Abstract
Introduction:
Non-conventional yeasts (NCYs) associated with spontaneous food fermentations represent an underexplored source of microorganisms with potential probiotic and biotechnological applications. Coffee cherry fermentations harbor diverse yeast communities; however, the functional properties of indigenous NCYs remain poorly characterized. This study identified NCYs isolated from coffee cherry fermentations and evaluated their probiotic-associated functional traits, including stress tolerance, biosafety, antimicrobial activity, cytocompatibility, and effects on epithelial cell migration.
Methods:
Five NCY isolates were identified by ITS rDNA sequencing and characterized using in vitro assays evaluating tolerance to simulated gastrointestinal conditions, osmotic and thermal stress, cell surface hydrophobicity (CSH), autoaggregation, extracellular enzyme production, hemolytic activity, susceptibility to antifungal agents, and antimicrobial activity of neutralized cell-free supernatants (CFS). Cytocompatibility of CFS was assessed in Caco-2 cells using LDH, MTT, and Live/Dead assays, whereas epithelial cell migration was evaluated using a scratch assay.
Results:
Four isolates were identified as Kurtzmaniella quercitrusa, whereas one isolate was identified as Meyerozyma caribbica. All isolates exhibited strain-dependent tolerance to simulated gastrointestinal and environmental stresses. Lev3 showed the highest tolerance to gastrointestinal conditions, with survival comparable to the reference probiotic Saccharomyces boulardii CNCM I-745 (SSb), whereas Lev10 exhibited superior tolerance to osmotic and thermal stress. CSH and autoaggregation were also strain dependent, with Lev10 and Lev5 exhibiting the most favorable adhesion-related profiles among the coffee-derived isolates. All isolates were catalase-positive and exhibited variable proteolytic activity. None showed hemolytic activity, and most were susceptible to fluconazole and nystatin. Neutralized CFS exhibited inhibitory activity against both Gram-positive and Gram-negative bacteria while displaying low cytotoxicity toward Caco-2 cells. Although CFS did not significantly enhance scratch closure relative to the untreated control, Lev11 and Lev13 maintained epithelial wound closure comparable to the control.
Conclusion:
Coffee cherry-derived NCYs exhibited strain-dependent probiotic-associated traits, favorable preliminary biosafety profiles, antimicrobial activity, and high cytocompatibility, supporting their potential for fermentation biotechnology and the development of next-generation probiotic or postbiotic products. Further genomic, metabolomic, and in vivo studies are required to validate their efficacy, safety, and mechanisms of action.
1 Introduction
Yeasts are important contributors to food and beverage fermentations, with Saccharomyces cerevisiae var. boulardii representing the best-characterized probiotic yeast (Pereira et al., 2025). Unlike bacterial probiotics, yeasts exhibit intrinsic resistance to antibacterial agents, enabling their concurrent use during antibiotic therapy (Hatoum et al., 2012). In addition to their tolerance to gastrointestinal conditions and interaction with host immune responses, many yeasts produce extracellular antimicrobial compounds that influence microbial ecology and contributed to food preservation. In addition, yeasts produce antimicrobial peptides and related compounds that influence microbial dynamics and contribute to biocontrol. For instance, S. cerevisiae CCMI 885 secretes low-molecular-weight peptides active against spoilage and pathogenic yeasts, including Hanseniaspora, Torulaspora, Kluyveromyces, Lachancea, and Brettanomyces spp. (Albergaria and Arneborg, 2016; Muccilli and Restuccia, 2015). Similarly, a ~ 5 kDa peptide from Candida intermedia inhibits Brettanomyces bruxellensis (Al-Sahlany et al., 2020), while killer toxins produced by S. cerevisiae display antimicrobial activity against Escherichia coli, Salmonella spp., Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans (Kumura et al., 2004; Hatoum et al., 2012). Together, these findings highlight yeasts as promising sources of antimicrobial compounds for microbial control and food preservation.
Recent advances in microbial biotechnology have intensified interest in non-conventional yeasts (NCYs), which are frequently isolated fruits and fermented foods and beverages (Tamang et al., 2016; Ye et al., 2019; Melini et al., 2021; Martins et al., 2025). Genera such as Meyerozyma, Pichia, and Torulaspora include species with diverse functional properties, including antimicrobial and antioxidant activities, gastrointestinal stress tolerance, and modulation of gut microbial communities (Arévalo-Villena et al., 2020). Among these, Meyerozyma caribbica and Kurtzmaniella quercitrusa, formerly classified within the polyphyletic genus Candida, are commonly associated with fruit fermentations and have attracted growing interest (Piskur and Langkjaer, 2004; El-Adawy et al., 2020). M. caribbica exhibits several probiotic-associated traits, including tolerance to acidic and bile conditions, auto-aggregation, adhesion to epithelial cells, antimicrobial activity, and antioxidant capacity (Amorim et al., 2018; Padilha et al., 2018; de Moraes et al., 2021; Simões et al., 2021). In contrast, K. quercitrusa remains largely unexplored, despite evidence of metabolic adaptability across diverse environments.
Beyond the effects of viable cells, NCYs may produce extracellular metabolites with biological activities that could contribute to host- or process-related benefits and therefore warrant investigation as potential postbiotic preparations. Yeast-derived compounds, including phenolic molecules, glutathione, vitamins, organic acids, and antioxidant enzymes such as superoxide dismutase and catalase, contribute to cellular protection against oxidative stress, a process associated with chronic disease development (Zago et al., 2022; Reddy, 2023). These functional properties are highly strain dependent and reflect differences in metabolic output, antimicrobial activity, and antioxidant potential. Consistent with this variability, microbial consortia isolated from fermented Coffea arabica cherries exhibited substantial heterogeneity in metabolite production and bioactivity among strains, including selected NCYs with promising functional characteristics (Cifuentes et al., 2025).
Increasing evidence indicates that yeast-derived extracellular metabolites may exert biological activities extending beyond antimicrobial effects, including modulation of epithelial integrity, oxidative stress, and tissue repair (Tadioto et al., 2023). Several NCYs have been investigated as microbial platforms for producing bioactive molecules with potential applications in biotechnology and regenerative medicine. For example, Yarrowia lipolytica synthesizes lipids, organic acids, and enzymes that have been explored for tissue repair and wound healing applications (Fickers et al., 2020; Lee et al., 2025). These findings support evaluating the biological activity of yeast-derived cell-free supernatants in epithelial models.
Despite increasing interest in NCYs from fermented foods (Staniszewski and Kordowska-Wiater, 2021; Yan et al., 2021), limited information is available regarding the functional characteristics and bioactive extracellular metabolites of yeasts isolated from fermented C. arabica cherries. Accordingly, the present study evaluated the probiotic-associated traits and biological activities of five NCYs isolated from three fermented C. arabica cherry varieties from Intag, Ecuador. The study included assessment of tolerance to simulated gastrointestinal conditions, osmotic and thermal stress, cell surface properties (hydrophobicity and autoaggregation), biosafety-related characteristics, and antimicrobial activity of extracellular metabolites present in yeast-derived cell-free supernatants (CFS). Their biological activity was further assessed through cytotoxicity and scratch assays using Caco-2 cells to evaluate epithelial cell compatibility and restitution. Together, these analyses aimed to identify strain-dependent functional traits and extracellular bioactive products with potential applications in food biotechnology and the development of next-generation probiotic candidates and postbiotic preparations.
2 Materials and methods
2.1 Isolation and selection of yeasts
Yeast isolates were obtained from spontaneous fermentations of coffee cherries conducted for 6 days at room temperature. Cherries were collected at two ripening stages (immature green and mature red/ yellow) from three Coffea arabica varieties: Typica (TYP), Yellow Caturra (CATY), and Red Caturra (CATR). The physicochemical characteristics and yeast populations of the fermented cherries are presented in Supplementary Table S1. Serial dilutions of the fermented cherries were plated on yeast extract peptone dextrose (YPD) agar, and colonies exhibiting distinct colony characteristics (e.g., size, shape, and color) were purified and examined by light microscopy to confirm yeast morphology. The purified isolates were subsequently screened for fermentative capacity (highest CO₂ production) in YPD broth. When multiple colonies with similar morphological characteristics were recovered from the same sample, only one representative isolate was retained to avoid duplicate characterization. Consequently, one representative isolate was selected from each variety-ripening stage. No yeast colonies were isolated from fermented immature green cherries of Red Caturra, consistent with the absence of detectable viable yeasts in this sample (Supplementary Table S1). The five selected isolates were designated “Lev” (from the Spanish word levadura, meaning yeast): TYPVLev13 (Lev13), isolated from fermented immature green Typica cherries; TYPRLev10 (Lev10), from mature red Typica cherries; CRYVLev5 (Lev5) and CRYALev3 (Lev3), from fermented immature, green and mature yellow, Yellow Caturra cherries, respectively; and CATRLev11 (Lev11), from fermented mature red Red Caturra cherries. Cultures were preserved in YPD broth supplemented with 20% (w/v) glycerol at −80 °C and reactivated in fresh YPD medium prior to further analyses.
