A. defluvii (7.65%), and A. ellisii (2.78%), which may have been introduced from environmental or water-related sources. The wine samples from clay vessels showed a shift toward Oenococcus spp. (22.72%) and Oenococcus oeni (19.48%), reflecting ongoing malolactic fermentation. Burkholderia spp. (16.5%) were also prominent, suggesting an environmental reservoir or an association with the clay material. The presence of Pediococcus parvulus in unfiltered wine from clay vessels (6.11%), which is a species known for exopolysaccharide production, may have implications for wine texture and stability. In the bottled wine, lactic acid bacteria became dominant, with Lactobacillus spp. accounting for 29% of the total reads. Notably, Lentilactobacillus. parafarraginis (25.1%) and Lentilactobacillus diolivorans (13.09%) were well represented, likely due to their stress tolerance and role in continued malolactic activity. A small proportion of Oenococcus oeni (1.37%) and Burkholderiaceae (3.14%) persisted, indicating selective survival in the bottled environment.
Table 5 presents the microbial composition of wine samples fermented in stainless-steel (inox) vessels, revealing distinct profiles between the wine from the vessel and bottled wine stages based on 16S rRNA gene sequencing. In the wine sample from the vessel, Acetobacter spp. dominated (39%), followed by Burkholderia spp. (23.73%), with minor contributions from Rhizobium spp. (5.30%) and Pseudoalteromonas spp. (3.92%), indicating potential environmental introduction. In the bottled wine, Burkholderia remained prevalent (34%), while the presence of Acetobacter decreased to 17%, possibly due to reduced oxygen exposure or antimicrobial treatments. Notably, Oenococcus oeni appeared at 5.53%, suggesting post-fermentation malolactic activity. Low levels of Bacillus (1.91%) and Lentilactobacillus diolivorans (1.5%) were also detected, likely representing residual or stress-tolerant populations.
In order to compare successful and less successful spontaneous fermentations of natural wine (with either typical–desirable, or atypical–undesirable sensory and especially flavor characteristics) and the corresponding microflora that prevails, the taxonomic composition of microbial communities in wine samples from stainless-steel (inox) fermentation vessels of the previous vintage (2024) was assessed using 16S rRNA gene sequencing, revealing the clear dominance of Lactobacillus taxa across all samples (Table 6). In the standard bottled wine with typical sensory characteristics (no off-flavors), Lactobacillus spp. constituted 39% of valid reads, followed by Lentilactobacillus parafarraginis (19.19%) and Lentilactobacillus diolivorans (14.11%), with minor representation of Oenococcus oeni (1.97%) and Burkholderia spp. (1.68%). The bottled Limniona sample, exhibiting atypical sensory characteristics, displayed a comparable microbial profile, with high abundances of Lactobacillus spp. (30.6%), Lentilactobacillus parafarraginis (28.16%), and Lentilactobacillus diolivorans (10.17%), along with Burkholderia spp. (2.37%). In contrast, the sample of bottled Malagouzia white wine with sensory deviation was strongly dominated by L. parafarraginis (63.16%), accompanied by Acetobacter spp. (11%), Lactobacillus spp. (2%), and O. oeni (2.07%). These findings suggest that elevated levels of Lentilactobacillus parafarraginis, particularly in conjunction with other Lactobacillus species, may be associated with the emergence of atypical sensory profiles, underscoring the potential influence of microbial dynamics on wine stability and sensory quality.
The 3D PCoA plot shown in Figure 2, derived from Bray–Curtis dissimilarity, highlights clear differences in microbial community structure among wine samples, with the first three axes explaining a combined 91.45% of the variation (PC1: 55.16%, PC2: 23.05%, PC3: 13.24%). A tight cluster of samples on the right side of the plot—comprising the green, purple, pink, and yellow points—represents bottled wines, including those with atypical sensory characteristics (Limniona and Malagouzia). Their proximity indicates a high degree of similarity in microbial composition, despite sensory deviations. This suggests that sensory faults may not always correspond to major shifts in the overall microbial community. In contrast, the red (bottled wine from inox vessel), blue (sediment from clay vessel), orange (inox wine), and cyan (clay wine) samples are distinctly separated from the cluster, especially along PC1 and PC2. This reflects substantial microbial divergence, likely driven by fermentation vessel (clay vs. inox) and wine stage (sediment, wine from vessel, bottled). The red and orange samples (from inox) show strong separation, consistent with a different microbial signature influenced by higher oxygen exposure. Similarly, the blue and cyan samples (from clay) appear independently positioned, highlighting the influence of clay vessel microenvironments and sediment-associated microbiota. Overall, the analysis confirms that fermentation environment and processing stage strongly influence microbial diversity, while samples with atypical sensory traits (off-flavors) do not differ drastically in overall microbial communities from other bottled wines of desirable sensory properties. Due to the limited number of samples, the PCoA should be viewed as an exploratory visualization of microbial trends rather than a test of statistical significance.

