Skip to main content
NCBI home page
MicrobiologyOpen logoLink to MicrobiologyOpen
. 2026 Oct 11;15(5):e70439. doi: 10.1002/mbo3.70439

Antifungal Activity Assessment of Postbiotic Mediators Derived From Lactobacillus rhamnosus GG and Lactobacillus reuteri Against Candida albicans

Fatemeh Amirinia 1,2, Morteza Banakar 1,3,4,✉, Mojgan Alaeddini 1,✉, Shahroo Etemad‐Moghadam 1, Maryam Pourhajibagher 1, Maryam Noori 5
PMCID: PMC13655302  PMID: 42860635

ABSTRACT

The rise of antimicrobial resistance has increased interest in safe alternative therapeutic agents, including postbiotic mediators (PMs), which are sterile, cell‐free, filter‐sterilized preparations derived from probiotics. This study investigated the antifungal and antibiofilm activity of PMs derived from Lactobacillus rhamnosus GG (LGG) and Lactobacillus reuteri (LR) against Candida albicans, an opportunistic pathogen implicated in oral infections. PMs were prepared from LGG and LR cultures. The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) against C. albicans were determined using broth microdilution and colony‐forming unit (CFU) counts. Antibiofilm activity at sub‐MIC concentrations was evaluated by assessing metabolic activity (XTT assay) and biofilm architecture (scanning electron microscopy, SEM). The MIC and MFC of PMs from both LGG and LR were 25% (v/v). Both PMs significantly reduced the metabolic activity of C. albicans biofilms as measured by the XTT assay (p ≤ 0.002); however, a statistically significant reduction in viable biofilm counts was observed only with LGG PMs (p = 0.0038), whereas LR PMs showed a non‐significant reduction (p = 0.1537). SEM showed marked changes in biofilm architecture. PMs from LGG and LR showed in vitro antifungal activity against C. albicans, with both preparations suppressing biofilm metabolic activity and LGG PMs additionally producing a statistically significant reduction in viable biofilm counts. These findings support further investigation of Lactobacillus‐derived PMs as candidate adjunctive antifungal approaches; however, validation in clinical and antifungal‐resistant isolates, chemical and mechanistic characterization, cytotoxicity testing, and in vivo studies are required before clinical application.

Keywords: antifungal effects, biofilm inhibition, Candida albicans, Lactobacillus, oral candidiasis, postbiotics

Postbiotic metabolites derived from Lactobacillus rhamnosus GG and Lactobacillus reuteri demonstrate potent antifungal and anti‐biofilm activity against Candida albicans. By significantly impairing fungal viability, metabolic activity, and biofilm structural integrity, these non‐viable compounds offer a promising, safe alternative for managing oral candidiasis and preventing dental caries.

graphic file with name MBO3-15-e70439-g004.webp

1. Introduction

Candida albicans (C. albicans) is a naturally occurring oral yeast that can contribute to dental caries and oral infection (Cao et al. 2025; Ev et al. 2020; Li et al. 2014). This yeast colonizes various surfaces, including the tongue and dental plaque (Rawnuck et al. 2025). However, under conditions that disrupt the oral microbial balance, it can proliferate excessively and lead to infection (Cao et al. 2025). C. albicans can be present in decayed teeth, particularly in dentin, and may contribute to tissue degradation by secreting collagenolytic enzymes (Lu et al. 2023). These enzymes can break down dentinal collagen, increasing permeability and causing damage to the dental matrix (Du et al. 2021). C. albicans can also promote demineralization of hard dental tissues by lowering the pH of its environment (Eidt et al. 2019). Therefore, this yeast may contribute to the progression of dental caries in conjunction with other microorganisms (Regis et al. 2025).

The overuse and misuse of antifungal agents have contributed to the emergence of resistant C. albicans strains, posing significant therapeutic challenges (Abu‐Humaidan et al. 2025; Mudenda 2024). Studies have reported resistance of C. albicans to drugs such as fluconazole and itraconazole, which can contribute to chronic and recurrent infections (Mudenda 2024; Rawnuck et al. 2025; Seyedjavadi et al. 2024). In addition, non‐albicans Candida species are increasingly recognized as treatment challenges (Raposa and Vazquez 2025; Rawnuck et al. 2025). Given the growing resistance of C. albicans to conventional antifungals, alternative therapeutic approaches, including postbiotic metabolites, warrant investigation. However, studies evaluating the direct effects of PMs on fungal biofilms remain limited.

As live microorganisms, probiotics may play a role in managing oral candidiasis (Rajão et al. 2025). They can suppress the growth and colonization of C. albicans and help maintain oral microbial balance by producing antifungal compounds such as lactic acid and bacteriocins (Homayouni Rad et al. 2023; Pallavi et al. 2024; Rajão et al. 2025; Sharifzadeh et al. 2023). Potential benefits of probiotics include reduced adverse effects associated with antifungal medications and immunomodulatory effects (Wu et al. 2022). However, potential drawbacks include delayed effects, variable efficacy among individuals, and an increased risk of infection in immunocompromised patients (Wu et al. 2022).