2.2 Identification of selected isolates
Genomic DNA extraction, polymerase chain reaction (PCR) amplification, and sequencing were performed by Macrogen Inc. (Seoul, Korea) following their standard protocols. Molecular identification was performed by PCR amplification of the internal transcribed spacer (ITS) region, encompassing the 5.8S rRNA gene. Amplification was carried out using the universal primers ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) and ITS5 (5′-GGAAGTAAAAGTCGTAACAAGG-3′). Sequencing reactions were conducted using the PRISM BigDye™ Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). The extension products were combined with Hi-Di™ formamide (Applied Biosystems, Foster City, CA, United States), denatured at 95 °C for 5 min, rapidly cooled on ice for 5 min, and analyzed on an ABI Prism 3730XL DNA Analyzer (Applied Biosystems). Putative species-level identification and phylogenetic placement were achieved by comparing the obtained sequences against the National Center for Biotechnology Information (NCBI) database using the nucleotide Basic Local Alignment Search Tool (BLASTn).
2.3 Phylogenetic analysis
The ITS-rRNA gene sequences obtained from the yeast isolates were compared with reference sequences retrieved from GenBank to determine their taxonomic affiliation. Reference sequences corresponding to K. quercitrusa (PP589185), Candida quercitrusa (KF747756), and M. caribbica CBS 9966 were included to support species-level identification. The ITS sequence of Hyphopichia sp. (GenBank accession no. ON508865) and Pichia caribbica (GenBank accession no. FN428887) were included as an outgroup to root the phylogenetic tree. Sequences were edited and assembled using MEGA v.11 (PSU, United States). Multiple sequence alignment was performed using the MUSCLE algorithm with default parameters. Phylogenetic relationships were inferred using the Neighbor-Joining (NJ) method based on the Kimura two-parameter (K2P) nucleotide substitution model. Branch support was assessed by bootstrap analysis with 1,000 replicates, and bootstrap values greater than 50% are shown at the corresponding nodes. Branch lengths represent the estimated number of nucleotide substitutions per site.
2.4 Probiotic-associated traits evaluation
Three reference yeast strains were included in the probiotic-associated traits assays: the commercial probiotic strain S. boulardii CNCM I-745 (SSb), and two laboratory strains Saccharomyces cerevisiae Lev30 (Lev30), and Candida intermedia Lev9 (Lev9). The selection of reference strains depended on the experimental endpoint evaluated and is described in the respective assay.
2.4.1 Tolerance to modified simulated gastric juice and bile salts
Tolerance to modified simulated gastric conditions and bile salts was assessed as previously described by Tenea et al. (2022). Yeast cultures were centrifuged at 5,000 × g for 5 min at 4 °C, washed twice with sterile Ringer’s solution (pH 7.2), and resuspended in simulated gastric juice adjusted to pH 2.5 and pH 3.0. Suspensions were incubated at 37 °C for 4 h, and viability was determined hourly by plating on YPD agar. The simulated gastric juice contained glucose (3.5 g/L), NaCl (2.05 g/L), KH₂PO₄ (0.60 g/L), CaCl₂ (0.11 g/L), KCl (0.37 g/L), pepsin (3 g/L), and lysozyme (0.1 g/L), according to Corcoran et al. (2005). For bile tolerance, overnight cultures (1 × 108 CFU/mL) were incubated in YPD broth supplemented with 0.3 and 1% (w/v) oxgall bile salts at 37 °C for 4 h. Cell survival was calculated as follows: survival (%) = (CFU final / CFU initial) × 100. The probiotic reference strain SSb was included for comparison. All assays were performed using independent biological triplicates.
2.4.2 Tolerance to sodium chloride and temperature
Sodium chloride tolerance was evaluated by culturing yeast strains in YPD broth containing 1 and 4% (w/v) NaCl. Cultures were incubated at 37 °C for 24 h, and growth was evaluated by viable plate counts on YPD agar. Temperature tolerance was assessed at 4, 37, and 45 °C under identical conditions. Survival percentages were calculated as described in Section 2.4.1. Unmodified YPD broth served as the control. The probiotic reference strain SSb was included for comparison. All assays were performed using independent biological triplicates.
2.4.3 Cell surface hydrophobicity (CSH) and autoaggregation
CSH was determined according to the microbial adhesion to hydrocarbons method described by Shazad et al. (2025). Yeast suspensions (1 × 108 CFU/mL) were prepared in phosphate-buffered saline (PBS; pH 7.2) following centrifugation at 5,000 × g for 5 min at 4 °C. The initial absorbance (A₀) was measured at 600 nm. Cell suspensions were mixed with 1 mL of hexane, ethyl acetate, or chloroform and vortexed for 1 min. After phase separation (10, 30, 60, and 180 min), the absorbance of the aqueous phase (A₁) was measured. Hydrophobicity was calculated as follows: Hydrophobicity (%) = [1-(A₁/A₀)] × 100. Autoaggregation ability was evaluated using a modified method described by Shazad et al. (2025). Yeast cells (1 × 108 CFU/mL) were washed, resuspended in PBS (pH 7.2), vortexed for 10 s, and the initial absorbance (A₀) was measured at 600 nm. Samples were incubated at 37 °C and absorbance values were recorded at 2, 4, 6, 8, and 24 h. Autoaggregation was calculated as follows: Autoaggregation (%) = [1 − (At/A0)] × 100 (Shazad et al., 2025). The results were compared with the reference strains SSb, Lev30, and Lev9. Mean values of hydrophobicity and autoaggregation were subjected to hierarchical cluster analysis using Euclidean distance as the similarity metric and complete linkage as the clustering algorithm (Borcard et al., 2018). The resulting dendrograms and heatmaps were used as exploratory tools to visualize similarities in adhesion-related phenotypes among strains rather than for hypothesis testing or inferential statistical analysis.
2.4.4 Qualitative protease and catalase activity
Extracellular protease production was assessed following the method described by Hanane et al. (2022). Yeast isolates were inoculated onto YPD agar supplemented with 10% skim milk powder and incubated at 30 °C for 7 days. Proteolytic activity was indicated by the formation of a clear halo surrounding colonies due to casein hydrolysis. Catalase activity was determined by adding 3% (v/v) hydrogen peroxide directly onto 48 h-old yeast cultures (Hanane et al., 2022). Immediate bubble formation was considered indicative of catalase activity. Results were compared with SSb, Lev30, and Lev9.
2.5 Biosafety evaluation
2.5.1 Hemolytic activity
Hemolytic activity was evaluated using Columbia agar supplemented with 5% sheep blood according to Yasmin et al. (2020). Plates were incubated at 37 °C for 48 h, and hemolytic patterns were classified as α-hemolysis (greenish halo), β-hemolysis (clear halo), or γ-hemolysis (absence of halo). Strains exhibiting γ-hemolysis were considered non-hemolytic. Results were compared with SSb, Lev30, and Lev9.
2.5.2 Susceptibility to antifungal agents
Susceptibility to antifungal agents was evaluated using an agar well diffusion assay following the CLSI M44 disk diffusion guideline (Clinical and Laboratory Standards Institute [CLSI], 2018). Overnight yeast cultures were spread uniformly onto YPD agar plates. Wells were aseptically prepared using a sterile cork borer and filled with antifungal solutions containing amphotericin B (32 μg/mL), voriconazole (2 μg/mL), fluconazole (64 μg/mL), or nystatin (1.25 μg/mL) (Merck, United States). Plates were incubated at 37 °C for 48 h, after which inhibition zones were measured in millimeters using an automatic plate scanner (Scan500, Interscience, France). Larger inhibition zones were interpreted as indicative of greater antifungal susceptibility under the tested conditions. All assays were performed using independent biological triplicates.