4. Discussion

4.1. Microbial Succession and Community Dynamics During Winemaking: Insights from Selective Media Profiling

Microbial profiling of immature grapes revealed low but detectable microbial counts, with some differences between varieties (Table 1), supporting the idea that grape microbiota are shaped by cultivar traits, vineyard conditions, and environmental exposure [24,25,26,27]. According to Renouf et al. (2007), yeast and lactic acid bacteria (LAB) populations increase notably during ripening, especially at veraison, under the influence of grape variety and vineyard management [28].
Contrary to general assumptions, fermentative Saccharomyces species (e.g., S. cerevisiae) are rarely abundant on intact, healthy grapes and are seldom isolated from undamaged berries or vineyard soils [24,29]. Typically, only 10–103 CFU/g of yeasts is found on immature berries, increasing to 104–106 CFU/g as ripening progresses [30]. According to Martini, Ciani, and Scorzetti [29], different grape varieties exhibit distinct yeast communities in terms of population size, typically ranging from 1 × 104 to 1 × 106 cells/mL [31]. Similarly, mature grape berries generally support dense microbial populations of 104–106 CFU/g, primarily consisting of yeasts, lactic acid bacteria, and acetic acid bacteria [27].
The population lactic acid bacteria as well as yeasts differed between Limniona Zisi and Limniona Polia regions, as well as between MRS and M17 agar (for LAB) or PDA and Wort agar (for yeasts), as shown in Table 1. This underlines the variance in the microbial composition of grapes of the same variety from different regions and the importance of using multiple culture media in enological microbiological profiling [29].
Microbial enumeration throughout the vinification process using four selective agars revealed clear shifts in population levels, illustrating the dynamic succession of microbial communities. Counts ranged from 4.2 to 7.7 log CFU/g (Table 2), with statistically significant differences depending on the sample stage and culture medium (p < 0.05, p < 0.01).
As expected, fermenting must yield the highest microbial counts, reaching 7.68 ± 0.27 log CFU/g on MRS and 7.65 ± 0.39 on Wort agar (Table 2), reflecting the rapid expansion of fermentative yeasts and associated taxa, driven by high sugar content, warm temperatures, and anaerobic conditions [24,25,28,31]. Saccharomyces cerevisiae and related fermentative yeasts ultimately prevail during alcoholic fermentation by outcompeting oxidative and non-fermentative species. This dominance is attributed to their high ethanol and stress tolerance, rapid sugar uptake, and superior fermentative capacity under anaerobic conditions [29,32].
By contrast, initial must and ripe grape samples exhibited lower microbial levels (4.24 ± 0.06 to 5.35 ± 0.06 log CFU/g) (Table 2), consistent with a diverse but sparse epiphytic community on freshly harvested grapes. S. cerevisiae and LAB are usually scarce on intact berries but increase during processing, mainly due to winery-associated contamination [24,28]. Yeast populations on grape surfaces have been observed to vary significantly among cultivars, typically ranging from 104 to 107 cells/mL. This natural variation is influenced by several factors, including vineyard microclimate and terroir, viticultural management practices, and the timing of harvest and grape maturity, all of which affect microbial colonization and growth dynamics [24,25,33,34].
The unfiltered wine samples showed intermediate microbial levels (Table 2), indicating a decline in viable populations post-fermentation, likely due to nutrient depletion, ethanol accumulation, and other inhibitory conditions [29]. Statistical analysis for presumptive lactococci enumerated on M17 agar confirmed significant differences among production stages (p < 0.01), while presumptive lactobacilli on MRS agar also showed strong variability—except between grapes and must or grapes and unfiltered wine—suggesting gradual shifts in LAB populations. Yeast counts on PDA agar increased from grape to fermenting must, in a statistically significant manner, although yeast populations declined at the end of fermentation, having little difference in the final (unfiltered) wine compared to initial must. Wort agar displayed a similar pattern, but with no significant differences between grape, must, and final (unfiltered) wine samples, indicating lower selectivity or overlapping yeast viability (Table 2).
Overall, these results support the concept of microbial succession, where the community transitions from diverse, low-abundance epiphytes to a dominant fermentative microbiota during winemaking [33,35]. Moreover, they highlight the importance of using multiple culture media to capture the full spectrum of microbial diversity and reinforce the relevance of microbiological monitoring to optimize fermentation outcomes.