2.6 Cell-free supernatant (CFS) characterization
2.6.1 CFS extraction
An overnight yeast culture was inoculated into 100 mL of YPD broth supplemented with 10% sucrose and incubated at 30 °C for 48 h with agitation at 200 rpm. The medium was supplemented with sucrose because elevated sugar concentrations have been reported to enhance the production of extracellular bioactive metabolites by several yeast species (Tenea et al., 2023), thereby increasing the recovery of compounds for CFS characterization. Cultures were centrifuged at 13,000 × g for 20 min at 4 °C, and the resulting supernatants were sequentially filtered through 0.45 and 0.22 μm syringe filters (Chemlab Group, Washington, DC, United States). Filtered CFS samples were lyophilized and stored until analysis.
2.6.2 Antimicrobial activity toward bacterial pathogens
Antimicrobial activity was evaluated using the indicator bacterial strains: Escherichia coli ATCC 25922, E. coli L1PEag1, Staphylococcus aureus ATCC43300, S. aureus ATCC1026, Salmonella enterica ATCC51741, S. enterica ATCC35640, Shigella sonnei ATCC25931, Enterobacter sp. dFMcEag13, Kluyvera intermedia dfMPEag15, Ewingella sp. UMEag21, Pantoea sp. UMEag23, Serratia liquefaciens P4StpC1, Listeria monocytogenes ATCC18115, S. epidermidis L4MStp5, S. xylosus FFCShyA4. Bacterial suspensions (1 × 107 CFU/mL) were mixed with 0.75% nutrient soft agar and overlaid onto nutrient agar plates. Prior to testing, yeast-derived CFS were adjusted to pH 7.0 using sterile NaOH to eliminate the inhibitory effect associated with organic acids and low pH. Wells (6.5 mm diameter) were aseptically prepared in the agar and filled with 100 μL of neutralized CFS. Plates were incubated at 37 °C for 24–48 h, after which inhibition zones were measured using an automatic plate scanner (Scan500, Interscience, France). All assays were performed in triplicate (independent biological replicates). YPD broth supplemented with 10% sucrose and adjusted to the same pH served as the negative control.
2.6.3 Cytotoxicity and viability assessment
Cytotoxicity of yeast-derived CFS preparations was evaluated in Caco-2 cells using a lactate dehydrogenase (LDH) release assay (Roche, cat. no. 11644793001, Roche, Basel, Switzerland). Caco-2 cells were exposed to CFS samples (1:10 dilution) for 24 h. Subsequently, 50 μL of cell culture supernatant was mixed with an equal volume of LDH reaction mixture and incubated for 30 min at room temperature in the dark. Absorbance was measured at 490 nm using 620 nm as the reference wavelength. Cytotoxicity percentages were calculated according to the manufacturer’s instructions. Cell viability was further assessed using the MTT assay (Roche, cat. no. 475989). Following 24 h exposure to CFS samples, 10 μL of MTT reagent was added to each well and plates were incubated for 4 h at 37 °C. Formazan crystals were dissolved overnight in 100 μL of solubilization buffer, and absorbance was measured at 570 nm with a reference wavelength between 630 and 690 nm. Viability was expressed as percentage relative to untreated controls. Cell viability was additionally confirmed using the LIVE/DEAD Viability/Cytotoxicity Kit for mammalian cells (Invitrogen, Thermo Fisher Scientific; Cat. No. L3224). Cells were incubated with staining solution containing 2 μM calcein AM and 4 μM ethidium homodimer-1 for 30 min at room temperature in the dark. Representative fluorescence micrographs were acquired using a Zeiss Axiocam 305 mono camera equipped with a 1.0× camera adapter and a 10× objective.
2.6.4 Scratch wound-healing assay
The wound-healing potential of yeast-derived CFS was evaluated using an in vitro scratch assay following the method described by Liang et al. (2007) with minor modifications. Briefly, Caco-2 cells were cultured in complete medium until reaching approximately 90% confluence in 24-well plates. A linear scratch was generated across the cell monolayer using a sterile pipette tip, and detached cells were removed by washing with phosphate-buffered saline (PBS). Cells were then exposed to CFS (1:10 dilution in cell culture media) preparations and incubated at 37 °C under 5% CO₂. Wound closure was monitored by phase-contrast microscopy at 0 and 24 h, and images were analyzed to determine the percentage of wound closure relative to the initial scratch area. Untreated cells cultured in fresh medium served as the control. Images of the wound area were acquired immediately after scratching (0 h) and after 24 h of incubation using an inverted microscope. Wound closure was quantified using ImageJ software (National Institutes of Health, Bethesda, MD, United States). The wound area was manually outlined and measured at each time point. The percentage of wound closure was calculated according to the following formula: Wound closure (%) = [(A₀ − A₂₄)/A₀] × 100, where A₀ represents the wound area at 0 h and A₂₄ represents the wound area after 24 h of treatment. Experiments were performed in triplicate.
2.7 Statistical analysis
Data are presented as the mean ± standard deviation (SD). Each experiment was performed using three independent biological replicates, with measurements recorded in technical triplicate where applicable. Statistical analysis was conducted using GraphPad Prism version 10 (GraphPad Software, San Diego, CA, United States). Data normality was assessed using the Shapiro–Wilk test. Datasets that met the assumptions of normality were analyzed using one-way ANOVA followed by Tukey’s multiple comparisons test, whereas datasets that did not satisfy normality were analyzed using the Kruskal–Wallis test followed by Dunn’s multiple comparisons test. Statistical significance was determined at a confidence level of p < 0.05.
3 Results and discussion
3.1 Yeasts taxonomy and phylogenetic relationship
Among the selected isolates, four were identified as Kurtzmaniella quercitrusa and one as Meyerozyma caribbica (Table 1). K. quercitrusa is commonly linked to insect-associated and plant-derived environments, such as decaying organic material and plant exudates (Kurtzman et al., 2011; Arrey et al., 2021), which aligns with its occurrence in coffee fermentations. Similar findings have been reported in other coffee-producing regions, where non-Saccharomyces yeasts play an active role in fermentation processes (Haile and Kang, 2019). NJ phylogenetic analysis based on ITS rRNA gene sequences clustered the four isolates (Lev3, Lev5, Lev11, and Lev13) within a well-supported K. quercitrusa clade, clearly separated from the reference sequences (Figure 1A). Short branch lengths and the presence of two closely related subclades indicate limited sequence divergence among the isolates at the ITS locus (Figure 1A). The isolate Lev10 clustered with reference strains of M. caribbica with strong bootstrap support, confirming its taxonomic assignment (Figure 1B). The presence of M. caribbica is consistent with its recognized role in coffee fermentation, where it contributes to carbohydrate metabolism and the production of volatile compounds that affect flavor (De Bruyn et al., 2017; Silva et al., 2024). Its frequent co-occurrence with other non-Saccharomyces yeasts and its persistence in sugar-rich environments further support its ecological relevance (Shen et al., 2025). In addition, its use as a starter culture has been associated with improved sensory characteristics, particularly fruity and floral notes (Evangelista et al., 2014).
Table 1
| Group origin | Description | Sequence ID | Identification | Sample code | #Accession |
|---|---|---|---|---|---|
| Coffea arabica var. Typica | fermented green cherries | TYPVLev13 | Kurtzmaniella quercitrusa | Lev13 | PX806287 |
| fermented red cherries | TYPRLev10 | Meyerozyma caribbica | Lev10 | PX806286 | |
| C. arabica var. Yellow Caturra | fermented green cherries | CRYVLev5 | Kurtzmaniella quercitrusa | Lev5 | PX806285 |
| fermented yellow cherries | CRYALev3 | Kurtzmaniella quercitrusa | Lev3 | PX806284 | |
| C. arabica var. Red Caturra | fermented red cherries | CATRLev11 | Kurtzmaniella quercitrusa | Lev11 | PX806283 |
Taxonomic identification of yeast strains isolated from fermented cherries of different Coffea arabica varieties.