4.2. Microbial Diversity Across Winemaking Stages

While culture-dependent methods remain valuable for isolating and characterizing grape-associated microbes and assessing their metabolic functions in vitro, they capture only a fraction of the total microbiome, overlooking 95–99% of non-culturable organisms [28,30]. However, culture-dependent methods for microbial characterization, like MALDI-TOF MS, are rapid, reliable, and have the benefit of identifying readily culturable species that could be used as potential starter cultures, or as quality indicators when monitoring the vinification process of (natural) wine.
MALDI-TOF MS profiling in this study provided important insights into the shifting microbial communities during different stages of the winemaking process, capturing both bacterial and yeast populations. Analysis of 72 isolates led to the identification of seven distinct microbial species, with a range of identification scores highlighting both the capabilities and limitations of the technique (Table 3).
Grapes host a diverse microbiome shaped by environmental conditions, location, grape variety, and vineyard phytochemicals, all of which affect fermentation outcomes and microbial composition [34]. The microbial community present on ripe Limniona grapes included both yeasts and bacteria, such as Candida lusitaniae, C. krusei, Staphylococcus epidermidis, and Bacillus amyloliquefaciens subsp. plantarum, reflecting a complex epiphytic microbiota inhabiting the grape surface. Notably, C. krusei consistently yielded high MALDI-TOF MS scores (>2.0), indicating reliable species-level identification. In contrast, Candida lusitaniae and Staphylococcus epidermidis exhibited scores below 2.0 in several isolates, indicating reduced confidence in species-level identification (Table 3).
Studies indicate that Saccharomyces cerevisiae is uncommon on healthy grapes, which are primarily colonized by oxidative yeasts like Rhodotorula and alcohol-sensitive species such as Kloeckera apiculate. These species often dominate the yeast community [35]. Yeasts are ubiquitous in nature, typically forming structured communities within specific ecological niches. In the context of winemaking, the surface of grape berries represents a primary natural reservoir for diverse yeast populations [29]. Kloeckera apiculata is the most frequently isolated native yeast, comprising over 50% of grape skin isolates, followed by Candida species (~30%), alongside Aureobasidium, Cryptococcus, Kluyveromyces, Metschnikowia, Pichia, and Rhodotorula [35]. The sugar-rich grape surface favors oxidative or weakly fermentative yeasts such as Candida, Hanseniaspora, Metschnikowia, and Pichia [35]. Candida species are common members of the natural microflora found on grape berries. In another study, five Candida species—C. valida, C. utilis, C. sorbosa, C. krusei, and C. saitoana—were identified along with four Saccharomyces cerevisiae strains, emphasizing the dominance of Candida among the isolates [34].
According to Koulougliotis and Eriotou [36], Rhodotorula glutinis (27.4%) was the most frequently detected yeast on grape samples, followed by Candida lusitaniae (18.3%) and Cryptococcus laurentii (13.3%) [37]. Candida lusitaniae was also found with related species, including Clavispora santaluciae, C. fructus, and several other Candida spp. [38]. Clavispora lusitaniae (previously Candida lusitaniae) was initially isolated from the gastrointestinal tract of warm-blooded animals and has since been recovered from a variety of environmental and food-related sources, including cornmeal, citrus peel, fruit juices, and milk from cows affected by mastitis [39]. Although GenBank holds grape-associated C. lusitaniae sequences, no peer-reviewed studies have confirmed these findings, suggesting the need for further investigation [36]. Non-Saccharomyces yeasts contribute to ester formation but can also cause spoilage, particularly during early fermentation. Species of Candida are known to oxidize ethanol, producing elevated levels of acetaldehyde, volatile acids, and esters. Ethyl acetate concentrations above 200 mg/L and acetic acid above 0.6 g/L are associated with sensory defects in wine [40].
A survey of grape samples revealed that Staphylococcus spp. constituted 11% of bacterial isolates, alongside Leuconostoc mesenteroides, Micrococcus luteus, Bacillus megaterium, and Lactobacillus paracasei, indicating notable species diversity [25]. Recently, uncommon bacteria such as Citrobacter freundii, Klebsiella oxytoca, Enterobacter ludwigii, Serratia marcescens, Enterococcus spp., and Staphylococcus spp. were detected on grapes and persisted into alcoholic fermentation [41]. Staphylococcus epidermidis, S. equorum, S. hominis, and S. warneri have been isolated from table grapes, confirming the presence of diverse Staphylococcus species in the grape surface microbiota [30,42]. Staphylococcus epidermidis, a common member of the human skin microbiota, is not typically associated with wine fermentation but may appear as a contaminant due to poor hygiene, handling practices, or inadequate equipment sanitation [43].