Figure 1
3.2 Yeasts probiotic-associated traits
3.2.1 Strain-dependent adaptation of yeasts to gastrointestinal stress conditions
The survival rates of the tested strains varied significantly under the simulated gastrointestinal conditions (Figure 2), with significant differences among strains observed within each treatment (p < 0.05). Overall, all isolates maintained high viability, with survival rates exceeding 68% under all conditions. Survival was generally lower at pH 2.5 than at pH 3.0, reflecting the greater inhibitory effect of severe acid stress. At pH 2.5, Lev3 exhibited significantly higher survival than the other test isolates and was statistically comparable to the reference strain (SSb). At pH 3.0, all isolates showed improved survival, with Lev3, Lev5, and Lev10 exhibiting survival rates comparable to the reference strain. Under 0.3% bile, Lev3 achieved the highest survival (95.1%), significantly exceeding both the reference strain and the remaining isolates. Increasing the bile concentration to 1% resulted in a moderate reduction in survival across all strains; however, Lev3 and Lev10 maintained survival comparable to the reference strain. Overall, these findings demonstrate strain-dependent variation in gastrointestinal stress tolerance, with Lev3 showing the greatest overall tolerance across the tested conditions and Lev10 exhibiting consistently high survival, identifying these two isolates as the most promising candidates for further probiotic characterization. Such variability is consistent with previous studies showing that tolerance to gastrointestinal-like conditions is highly strain-dependent among yeasts (Alkalbani et al., 2022a; Zheng et al., 2024). Similar observations have been reported for Saccharomyces boulardii, whose resistance varies according to environmental conditions (Czerucka et al., 2007), as well as for non-conventional yeasts including Zygosaccharomyces rouxii, Schizosaccharomyces pombe, Metschnikowia chrysoperlae, and Meyerozyma caribbica (Rodríguez Machado et al., 2024; Vu Thi Thanh and Ton That Huu, 2025).
Figure 2
3.2.2 Sodium chloride and temperature tolerance
The survival rates of the isolates under osmotic and thermal stress are presented in Figure 3. Statistical analysis revealed significant differences among the strains across the treatment conditions (p < 0.05). All isolates maintained relatively high survival at 1% NaCl (66.2–82.7%) and 4 °C (98.3–100%). Increasing NaCl concentration to 4% reduced survival in all strains, although Lev10 retained the highest survival among the test isolates and was statistically comparable to the reference strain (SSb). At 37 °C, complete survival was observed for Lev13 and Lev10, whereas Lev5, Lev3, and Lev11 showed significantly lower survival. Exposure to 45 °C caused the greatest reduction in viability, with survival ranging from 20.8 to 48.9%. Under this condition, Lev10 exhibited significantly higher survival than the other test isolates and remained comparable to the reference strain. These results highlight the strain-dependent nature of environmental tolerance, with Lev10 emerging as a highly resilient candidate capable of withstanding both osmotic and high-temperature conditions. The tolerance to NaCl observed in the evaluated yeast isolates is consistent with previous reports describing osmoadaptation mechanisms in halotolerant yeasts. Yeast survival under saline conditions may involve activation of the high-osmolarity glycerol (HOG) pathway, intracellular accumulation of glycerol, and regulation of ion transport systems that maintain osmotic balance and membrane integrity under hyperosmotic stress (Hohmann, 2002). Similar strain-dependent variability in salt tolerance has been reported in food-associated and halotolerant yeasts, where adaptation to NaCl is linked to differential regulation of osmolyte production and stress-response pathways (Stratford et al., 2019). The better adaptation of the Lev isolates to low-temperature environments may reflect differences in membrane adaptation mechanisms (Guyot et al., 2015). Besides, SSb likely possesses heat-adaptive mechanisms possibly related to enhanced heat stress responses (Wang et al., 2025; Zielińska et al., 2025).
Figure 3
3.2.3 Hydrophobicity and autoaggregation
Cell surface hydrophobicity (CSH), which is commonly used as an indicator of microbial surface interaction properties (Shazad et al., 2025), varied according to both the solvent and the yeast strain (Figure 4A). Hierarchical clustering based on Euclidean distance grouped strains according to the overall similarity of their hydrophobicity profiles across the three solvents and sampling times. The reference strain SSb formed a distinct cluster, reflecting its overall hydrophobicity profile across the evaluated solvents and sampling times. Lev30 clustered closely with SSb, whereas Lev10 and Lev5 formed a separate subgroup characterized by high affinity toward ethyl acetate and hexane. Lev13 and Lev11 showed intermediate profiles, while Lev3 and Lev9 clustered together due to their comparatively lower overall hydrophobicity. Across the evaluated strains, hydrophobicity was generally greatest in ethyl acetate and hexane, whereas lower values were observed with chloroform, indicating differences in cell surface physicochemical properties among isolates (Supplementary Table S2). Overall, Lev10 and Lev5 exhibited the most favorable hydrophobicity profiles among the coffee-derived isolates, suggesting more favorable adhesion-related surface characteristics than the remaining NCYs. These findings are consistent with previous reports that yeast hydrophobicity is strain-dependent (Alkalbani et al., 2022b). High CSH suggests enhanced adhesion, biofilm formation, and competitive fitness in microbial ecosystems, supporting the potential of isolates Lev10 and Lev5 for applications in fermentation and biocontrol. Overall, the observed variability underscores the importance of strain-level selection when targeting adhesion-related probiotic-associated traits among NCYs. While survival under gastrointestinal conditions is essential, the ability of yeasts to adhere to host surfaces remains a key determinant of probiotic-associated traits (Shazad et al., 2025). All NCYs exhibited a progressive increase in autoaggregation over time, with clear strain-dependent differences in aggregation kinetics (Figure 4B and Supplementary Table S3). During the first 2–6 h, Lev5, Lev3, and Lev11 aggregated more rapidly than Lev10 and Lev13, whereas the reference strains SSb, Lev30, and Lev9 exceeded 80% autoaggregation within 4 h. By 8 h, all coffee-derived isolates reached high aggregation levels (>86%), and after 24 h, all strains exhibited comparable autoaggregation (96.6–98.8%), indicating that the primary variation was in the rate rather than the final extent of aggregation. Hierarchical clustering based on Euclidean distance reflected these kinetic differences, separating the rapidly aggregating reference strains from the coffee-derived NCYs, with Lev10 and Lev13 showing the most similar profiles and Lev3 and Lev11 forming a closely related subgroup. Lev5 occupied an intermediate position, consistent with its relatively rapid early aggregation. These results indicate that autoaggregation is strain dependent, although all isolates ultimately achieved levels comparable to the probiotic reference strain (Alkalbani et al., 2022b). High autoaggregation has been associated with enhanced biofilm formation, colonization, and competitive exclusion of pathogens (Isenring et al., 2021). CSH is often positively correlated with aggregation behavior, as both are influenced by cell wall composition and surface properties (Wu et al., 2022). Consistent with this, Lev10 and Lev5 exhibited both high hydrophobicity and strong autoaggregation, suggesting favorable adhesion-related surface properties. In contrast, although Lev3 exhibited comparatively lower hydrophobicity and slower aggregation kinetics, adhesion may also involve alternative mechanisms, such as electrostatic interactions or specific adhesins (Alkalbani et al., 2022a). Overall, these findings demonstrate marked strain-dependent variation in adhesion-related properties among the evaluated NCYs. While Lev10 and Lev5 combined high hydrophobicity with strong autoaggregation, other isolates displayed distinct surface interaction profiles, emphasizing that probiotic-associated adhesion characteristics are multifactorial and strain-specific.
Figure 4
3.2.4 Catalase and protease activity
Catalase plays a central role in decomposing hydrogen peroxide into water and oxygen, thereby preventing the accumulation of reactive oxygen species (ROS) and protecting cellular macromolecules from oxidative damage (Gómez et al., 2019). All isolates exhibited positive catalase activity, evidenced by immediate oxygen evolution upon hydrogen peroxide exposure (Supplementary Figure S1A). This uniform response indicates a conserved oxidative stress defense mechanism, which is essential for maintaining cellular redox homeostasis under gastrointestinal and fermentation-associated stress conditions. Recent studies confirm that catalase-positive yeasts exhibit enhanced resilience and technological performance, particularly among non-Saccharomyces species, where higher catalase activity correlates with improved stress tolerance and viability during fermentation processes (Garofalo et al., 2020). Moreover, catalase activity is increasingly recognized as a potentially beneficial stress-response trait, contributing to antioxidant capacity and facilitating survival under gastrointestinal oxidative stress, thereby may contributing to host health and microbial persistence (Yang et al., 2025; Antimanon et al., 2026). Many yeast species, particularly within Saccharomyces and Candida, are known to produce extracellular proteases with important biotechnological and pathogenic roles (Ogrydziak, 1993). In the present study, the NCYs exhibited variable protease activity based on the qualitative plate assay (Supplementary Figure S1B). The reference strains (e.g., SSb, Lev9, and Lev30) showed clear zones of hydrolysis, confirming active protease secretion. Notably, Lev10 also displayed a pronounced zone of clearing, indicating strong proteolytic activity, comparable to the reference strains. In contrast, Lev11, Lev3, and Lev5 showed moderate substrate degradation, while Lev13 exhibited weak or negligible hydrolysis. These observations demonstrate that proteolytic capacity is strain-dependent among NCYs, rather than uniformly expressed. Similar variability has been reported in non-Saccharomyces genera such as Meyerozyma, Pichia, and Hanseniaspora, where extracellular enzyme production differs across strains and environmental conditions (Buzzini et al., 2012; Padilla et al., 2016). The isolates exhibiting strong proteolytic activity suggest a potential role in protein degradation and nitrogen turnover during fermentation, potentially enhancing free amino nitrogen availability and aroma precursor formation, whereas weakly active strains may rely on alternative nitrogen sources or metabolic pathways. Further analyses of free amino nitrogen, amino acid profiles, and volatile compounds are needed to validate these functional roles.