Bacillus spp. are resilient environmental bacteria capable of surviving extreme conditions via protective structures. Bacillus amyloliquefaciens is a soil-dwelling bacterium that is widely distributed in natural and agricultural environments. It is widely applied in agriculture for its antimicrobial enzymes and bioactive compounds [44,45,46]. Bacillus mycoides has been associated with environmental contamination in vineyard regions [44]. In contrast, Bacillus amyloliquefaciens has been isolated from withered grapes, with 41 out of 50 bacterial isolates identified as Bacillus, indicating dominance [45]. Bacillus amyloliquefaciens subsp. plantarum and strain G1, isolated from grapes, show strong biocontrol potential against plant diseases and downy mildew [46,47,48]. Additionally, table grapes have been found to harbor Bacillus megaterium, B. niacini, and B. cereus, highlighting the diversity of Bacillus species present on grape surfaces [34]. Bacillus amyloliquefaciens does not play a beneficial role in wine fermentation, and its presence is primarily associated with environmental contamination. Although not among the most common spoilage organisms in wine, it may contribute to sensory or visual defects under certain conditions [37].
Spontaneous wine fermentation is driven by the indigenous microbiota naturally present on grapes and within the winery environment. At the onset of fermentation, yeasts from the genera Metschnikowia, Candida, Hanseniaspora, Pichia, Lachancea, and Saccharomyces are prevalent. As ethanol increases, Saccharomyces cerevisiae becomes dominant and drives alcoholic fermentation. Non-Saccharomyces yeasts are typically grouped as (1) aerobic species (e.g., Pichia, Debaryomyces, Rhodotorula, Candida, Cryptococcus), (2) weakly fermentative apiculate yeasts (e.g., Hanseniaspora uvarum), and (3) fermentative species (e.g., Kluyveromyces marxianus, Torulaspora delbrueckii). These yeasts appear early—especially H. uvarum, which often appears in red musts—but often decline due to stress factors and competition from S. cerevisiae. Beyond their impact on wine aroma and composition, non-Saccharomyces yeasts can also affect lactic acid bacteria (LAB). During early fermentation, they may deplete nutrients and produce inhibitory metabolites that hinder LAB growth and malolactic fermentation [30,38]. However, some yeast by-products may also promote LAB activity, highlighting their complex role in microbial dynamics [30,38]. Saccharomyces cerevisiae strains are key to wine quality, with their diversity during spontaneous fermentation impacting flavor and composition. Selected strains are widely used as starters to ensure consistent and successful alcoholic fermentation [34].
In the must of Limniona red grapes, Saccharomyces cerevisiae was identified with low confidence (all isolates < 2.0), despite its central role in fermentation, but as fermentation progressed, S. cerevisiae became the dominant species, reflecting selective pressures favoring ethanol-tolerant yeasts (Table 3). Yet, the majority of isolates continued to score below the 2.0 threshold, confirming the technique’s limited strain-level resolution within this species. In the unfiltered wine sample, Saccharomyces cerevisiae was recovered from 23 isolates, with MALDI-TOF MS scores ranging from 1.793 to 2.004. Of these, 21 isolates yielded scores below the species-level confidence threshold of 2.000, suggesting limited strain differentiation and the need for complementary identification methods (Table 3).
These findings are consistent with those of Jeune [49], and they challenge earlier claims by Martini [50] that Saccharomyces cerevisiae is restricted to the winery environment and that winery-associated strains consistently outcompete indigenous yeasts. Additionally, S. cerevisiae strains from 12 Apulian musts showed strong genetic similarity, indicating that vineyard-specific environmental factors (terroir) influence native yeast populations more than grape variety [51].
Bacterial isolates from the initial must and fermenting must, such as Serratia marcescens and Klebsiella aerogenes, scored above 2.2, confirming the method’s robustness in bacterial taxonomy. The detection of S. marcescens and K. aerogenes during fermentation further suggests microbial persistence or adaptation, despite increasing ethanol concentrations. These opportunistic species, although not typical wine-associated microbes, may originate from winery equipment or grape handling practices [6]. Klebsiella oxytoca and Serratia marcescens have been isolated from grapes but were not detected during the middle and final stages of the fermentation process [42].