3.3 Biosafety of NCYs
3.3.1 Hemolytic activity
Hemolysis is considered a key virulence factor because iron acquisition is essential for microbial survival in the host, where free iron is limited and must be obtained from hemoglobin via hemolysin-mediated mechanisms (Luo et al., 2001). In this study, none of the NCY isolates showed hemolytic activity on blood agar, except the reference Candida strain Lev9 (Supplementary Figure S2). Hemolytic responses in yeasts are typically classified as alpha, beta, or gamma, although these categories are mainly descriptive and their mechanisms are not fully understood. In a recent study it has been shown that M. caribbica did not present hemolytic activity (Vu Thi Thanh and Ton That Huu, 2025). Furthermore, recent safety assessment indicates non-hemolytic activity of K. quercitrusa (Arrey et al., 2021; Liao et al., 2025). Thus, the lack of hemolytic activity in NCY isolates suggests a reduced virulence potential and supports their preliminary biosafety profile (Luo et al., 2001).
3.3.2 Inhibition by antifungal agents
The tested yeast isolates showed variable inhibition zones, relevant for evaluating their potential safety and technological applicability (Supplementary Table S4). Most isolates exhibited clear inhibition zones in response to fluconazole and nystatin, indicating sensitivity under the tested conditions. In contrast, amphotericin B and voriconazole produced little or no detectable inhibition, suggesting reduced sensitivity at the tested concentrations. Fluconazole exhibited the strongest inhibitory activity overall, with inhibition zones exceeding 25 mm in several isolates. In contrast, Lev9 exhibited low sensitivity to fluconazole under the tested conditions, indicating reduced susceptibility to azole antifungals. This observation is relevant from a probiotic safety perspective (FAO/WHO, 2002), since antifungal-resistant Candida strains may represent a potential opportunistic risk and should be carefully evaluated before considering biotechnological or probiotic applications. Conversely, the remaining susceptible isolates may represent safer candidates for further functional characterization. The activity of nystatin against all strains is consistent with its membrane-targeting mechanism through ergosterol binding (Diguță et al., 2022; Ngece et al., 2024). Antifungal susceptibility profiling is considered an important criterion in the safety assessment of yeasts exhibiting probiotic-associated properties and non-conventional microbial candidates intended for food or biomedical use (Kumura et al., 2004; EFSA Panel on Dietetic Products, Nutrition and Allergies [NDA], 2011).
3.4 Biological activity of NCY-derived CFS
3.4.1 Antimicrobial activity
The antimicrobial activity of neutralized NCY-derived CFS varied according to both the producing yeast strain and the target bacterial species (Supplementary Figure S3). Overall, all coffee-derived NCYs inhibited both Gram-negative and Gram-positive bacteria, although inhibition zones were generally moderate (approximately 8–11 mm). Gram-positive bacteria tended to be more susceptible than Gram-negative bacteria, particularly Staphylococcus saprophyticus, whereas activity against Gram-negative bacteria was more uniform across the tested isolates. Statistical analysis (Kruskal–Wallis followed by Dunn’s multiple comparisons test) confirmed significant strain-dependent differences for several bacterial species (Supplementary Figure S3). Because the CFS samples were neutralized to pH 7.0 before testing, the observed antimicrobial activity cannot be attributed solely to organic acids, suggesting the contribution of other extracellular antimicrobial metabolites. The bioactive compounds responsible for this activity were not characterized in the present study. Previous studies have shown that NCYs can produce diverse antimicrobial compounds, including killer toxins, siderophores such as pulcherriminic acid, hydrolytic enzymes, and volatile organic compounds that inhibit competing microorganisms (Oro et al., 2014; Melvydas et al., 2020). Further biochemical and metabolomic characterization will be necessary to identify the active compounds and clarify their mechanisms of action. Although the inhibition zones were relatively limited, the consistent inhibitory activity observed against both Gram-positive and Gram-negative bacteria suggests that these yeast isolates merit further investigation as potential sources of natural antimicrobial agents.
3.4.2 Cytotoxicity and cell viability assessment
The LDH assay revealed that all tested NCY-derived CFS samples induced low levels of cytotoxicity, indicating minimal membrane damage under the experimental conditions (Figure 5A). Among the strains, Lev5 exhibited the highest cytotoxicity, followed by Lev3, whereas Lev10, Lev11, and Lev13 showed consistently lower levels. Importantly, even the highest LDH release observed remained substantially below that of the positive lysis control, which approached complete cytotoxicity, confirming that overall membrane disruption was limited. These results suggest that the tested strains are generally well tolerated at the evaluated concentrations. The low LDH release in Lev10, Lev11, and Lev13 indicates preservation of membrane integrity, while the slightly higher values observed for Lev3 and particularly Lev5 may reflect a mild cytotoxic effect, although still far from levels associated with severe cellular damage (Figure 5A). The negative control showed negligible cytotoxicity, confirming low spontaneous LDH release, whereas the positive control validated the assay by inducing near-complete cell lysis. Consistent with these findings, the MTT assay demonstrated that all NCYs maintained high levels of metabolic activity, indicating preserved cell viability across conditions (Figure 5B). The untreated control exhibited maximal viability, while the cytotoxic control approached complete loss of viability, confirming the robustness and dynamic range of the assay. Among the tested strains, Lev10 showed the highest viability, followed by Lev13 and Lev11, whereas Lev3 and Lev5 displayed slightly reduced values. Nevertheless, differences among strains were modest, and overall viability remained well above levels associated with marked cytotoxicity. The Live/Dead fluorescence assay further confirmed the favorable cytocompatibility profile of the tested NCY-derived CFS (Figure 6). In all experimental groups, most cells displayed intense green fluorescence, indicating preserved membrane integrity and high cellular viability following exposure to the NCY-derived CFS. Only a limited number of red fluorescent cells, corresponding to membrane-compromised or dead cells, were observed across samples, supporting the low cytotoxicity profile previously identified through LDH and MTT assays. Among the tested isolates, Lev10 and Lev11 exhibited the highest proportion of viable, green-stained cells with very sparse red fluorescence, suggesting high cytocompatibility with the cellular model and minimal induction of membrane damage. The fluorescence pattern observed for these strains was highly comparable to the untreated control, indicating that exposure to these isolates did not substantially alter cell viability or morphology. These findings reinforce their potential suitability as postbiotic candidates.
Figure 5
Figure 6
3.4.3 Wound healing properties
Quantitative analysis of the scratch assay demonstrated strain-dependent differences in epithelial wound closure after 24 h of treatment with NCY-derived CFS (Figure 7), while representative micrographs illustrating these effects are shown in Supplementary Figure S4. Comparable initial scratch widths confirmed uniform baseline conditions across treatments. Among the evaluated isolates, Lev13 exhibited the greatest extent of scratch closure, followed by Lev11, whereas Lev3 and Lev5 displayed comparatively reduced closure. Lev11 and Lev13 maintained epithelial restitution comparable to the untreated control, while Lev10 produced an intermediate response. In contrast, wider residual wound areas and less cohesive wound fronts were observed following treatment with Lev3 and Lev5. Moreover, rounded and partially detached cells were evident in the Lev3-treated cultures, consistent with the moderate cytotoxicity detected in the LDH assay and suggesting a modest effect of this CFS on epithelial integrity. Overall, the results indicate that NCY-derived CFS exert strain-dependent effects on epithelial scratch closure without completely inhibiting wound repair or causing extensive cell detachment, supporting their general biocompatibility. The greater scratch closure observed for Lev13 and Lev11 suggests that these isolates produce extracellular metabolites that may contribute to epithelial restitution under the experimental conditions (Maurício et al., 2024). However, because the scratch assay does not distinguish between cell migration and proliferation, the mechanisms underlying wound closure cannot be established from the present data. Furthermore, only a single CFS concentration (1:10 dilution) was evaluated; therefore, dose-dependent effects remain to be determined in future studies together with in vivo validation. Comparable epithelial-protective effects have been reported for the probiotic yeast S. boulardii CNCM I-745, which contributes to intestinal barrier integrity and epithelial regeneration (Terciolo et al., 2019). Collectively, these findings indicate that selected coffee cherry-derived NCYs produce extracellular metabolites that may contribute to epithelial restitution and support their further investigation particularly as postbiotic candidates, while the probiotic potential of the producing strains requires further validation. Taken together, the cytocompatibility and scratch assay results demonstrate that the evaluated NCYs exhibit desirable characteristics for next-generation probiotic development. NCYs have recently attracted increasing attention because of their ability to tolerate harsh environmental conditions, produce bioactive metabolites, modulate host immune responses, and contribute to intestinal barrier homeostasis (Duysburgh et al., 2024; Calabrese et al., 2026).