4.3. Intraspecific Variation in Saccharomyces Cerevisiae

The MSP dendrogram analysis provided enhanced insight into the diversity of Saccharomyces cerevisiae. In the initial must (Figure 1a), clustering patterns indicated low strain diversity, supporting the view that early fermentation stages are typically dominated by a limited number of strains originating from the vineyard or winery environment [7,52]. In contrast, samples from the active fermentation phase (Figure 1b) revealed a dominant strain cluster alongside several divergent isolates [41,42], reflecting the dynamic nature of spontaneous fermentations and suggesting the involvement of wild-type strains [53].
In the unfiltered wine (Figure 1c), a greater diversity of strains was observed, with multiple isolates forming distinct clusters. This pattern aligns with previous findings showing that spontaneous fermentations promote a succession of S. cerevisiae strains, shaped by gradients in ethanol concentration, nutrient and oxygen availability [31,52]. The emergence of proteomically distinct isolates at later stages may reflect adaptive responses of minority strains to environmental stressors or indicate the survival of diverse populations maintained through mechanisms such as biofilm formation or spatial separation [53,54,55,56].

4.4. Taxonomic Profiling and Microbial Diversity in Wine Samples via 16S rRNA NGS

Spontaneous wine fermentation is driven by indigenous microbiota, with species of Metschnikowia, Candida, Hanseniaspora, Pichia, Lachancea (formerly Kluyveromyces), and Saccharomyces dominating the initial stages. As ethanol levels rise, Saccharomyces cerevisiae becomes predominant, completing alcoholic fermentation. Concurrently, malolactic fermentation (MLF), carried out by lactic acid bacteria (Oenococcus, Pediococcus, Lactobacillus, and Leuconostoc), converts malic acid into lactic acid, enhancing the wine’s sensory properties and stability. Conversely, acetic acid bacteria (AAB) can negatively impact wine quality by producing acetic acid and other spoilage metabolites [57].
The evolution of microbial and, in particular, bacterial communities during wine fermentation is marked by dynamic successions that shape both the biochemical profile and sensory characteristics of the final product. Our findings, derived from 16S rRNA gene sequencing, reveal distinct microbial trajectories in traditional clay vessels compared to stainless-steel (inox) fermenters, with clear implications for wine stability, quality and the risk of spoilage.
In clay vessels, the fermentation process exhibited a well-structured bacterial succession, transitioning from environmental taxa to communities dominated by lactic acid bacteria (LAB). Oenococcus oeni emerged as the predominant taxon in both early (30.6%) and mid-fermentation (19.5%) stages. This aligns with its well-documented role as the primary driver of malolactic fermentation (MLF) due to its resilience in the acidic, ethanol-rich conditions characteristic of wine [31]. The concurrent detection of Pediococcus spp. (22.7%) in wine sampled in the middle of the fermentation highlights the potential for co-occurrence with O. oeni, although this genus is also associated with wine spoilage through the production of diacetyl, exopolysaccharides, and biogenic amines [58].
By the bottling stage, the microbial profile was dominated by LAB, particularly Lentilactobacillus parafarraginis (25.1%) and Lentilactobacillus diolivorans (13.09%), suggesting the successful completion of MLF and microbial stabilization [51,59]. These species are known for their heterofermentative metabolism, contributing to aromatic complexity but also capable of generating undesirable compounds such as acetic acid and diacetyl when overrepresented [31].
In contrast, fermentations conducted in inox vessels showed the early dominance of Acetobacter spp. (39%) and Burkholderia spp. (23.73%), indicating the likely influence of oxygen presence and the lack of anaerobic conditions, or the surface-related contamination of inox vessels—conditions favorable for the undesirable acetic acid bacteria [60], as well as the contamination of equipment of winery environment from soil microorganisms or microorganisms naturally dwelling in healthy grapes like Burkholderia [61]. These are not linked to wine spoilage; instead, they may contribute to grape bioprotection from fungal pathogens [62]. The continued presence of Burkholderia at 34% in bottled samples suggests a less efficient microbial transition from grape microbiota towards a wine-adapted community, potentially due to the reduced porosity and inert surface of stainless steel, which limits microbial niche development [60].
In bottled wines from previous vintages, LAB remained prominent, particularly Lentilactobacillus parafarraginis, which was notably abundant in wines with atypical sensory profiles, reaching 63.16% in Malagouzia and 28.16% in Limniona wine. This overrepresentation raises concerns, as Lentilactobacillus parafarraginis is associated with the production of acetic acid and diacetyl, leading to off-flavors such as buttery or vinegary notes [59,60,61,62,63,64,65]. The sporadic presence of Acetobacter and Burkholderia in these wines further supports the possibility of oxidative stress or microbial imbalance contributing to quality deterioration [63].
Our observations are in line with previous research indicating that elevated LAB populations—particularly heterofermentative species—can result in the accumulation of undesirable metabolic byproducts such as acrolein (contributing to bitterness), ethyl carbamate precursors, and “mousy” off-flavors [59,64].
Compared to studies on spontaneous fermentations from six Portuguese appellations, where Proteobacteria accounted for over 60% and Firmicutes were secondary [31], our results suggest a more balanced distribution of these phyla. Notably, Lactobacillus and other Lactobacillaceae dominated fermentations conducted without sulfite additions or starter cultures, reflecting a trend toward indigenous microbial influence in the absence of enological intervention. Previous studies have consistently reported the dominance of O. oeni during MLF, with other LAB genera such as Lactobacillus, Lactococcus, Leuconostoc, and Pediococcus also commonly present in wine ecosystems.
The microbial ecology observed in clay vessels appears to support a more stable and balanced succession. The early dominance of O. oeni and later establishment of LAB with minimal oxidative or spoilage taxa suggests that the porous and micro-oxygenating nature of clay may foster a conducive environment for beneficial microbial development [31]. In contrast, inox fermentation systems exhibited delayed microbial stabilization, with persistent environmental taxa and increased risk of LAB overgrowth, particularly as regards L. parafarraginis.
These findings underscore the significant influence of vessel type on fermentation microbiota. While clay vessels promote microbial adaptation and succession aligned with traditional wine fermentation trajectories, the inert conditions of inox vessel may impede this process, enhancing susceptibility to spoilage and off-flavor development, especially if sulphites are absent and anaerobic conditions are not fully guaranteed [60,65]. This finding has not been previously reported, to our knowledge, and may point to a preference for clay vessels for the production of natural wines without the use of sulphites.

5. Conclusions

The microbial dynamics observed during fermentation in traditional clay vessels suggest the potential of spontaneous fermentation to foster a stable and beneficial microbiota dominated by lactic acid bacteria, particularly Oenococcus oeni, with minimal presence of spoilage-associated taxa. In contrast, stainless-steel fermentations appeared to exhibit a higher prevalence of oxidative bacteria and environmental contaminants, indicating that vessel material may influence microbial succession. The natural progression observed in clay vessels appears more conducive to the development of well-balanced wine microbiota with reduced risk of microbial faults.
A comprehensive understanding of how fermentation vessels influence microbial ecology is essential for optimizing wine quality. To ensure consistent and controlled outcomes, especially in stainless-steel systems where microbial instability may be more likely, the use of well-characterized O. oeni starter cultures could be recommended. Additionally, sanitation protocols must be adapted to vessel characteristics—addressing the porous, micro-oxygenating environment of clay and the inert nature of inox—to minimize contamination and promote beneficial microbial growth. Molecular monitoring techniques such as 16S rRNA gene sequencing may provide early insights into shifts in microbial communities, enabling timely interventions when necessary.
Of particular concern is the management of Lentilactobacillus parafarraginis, whose overrepresentation in stainless-steel fermentations has been associated in previous studies, with excessive heterofermentation and the formation of off-flavors, including acetic acid and diacetyl. Targeted monitoring and control of this species may therefore be important for preserving the sensory integrity of the wine.
Overall, these findings provide preliminary insights into the potential challenges and support the value of spontaneous wine fermentation, especially when performed under hygienic conditions with appropriate monitoring and equipment. The gradual prevalence and increased diversity of desirable Saccharomyces cerevisiae strains at the late stages of fermentation, despite their initial scarcity in the must, and the adaptation of beneficial microbiota of malolactic fermentation, especially in clay-fermented wines, suggest that these processes may contribute to the expression of unique sensory profiles, which characterize natural wines. When guided by microbial understanding and careful process control, spontaneous fermentation with minimum interventions can potentially serve as both a viable and desirable approach to producing wines that authentically reflect origin and terroir.

Author Contributions

Formal analysis, methodology, writing—original draft preparation, P.B.; formal analysis, data curation, C.M.; conceptualization, field work, A.T.B., conceptualization, supervision-project administration, review and editing, I.G. All authors have read and agreed to the published version of the manuscript.

Funding

Funding for this research was provided by the Greek Ministry of Agricultural Development and Food under the framework of the EU Rural Development Programme (RDP) 2014–2020 (Μ16ΣΥΝ2-00421), Measure 16 ‘Cooperation’, Submeasure 16.1–16.2 ‘Establishment and operation of Operational Groups of the EIP-AGRI’, co-financed by the European Agricultural Fund for Rural Development (EAFRD) and national resources. The funder was not involved in the study design, collection, analysis and interpretation of data, the writing of this article or the decision to submit it for publication.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original data presented in the study are openly available at www.preprints.org/manuscript/202508.1970/v1 (accessed on 15 August 2025).