Figure 7
3.5 Study limitations and future perspectives
The present study evaluated a limited collection of NCY isolates selected according to the criteria described in the Methods. Because Meyerozyma caribbica was represented by a single isolate (Lev10), the functional traits reported for this strain should not be extrapolated to the species level. The neutralized CFS displayed antimicrobial activity against the tested pathogens; however, the extracellular bioactive metabolites responsible for these effects were not characterized. In addition, epithelial restitution was assessed using a single concentration of CFS in an in vitro scratch assay, providing an initial indication of biological activity without allowing evaluation of concentration-dependent responses or validation under more physiologically relevant conditions.
Future studies should expand the diversity of coffee-associated NCY isolates included in the analysis and combine biochemical and metabolomic characterization of extracellular bioactive metabolites with functional validation to identify the compounds responsible for the observed activities.
4 Conclusion
Coffee cherry spontaneous fermentations represent a valuable source of non-conventional yeasts with promising functional properties. Molecular identification showed that the selected isolates belonged to Kurtzmaniella quercitrusa and Meyerozyma caribbica, two species that remain comparatively underexplored as candidates for probiotic- and postbiotic applications. The evaluated isolates exhibited marked strain-dependent variation in tolerance to simulated gastrointestinal conditions, osmotic and thermal stress, cell surface hydrophobicity, autoaggregation, and extracellular enzyme production. Among the isolates evaluated, Lev10 displayed the most balanced functional profile, combining high tolerance to environmental stresses with favorable adhesion-related characteristics and proteolytic activity, whereas Lev3 exhibited the greatest tolerance to simulated gastrointestinal conditions. The selected isolates also displayed a favorable preliminary biosafety profile, characterized by the absence of hemolytic activity, low cytotoxicity toward Caco-2 cells, and susceptibility to the antifungal agents evaluated under the tested conditions. In addition, their cell-free supernatants inhibited both Gram-positive and Gram-negative bacteria and maintained epithelial wound closure at levels comparable to the untreated control without affecting cell viability, supporting their potential as sources of bioactive postbiotic metabolites. Overall, the results identify selected coffee cherry-derived non-conventional yeast isolates as promising candidates for further investigation in functional food and related biotechnological applications. Future studies integrating whole-genome sequencing, metabolomic characterization of extracellular bioactive compounds, quantitative adhesion assays using intestinal epithelial cell models, and in vivo validation will be necessary to confirm safety, elucidate the mechanisms underlying the observed biological activities, and establish the probiotic- and postbiotic-associated potential of these isolates.
Statements
Data availability statement
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary material.
Ethics statement
Ethical approval was not required for the studies on humans in accordance with the local legislation and institutional requirements because only commercially available established cell lines were used.
Author contributions
VC: Writing – original draft, Investigation. GP: Writing – original draft, Investigation, Software, Writing – review & editing. GNT: Methodology, Validation, Data curation, Supervision, Writing – review & editing, Investigation, Software, Writing – original draft, Visualization, Formal analysis, Resources, Conceptualization, Funding acquisition, Project administration.
Funding
The author(s) declared that financial support was received for this work and/or its publication. This research was funded by Universidad Técnica del Norte under grant InvestigaUTN-2024-1432 awarded to GNT. Additionally, GT received partial support through the Scientific Visitor Fellowship Grant No. 147/2025 from the Research Institute of the University of Bucharest (ICUB), Romania.
Acknowledgments
The authors are thankful to Universidad Técnica del Norte for funding this research.
Conflict of interest
The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Generative AI statement
The author(s) declared that Generative AI was not used in the creation of this manuscript.
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Supplementary material
The Supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2026.1920410/full#supplementary-material
References
1
AlbergariaH.ArneborgN. (2016). Dominance of Saccharomyces cerevisiae in alcoholic fermentation processes: role of physiological fitness and microbial interactions. Appl. Microbiol. Biotechnol.100, 2035–2046. doi: 10.1007/s00253-015-7255-0,
2
AlkalbaniN. S.OsailiT. M.Al-NabulsiA. A.ObaidR. S.OlaimatA. N.LiuS. Q.et al. (2022a). In vitro characterization and identification of potential probiotic yeasts isolated from fermented dairy and non-dairy food products. J. Fungi8:544. doi: 10.3390/jof8050544,
3
AlkalbaniN. S.OsailiT. M.Al-NabulsiA. A.OlaimatA. N.LiuS.-Q.ShahN. P.et al. (2022b). Assessment of yeasts as potential probiotics: a review of gastrointestinal tract conditions and investigation methods. J. Fungi8:365. doi: 10.3390/jof8040365,
4
Al-SahlanyS. T. G.AltemimiA. B.Al-ManhelA. J. A.NiamahA. K.LakhssassiN.IbrahimS. A. (2020). Purification of bioactive peptide with antimicrobial properties produced by Saccharomyces cerevisiae. Foods9:324. doi: 10.3390/foods9030324
5
AmorimJ. C.SchwanR. F.DuarteW. F. (2018). Functional properties of yeast strains isolated from fermented tropical fruits and the potential for probiotic applications. J. Funct. Foods40, 759–765. doi: 10.1016/j.jff.2017.11.011
6
AntimanonS.RattanaphanN.NopgasonR.DechpreechakulT.ChamkhuyW.KingchaY.et al. (2026). Potent probiotic yeast Saccharomyces cerevisiae TBRC 3616: production development for food and feed applications. ACS Omega11, 17652–17662. doi: 10.1021/acsomega.5c11536,
7
Arévalo-VillenaM.Briones PérezA.BenitoM. J. (2020). Non-Saccharomyces yeasts in the winemaking process: biotechnological role and quality improvement. Foods9:1239. doi: 10.3390/foods9091239
8
ArreyG.LiG.MurphyR.GuimaraesL.AlizadehS.PoulsenM.et al. (2021). Isolation, characterization, and genome assembly of Barnettozyma botsteinii sp. nov. and novel strains of Kurtzmaniella quercitrus isolated from the intestinal tract of the termite Macrotermes bellicosus. G311:jkab342. doi: 10.1093/g3journal/jkab342,
9
BorcardD.GilletF.LegendreP. (2018). Numerical Ecology with R. 2nd Edn Cham: Springer International Publishing.
10
BuzziniP.BrandaE.GorettiM.TurchettiB. (2012). Psychrophilic yeasts from worldwide glacial habitats: diversity, adaptation strategies and biotechnological potential. FEMS Microbiol. Ecol.82, 217–241. doi: 10.1111/j.1574-6941.2012.01348.x,
11
CalabreseF. M.VincentiniO.FilanninoP.De BattistisF.ProtaV.LatronicoR.et al. (2026). Non-conventional yeasts with the potential to bridge probiotic and parabiotic applications in foods and dietary supplements: genomics and human model interaction. Appl. Food Res.6:101811. doi: 10.1016/j.afres.2026.101811
12
CifuentesV.MarinasI. C.MarinescuG. C.PopescuR. G.ChifiriucM. C.TeneaG. N. (2025). Metabolomic profiling of indigenous lactic acid bacteria reveals functional traits shaping the flavor and bioactivity of Ecuadorian coffee. Front. Microbiol.16:1697280. doi: 10.3389/fmicb.2025.1697280,
13
Clinical and Laboratory Standards Institute [CLSI] (2018). Method for Antifungal Disk Diffusion Susceptibility Testing of Yeasts. 3rd Edn. CLSI standard M44Wayne, PA: Clinical and Laboratory Standards Institute.