Acknowledgments

The authors wish to thank Kontozisis Organic Vineyards for the provision of grapes and natural wines used in the present study.

Conflicts of Interest

Author A.T.B. was employed by the company Kontozisis Organic Vineyards which kindly offered samples of grapes and wines for the study, without funding the study. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Beverages 11 00160 g001aBeverages 11 00160 g001b
Figure 1. Main Spectrum Profile (MSP) dendrograms of Saccharomyces cerevisiae isolates from different winemaking stages, based on MALDI-TOF MS profiles. (a) Initial must: low diversity, two closely related clusters. (b) Fermenting must: one dominant cluster with distinct isolates (e.g., 47, 48), indicating strain-level variation. (c) Unfiltered wine: higher diversity and complex clustering, suggesting persistence or emergence of diverse strains. Distance levels represent proteomic dissimilarity; lower values indicate greater similarity.
Figure 1. Main Spectrum Profile (MSP) dendrograms of Saccharomyces cerevisiae isolates from different winemaking stages, based on MALDI-TOF MS profiles. (a) Initial must: low diversity, two closely related clusters. (b) Fermenting must: one dominant cluster with distinct isolates (e.g., 47, 48), indicating strain-level variation. (c) Unfiltered wine: higher diversity and complex clustering, suggesting persistence or emergence of diverse strains. Distance levels represent proteomic dissimilarity; lower values indicate greater similarity.
Beverages 11 00160 g001aBeverages 11 00160 g001b
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Figure 2. Principal coordinate analysis (PCoA) of wine microbial communities based on bray–curtis dissimilarity.
Figure 2. Principal coordinate analysis (PCoA) of wine microbial communities based on bray–curtis dissimilarity.
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Table 1. Mean counts (log CFU/g ± standard deviation) of bacterial groups from two immature grape samples.
Table 1. Mean counts (log CFU/g ± standard deviation) of bacterial groups from two immature grape samples.
Immature Grape Varieties and RegionsMicrobial Groups/Substrate
Lactococci (M17 Agar)Lactobacilli (MRS Agar)Yeasts (PDA Agar)Yeasts (WORT Agar)
Limniona Zisi1.39 ± 0.36 ᵃᵇ<DL1.44 ± 0.42 a<DL
Limniona Polia1.10 ± 0.74 a1.33 ± 0.28 a1.50 ± 0.87 a1.83 ± 1.44 a
Values presented are the mean value ± SD (n = 3). Abbreviation: DL (Below Detection Limit). DL was 1.0 log cfu/g. Values with different superscript letters (a, b) within a column, are statistically different from one another. Values sharing any common letter are not considered significantly different.
Table 2. Mean counts (log CFU/g ± standard deviation) of bacterial groups from sequential sampling stages during the winemaking process, specifically involving the Limniona grape variety.
Table 2. Mean counts (log CFU/g ± standard deviation) of bacterial groups from sequential sampling stages during the winemaking process, specifically involving the Limniona grape variety.
Sampling StagesMicrobial Groups
Lactococci (M17 Agar)Lactobacilli (MRS Agar)Yeasts (PDA)Yeasts (WORT Agar)
Mature grapes (Limniona)4.62 ± 0.024.59 ± 0.055.35 ± 0.065.25 ± 0.07
Initial must (Limniona, before filtration)4.24 ± 0.064.25 ± 0.074.28 ± 0.094.28 ± 0.07
Fermenting must (Limniona, before filtration)7.49 ± 0.097.68 ± 0.277.50 ± 0.047.65 ± 0.39
Wine sample (Limniona, after filtration and bottling)5.07 ± 0.134.95 ± 0.025.11 ± 0.134.40 ± 0.67
Values presented are the mean value ± SD (n = 3).
Table 3. MALDI-TOF identification results and range of identification score values for 68 isolates from key stages of the winemaking process.
Table 3. MALDI-TOF identification results and range of identification score values for 68 isolates from key stages of the winemaking process.
SampleIdentificationNumber of IsolatesRange of Identification Score Values
Mature grapes (Limniona)Candida lusitaniae61.850–2.084
(2 isolates < 2.000)
Bacillus amyloliquefaciens_ssp_plantarum31.834–1.99
Staphylococcus epidermidis21.934–2.077
(1 isolate < 2.000)