14
CorcoranB. M.StantonC.FitzgeraldG. F.RossR. P. (2005). Survival of probiotic lactobacilli in acidic environments is enhanced in the presence of metabolizable sugars. Applied and environmental microbiology, 71, 3060–3067. doi: 10.1128/AEM.71.6.3060-3067.2005
15
CzeruckaD.PicheT.RampalP. (2007). Review article: yeast as probiotics – Saccharomyces boulardii. Aliment. Pharmacol. Ther.26, 767–778. doi: 10.1111/j.1365-2036.2007.03442.x,
16
De BruynF.ZhangS. J.PothakosV.TorresJ.LambotC.MoroniA. V.et al. (2017). Exploring the impacts of postharvest processing on the microbiota and metabolite profiles during green coffee bean production. Appl. Environ. Microbiol.83, e02398–e02316. doi: 10.1128/AEM.02398-16,
17
de MoraesJ. P.FernandesL. L.MeloF. L.NicoliJ. R. (2021). Evaluation of Meyerozyma caribbica as a novel probiotic yeast with antimicrobial activity and tolerance to gastrointestinal conditions. Probiot. Antimicrob. Proteins13, 100–108. doi: 10.1007/s12602-020-09671-z
18
DiguțăC. F.MihaiC.TomaR. C.CîmpeanuC.MateiF. (2022). In vitro assessment of yeast strains with probiotic attributes for aquaculture use. Foods12:124. doi: 10.3390/foods12010124
19
DuysburghC.MiclotteL.GreenJ. B.WattsK. T.SardiM. I.ChakrabartiA.et al. (2024). Saccharomyces cerevisiae derived postbiotic alters gut microbiome metabolism in the human distal colon resulting in immunomodulatory potential in vitro. Front. Microbiol.15:1358456. doi: 10.3389/fmicb.2024.1358456,
20
EFSA Panel on Dietetic Products, Nutrition and Allergies [NDA] (2011). Scientific opinion on the substantiation of health claims related to microorganisms. EFSA J.9:1984. doi: 10.2903/j.efsa.2011.1984
21
El-AdawyM.El-SayedA. S. A.El-KadiS. (2020). Functional characterization of yeast strains from fermented fruits: potential for probiotic use. LWT Food Sci. Technol.123:109097. doi: 10.1016/j.lwt.2020.109097
22
EvangelistaS. R.SilvaC. F.MiguelM. G. P. C.CordeiroC. S.PinheiroA. C. M.DuarteW. F.et al. (2014). Improvement of coffee beverage quality by using selected yeast strains during fermentation in the dry process. Food Res. Int.61, 183–195. doi: 10.1016/j.foodres.2013.11.033
23
FAO/WHO (2002). Guidelines for the Evaluation of Probiotics in Food. Food and Agriculture Organization/World Health Organization.
24
FickersP.ChengH.Sze Ki LinC. (2020). Sugar alcohols and organic acids synthesis in Yarrowia lipolytica: where are we?Microorganisms8:574. doi: 10.3390/microorganisms8040574,
25
GarofaloC.TristezzaM.GriecoF.SpanoG.CapozziV. (2020). Basal catalase activity and high glutathione levels influence the performance of non-Saccharomyces active dry wine yeasts. Food Microbiol.92:103589. doi: 10.1016/j.fm.2020.103589
26
GómezS.Navas-YusteS.PayneA. M.RiveraW.López-EstepaM.BrangbourC.et al. (2019). Peroxisomal catalases from yeasts as models for oxidative damage. Free Radic. Biol. Med.141, 279–290. doi: 10.1016/j.freeradbiomed.2019.06.025,
27
GuyotS.GervaisP.YoungM.WincklerP.DumontJ.DaveyH. M. (2015). Surviving the heat: heterogeneity of response in Saccharomyces cerevisiae provides insight into thermal damage to the membrane. Environ. Microbiol.17, 2982–2992. doi: 10.1111/1462-2920.12866,
28
HaileM.KangW. H. (2019). Isolation, identification, and characterization of pectinolytic yeasts for starter culture in coffee fermentation. Microorganisms7:401. doi: 10.3390/microorganisms7100401,
29
HananeT.NajouaB.SalsabilH.AbdellatifJ. I.DalilaB.AhmadI.et al. (2022). Qualitative screening of yeast biodiversity for hydrolytic enzymes isolated from the gastrointestinal tract of a coprophage “Gymnopleurus sturmi” and dung of ruminants. Fermentation8:692. doi: 10.3390/fermentation8120692
30
HatoumR.LabrieS.FlissI. (2012). Antimicrobial and probiotic properties of yeasts: from fundamental to novel applications. Front. Microbiol.3:421. doi: 10.3389/fmicb.2012.00421,
31
HohmannS. (2002). Osmotic stress signaling and osmoadaptation in yeasts. Microbiol. Mol. Biol. Rev.66, 300–372. doi: 10.1128/MMBR.66.2.300-372.2002,
32
IsenringJ.GeirnaertA.LacroixC.StevensM. J. A. (2021). Bistable auto-aggregation phenotype in Lactiplantibacillus plantarum emerges after cultivation in in vitro colonic microbiota. BMC Microbiol.21:268. doi: 10.1186/s12866-021-02331-x,
33
KumuraH.TanoueY.TsukaharaM.TanakaT.ShimazakiK. (2004). Screening of dairy yeast strains for probiotic applications. J. Dairy Sci.87, 4050–4056. doi: 10.3168/jds.S0022-0302(04)73546-8,
34
KurtzmanC. P.FellJ. W.BoekhoutT. (2011). “Definition, classification and nomenclature of the yeasts,” In C. P. Kurtzman, J. W. Fell, & T. Boekhout (Eds.), The Yeasts, vol. 1 (Amsterdam, Netherlands: Elsevier), 3–5.
35
LeeS.LeeJ. H.ParkH. J.BaekS. H. (2025). Yarrowia lipolytica as a promising cell factory for microbial production of value-added nutraceuticals. Front. Bioeng. Biotechnol.13:1673169. doi: 10.3389/fbioe.2025.1673169,
36
LiangC. C.ParkA. Y.GuanJ. L. (2007). In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro. Nature protocols, 2, 329–333. doi: 10.1038/nprot.2007.30
37
LiaoB.ChenO.ZhangH.WangW.YiL.LiH.et al. (2025). Biocontrol yeast Kurtzmaniella quercitrusaBS-AY-S1 controls postharvest green mold of citrus fruit by producing metabolites with antifungal activity. Biol. Control200:105667. doi: 10.1016/j.biocontrol.2024.105667
38
LuoG.SamaranayakeL. P.YauJ. Y. (2001). Candida species exhibit differential in vitro hemolytic activities. J. Clin. Microbiol.39, 2971–2974. doi: 10.1128/JCM.39.8.2971-2974.2001,
39
MartinsP. M. M.FalcãoI. J.BatistaN. N.BernardesP. C.SchwanR. F. (2025). Selection and characterization of non-Saccharomyces yeast strains for potential use in Arabica and Conilon coffee fermentations. J. Food Sci.90:e70431. doi: 10.1111/1750-3841.70431,
40
MaurícioE. M.BrancoP.AraújoA. L. B.Roma-RodriguesC.LimaK.DuarteM. P.et al. (2024). Evaluation of biotechnological active peptides secreted by Saccharomyces cerevisiae with potential skin benefits. Antibiotics13:881. doi: 10.3390/antibiotics13090881,
41
MeliniF.MeliniV.LuziatelliF.FiccaA. G.RuzziM. (2021). Health-promoting components in fermented foods: an up-to-date systematic review. Nutrients13:1390. doi: 10.3390/nu13041390
42
MelvydasV.SvedieneJ.SkridlaiteG.VaiciunieneJ.GarjonyteR. (2020). In vitro inhibition of Saccharomyces cerevisiae growth by Metschnikowia spp. triggered by fast removal of iron. Braz. J. Microbiol.51, 1953–1964. doi: 10.1007/s42770-020-00357-3,
43
MuccilliS.RestucciaC. (2015). Bioprotective role of yeasts. Microorganisms3, 588–611. doi: 10.3390/microorganisms3040588,
44
NgeceK.NtondiniT. L.KhwazaV.PacaA. M.AderibigbeB. A. (2024). Polyene-based derivatives with antifungal activities. Pharmaceutics16:1065. doi: 10.3390/pharmaceutics16081065,
45
OgrydziakD. M. (1993). Yeast extracellular proteases. Crit. Rev. Biotechnol.13, 1–55. doi: 10.3109/07388559309069197,
46
OroL.CianiM.ComitiniF. (2014). Antimicrobial activity of Metschnikowia pulcherrima on wine yeasts. J. Appl. Microbiol.116, 1209–1217. doi: 10.1111/jam.12446,
47
PadilhaM.VilelaD.BrandãoR. L.NicoliJ. R. (2018). Probiotic properties of yeasts isolated from fermented foods and the gastrointestinal tract of healthy animals. World J. Microbiol. Biotechnol.34:92. doi: 10.1007/s11274-018-2470-7
48
PadillaB.GilJ. V.ManzanaresP. (2016). Past and future of non-Saccharomyces yeasts: from spoilage microorganisms to biotechnological tools for improving wine aroma complexity. Front. Microbiol.7:411. doi: 10.3389/fmicb.2016.00411,
49
PereiraK. N.de OliveiraA. C. D.de SouzaH. F.UllahS.NasirU.AliS.et al. (2025). Application of Saccharomyces cerevisiae var. boulardii for biological detoxification of chemical contaminants in foods: a comprehensive review. Foods14:4260. doi: 10.3390/foods14244260,
50
PiskurJ.LangkjaerR. B. (2004). Yeast genome sequencing: lessons from Candida and Saccharomyces. FEMS Yeast Res.4, 3–9. doi: 10.1016/S1567-1356(03)00159-1
51
ReddyV. P. (2023). Oxidative stress in health and disease. Biomedicine11:2925. doi: 10.3390/biomedicines11112925,
52
Rodríguez MachadoA.CaroC. M.Hurtado-MurilloJ. J.Gomes LoboC. J.ZúñigaR. N.FrancoW. (2024). Unconventional yeasts isolated from Chilean honey: a probiotic and phenotypic characterization. Foods13:1582. doi: 10.3390/foods13101582,
53
ShazadA.TlaisA. Z. A.CappelloC.Casagrande BacchiocchiS.FilanninoP.GobbettiM.et al. (2025). Comprehensive functional profiling of yeasts: gastrointestinal resistance, metabolic capacities, and psychobiotic potential. Appl. Food Res.5:101178. doi: 10.1016/j.afres.2025.101178
54
ShenX.WangQ.WangH.FangG.LiY.ZhangJ.et al. (2025). Microbial characteristics and functions in coffee fermentation: a review. Fermentation11:5. doi: 10.3390/fermentation11010005,
55
SilvaL. C. F.PereiraP. V. R.CruzM. A. D. D.CostaG. X. R.RochaR. A. R.BertariniP. L. L.et al. (2024). Enhancing sensory quality of coffee: the impact of fermentation techniques on Coffea arabica cv. Catiguá MG2. Foods13:653. doi: 10.3390/foods13050653,
56
SimõesL. R.PereiraG. V. M.SchwanR. F. (2021). Functional properties of Meyerozyma caribbica isolated from cocoa fermentation and its potential use in probiotics. J. Appl. Microbiol.130, 516–528. doi: 10.1111/jam.14715
57
StaniszewskiA.Kordowska-WiaterM. (2021). Probiotic and potentially probiotic yeasts-characteristics and food application. Foods10:1306. doi: 10.3390/foods10061306,
58
StratfordM.SteelsH.NovodvorskaM.ArcherD. B.AveryS. V. (2019). Extreme osmotolerance and halotolerance in food-relevant yeasts and the role of glycerol-dependent cell individuality. Front. Microbiol.9:3238. doi: 10.3389/fmicb.2018.03238,
59
TadiotoV.GiehlA.CadamuroR. D.GuterresI. Z.dos SantosA. A.BressanS. K.et al. (2023). Bioactive compounds from and against yeasts in the one health context: a comprehensive review. Fermentation9:363. doi: 10.3390/fermentation9040363
60
TamangJ. P.WatanabeK.HolzapfelW. H. (2016). Review: diversity of microorganisms in global fermented foods and beverages. Front. Microbiol.7:377. doi: 10.3389/fmicb.2016.00377,
61
TeneaG. N.Anrango CajasB.Carlosama SanchezB. (2023). Inhibitory-like substances produced by yeasts isolated from Andean blueberries: prospective food antimicrobials. Foods12:2435. doi: 10.3390/foods12132435,
62
TeneaG. N.GonzalezG. L.MorenoJ. L. (2022). Probiotic characteristics and antimicrobial potential of a native Bacillus subtilis strain Fa17.2 rescued from wild Bromelia sp. flowers. Microorganisms10:860. doi: 10.3390/microorganisms10050860,
63
TercioloC.DapoignyM.AndreF. (2019). Beneficial effects of Saccharomyces boulardii CNCM I-745 on clinical disorders associated with intestinal barrier disruption. Clin. Exp. Gastroenterol.12, 67–82. doi: 10.2147/CEG.S181590,
64
Vu Thi ThanhT.Ton That HuuD. (2025). Probiotic potential of the yeast strain Meyerozyma caribbica TV2 isolated from the wild bird Pycnonotus jocosus. Microbiol. Biotechnol. Lett.53, 520–529. doi: 10.48022/mbl.2510.10009
65
WangD.LiC.LaiJ.LiuS.ZhangY.XuZ.et al. (2025). Therapeutic potential of Saccharomyces boulardii in alleviating gastrointestinal stress through preservation of intestinal cell membrane integrity. BMC Microbiol.25:359. doi: 10.1186/s12866-025-03984-8,
66
WuD. Q.DingX. S.ZhaoB.AnQ.GuoJ. S. (2022). The essential role of hydrophobic interaction within extracellular polymeric substances in auto-aggregation of Pseudomonas stutzeri strain XL-2. Int. Biodeterior. Biodegrad.171:105404. doi: 10.1016/j.ibiod.2022.105404
67
YangL.WuX.LiuD. (2025). Mechanism and application of yeast and its culture in regulating intestinal antioxidant defense in ruminants. Front. Vet. Sci.12:1657244. doi: 10.3389/fvets.2025.1657244,
68
YanW.GaoH.QianX.JiangY.ZhouJ.DongW.et al. (2021). Biotechnological applications of the non-conventional yeast Meyerozyma guilliermondii. Biotechnol. Adv.46:107674. doi: 10.1016/j.biotechadv.2020.107674,
69
YasminI.SaeedM.KhanW. A.KhaliqA.ChughtaiM. F. J.IqbalR.et al. (2020). In vitro probiotic potential and safety evaluation (hemolytic, cytotoxic activity) of Bifidobacterium strains isolated from raw camel milk. Microorganisms8:354. doi: 10.3390/microorganisms8030354,
70
YeL.AmbergC.ShenW.LindnerS. N. (2019). Non-Saccharomyces yeasts: emerging biotechnological tools for health and food applications. Crit. Rev. Food Sci. Nutr.59, 1211–1223. doi: 10.1080/10408398.2017.1399146
71
ZagoM.BerettaM.RossettiL.GiraffaG. (2022). Antioxidant capacity and stress resistance of yeasts isolated from traditional foods. Food Microbiol.102:103898. doi: 10.1016/j.fm.2021.103898
72
ZhengM.SuQ.WuH.CaiC.NinhL. T.CaiH. (2024). Elucidating bile acid tolerance in Saccharomyces cerevisiae: effects on sterol biosynthesis and transport protein expression. Foods13:3405. doi: 10.3390/foods13213405,
73
ZielińskaD.KrawczykM.Neffe-SkocińskaK. (2025). Thermotolerant probiotic—the potential of improving the survivability of beneficial bacteria. Fermentation11:313. doi: 10.3390/fermentation11060313
Summary
Keywords
antimicrobial activity, biosafety, coffee fermentation, functional foods, Kurtzmaniella quercitrusa, Meyerozyma caribbica, non-conventional yeasts, postbiotics
Citation
Cifuentes V, Pircalabioru GG and Tenea GN (2026) Functional assessment of coffee cherry-derived non-conventional yeasts identifies promising candidates for probiotic and postbiotic applications. Front. Microbiol. 17:1920410. doi: 10.3389/fmicb.2026.1920410
Received
26 June 2026
Revised
24 July 2026
Accepted
27 July 2026
Published
12 August 2026
Volume
17 - 2026
Edited by
Massimo Iorizzo, University of Molise, Italy
Reviewed by
Amishi Bhatt, Darshan University, India
Hayrunisa İçen, Firat University, Türkiye
Mohamad Eshaghi Gorji, The Hong Kong Polytechnic University, Hong Kong SAR, China
Updates
Copyright
© 2026 Cifuentes, Pircalabioru and Tenea.
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Gabriela N. Tenea, gntenea@utn.edu.ec
Disclaimer
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.