Candida krusei22.043–2.079
Initial must (Limniona)Saccharomyces cerevisiae41.780–1.936
(4 isolates < 2.000)
Serratia marcescens22.131–2.298
Klebsiella aerogenes12.204
Fermenting must (Limniona)Saccharomyces cerevisiae141.754–2.011
(11 isolates < 2.000)
Serratia marcescens12.273
Klebsiella aerogenes12.046
Unfiltered wine (Limniona)Saccharomyces cerevisiae231.793–2.004
(21 isolates < 2.000)
Table 4. Microbial composition of Limniona wine from traditional clay fermentation vessel using 16S rRNA gene sequencing.
Table 4. Microbial composition of Limniona wine from traditional clay fermentation vessel using 16S rRNA gene sequencing.
Unfiltered Wine from Clay VesselFiltered and Bottled Wine from Clay VesselSediment from Clay Vessel
GenusSpecies% of Valid ReadsGenusSpecies% of Valid ReadsGenusSpecies% of Valid Reads
OenococcusND22.72LactobacillusND29Oenococcusoeni30.6
Oenococcusoeni19.48Lentilactobacillusparafarraginis25.1Arcobactervenerupis8.78
BurkholderiaND16.5Lentilactobacillusdiolivorans13.09Arcobacterdefluvii7.65
Pediococcusparvulus6.11BurkholderiaceaeND3.14Buttiauxellawarmboldiae5.21
ArcobacterND3Oenococcusoeni1.37Arcobacterellisii2.78
AcetobacterND2 Citrobactergillenii1.7
ND: Not determined.
Table 5. Microbial composition of Limniona wine from stainless-steel (inox) fermentation vessel using 16S rRNA gene sequencing.
Table 5. Microbial composition of Limniona wine from stainless-steel (inox) fermentation vessel using 16S rRNA gene sequencing.
Unfiltered Wine from Inox VesselFiltered and Bottled Wine from Inox Vessel
GenusSpecies% of Valid ReadsGenusSpecies% of Valid Reads
AcetobacterND39BurkholderiaND34
BurkholderiaND23.73AcetobacterND17
RhizobiumND5.30RhizobiumND6.8
PseudoalteromonasND3.92Oenococcusoeni5.53
BacillusND1.91
Lentilactobacillusdiolivorans1.5
ND: Not determined.
Table 6. Microbial composition of wine samples from stainless-steel fermentation vessel from the previous vintage (2024) with typical and atypical sensory characteristics, using 16S rRNA gene sequencing.
Table 6. Microbial composition of wine samples from stainless-steel fermentation vessel from the previous vintage (2024) with typical and atypical sensory characteristics, using 16S rRNA gene sequencing.
Bottled “Limniona” Red Wine with Typical Sensory CharacteristicsBottled “Limniona” Red Wine with Atypical Sensory CharacteristicsBottled “Malagouzia” White Wine with Atypical Sensory Characteristics
GenusSpecies% of Valid ReadsGenusSpecies% of Valid ReadsGenusSpecies% of Valid Reads
LactobacillusND39LactobacillusND30.6Lentilactobacillusparafarraginis63.16
Lentilactobacillusparafarraginis19.19Lentilactobacillusparafarraginis28.16AcetobacterND11
Lentilactobacillusdiolivorans14.11Lentilactobacillusdiolivorans10.17LactobacillusND2
Oenococcusoeni1.97BurkholderiaND2.37Oenococcusoeni2.07
BurkholderiaND1.68
ND: Not determined.
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Bouki, P.; Mitsagga, C.; Tousia Becker, A.; Giavasis, I. Characterization of Microbial Population of Organic Grapes, Must and Natural Wine During Spontaneous Vinification of Limniona Red Grape Variety. Beverages 2025, 11, 160. https://doi.org/10.3390/beverages11060160

AMA Style

Bouki P, Mitsagga C, Tousia Becker A, Giavasis I. Characterization of Microbial Population of Organic Grapes, Must and Natural Wine During Spontaneous Vinification of Limniona Red Grape Variety. Beverages. 2025; 11(6):160. https://doi.org/10.3390/beverages11060160

Chicago/Turabian Style

Bouki, Paraskevi, Chrysanthi Mitsagga, Aphrodite Tousia Becker, and Ioannis Giavasis. 2025. "Characterization of Microbial Population of Organic Grapes, Must and Natural Wine During Spontaneous Vinification of Limniona Red Grape Variety" Beverages 11, no. 6: 160. https://doi.org/10.3390/beverages11060160

APA Style

Bouki, P., Mitsagga, C., Tousia Becker, A., & Giavasis, I. (2025). Characterization of Microbial Population of Organic Grapes, Must and Natural Wine During Spontaneous Vinification of Limniona Red Grape Variety. Beverages, 11(6), 160. https://doi.org/10.3390/beverages11060160

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