Unlike probiotics, which are live microorganisms, postbiotics comprise non‐viable microbial preparations or their components that retain bioactive properties (Prajapati et al. 2023). Postbiotic mediators (PMs), defined herein as sterile, cell‐free, filter‐sterilized preparations derived from probiotic bacterial cultures and containing bioactive metabolites, are products of probiotic metabolic activity (Hill et al. 2021; Chakraborty et al. 2025; Samuel et al. 2024; Yeşilyurt et al. 2021). Compared with live probiotics, cell‐free PM preparations avoid administration of viable microorganisms and may offer practical advantages in storage and formulation; nevertheless, the safety and stability of each preparation require direct product‐specific evaluation (Ji et al. 2023; Prajapati et al. 2023). These characteristics make PMs of particular interest for populations in whom administration of live microorganisms may be undesirable, while formal biocompatibility assessment remains necessary (Fang et al. 2025; Ji et al. 2023).

Different Lactobacillus strains have long been recognized as effective probiotics (Chakraborty et al. 2025); however, the number of studies on their PMs remains limited. Lactobacillus rhamnosus GG (LGG) and Lactobacillus reuteri (LR) are well‐studied probiotic strains with reported antimicrobial activity. They are known for robust growth, adhesion, pathogen antagonism, and production of antimicrobial substances, and are therefore considered promising probiotic strains. The strain‐specific effects of various Lactobacillus isolates, their metabolites, and the mechanisms underlying activity against C. albicans remain unclear. Therefore, this study aimed to investigate the antifungal effects of PMs derived from LGG and LR by assessing their antibiofilm activity and effects on the metabolic activity of C. albicans.

2. Methods

2.1. Culture Strains and Growth Conditions

L. rhamnosus GG (ATCC 53103) and L. reuteri (ATCC 23272) were selected because of their well‐documented probiotic properties and prior reports of antimicrobial activity against oral pathogens (Banakar et al. 2023). They were maintained on MRS agar (de Man, Rogosa, and Sharpe agar) (Merck, Darmstadt, Germany), subsequently cultured in MRS broth (Merck, Darmstadt, Germany), and incubated in a CO2 incubator at 37°C ± 1°C for 48 h. C. albicans ATCC 10231 was obtained from the Iranian Biological Resource Centre (Tehran, Iran) and incubated on Sabouraud dextrose agar (SDA, Merck, Darmstadt, Germany) at 35°C ± 1°C for 24 h.

Five colonies with a diameter of approximately 1 mm were selected from a 24‐h culture. These colonies were suspended in 5 mL of sterile saline and vortexed for 15 s. The yeast cell density was standardized to 0.5 McFarland turbidity (1–5 × 106 CFU/mL) using sterile saline, as measured spectrophotometrically at 600 nm. The suspension was then diluted with RPMI 1640 medium (with glutamine and without bicarbonate) (QLab, Canada) buffered with 0.165 mol/L MOPS (3‐(N‐morpholino)propanesulfonic acid) to achieve a final concentration of 5.0 × 102 to 2.5 × 103 yeast cells/mL for MIC/MFC susceptibility testing, in accordance with CLSI M27‐A3 guidelines (Banakar et al. 2023; Cilo and Ener 2021; El‐Mashad et al. 2012; Rodríguez‐Tudela et al. 2003). For biofilm formation assays (biofilm destruction and XTT reduction assay), a higher inoculum of 1.5 × 106 CFU/mL was used, consistent with established protocols for in vitro C. albicans biofilm generation, which require a higher initial cell density to ensure confluent and reproducible biofilm formation within the incubation period (Matsubara et al. 2016; Rossoni et al. 2018).

2.2. Postbiotic Mediators

After 48 h of incubation, the tubes containing the culture medium and Lactobacillus species were centrifuged at 10,000g for 10 min at 4°C. The supernatant was then separated from the sediment and transferred to a new tube. All supernatants were filtered through a 0.22 μm polyethersulfone membrane syringe filter (Millipore, Burlington, MA, USA). The pH of the supernatants was adjusted to physiological pH (7.2–7.4) using 5 M sodium hydroxide before use (Banakar et al. 2023). This pH adjustment may alter the antimicrobial activity of organic acid constituents (e.g., lactic acid and acetic acid), as these compounds exert their primary antifungal effects in their protonated, acidic form; this consideration is discussed further in the Limitations section.

2.3. Evaluation of Antifungal and Antibiofilm Activity

2.3.1. Determination of Minimum Inhibitory Concentration and Minimum Fungicidal Concentration of Postbiotic Mediators

The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) were determined according to the Clinical and Laboratory Standards Institute (CLSI) M27‐A3 broth microdilution guidelines (Clinical and Laboratory Standards Institute 2008, 2017). Briefly, 100 μL of RPMI 1640 medium buffered with 0.165 mol/L MOPS was dispensed into wells 2–11 of each row in round‐bottom 96‐well microplates. One hundred microliters of undiluted PM (100% v/v) was added to well 1, and serial twofold dilutions were prepared by transferring 100 μL aliquots sequentially from well 1 through well 10, with thorough mixing at each step; the final 100 μL from well 10 was discarded. One hundred microliters of the standardized C. albicans inoculum (5.0 × 102 to 2.5 × 103 cells/mL in RPMI‐MOPS) was then added to all wells, resulting in a final assay volume of 200 μL and nominal PM concentrations ranging from 50% (v/v) downward. Plates were incubated at 37°C for 24 h. The MIC was recorded as the lowest PM concentration showing no visible turbidity. Appropriate positive (C. albicans in medium alone) and negative (PMs in medium alone) controls were included. All determinations were performed in triplicate.

Subcultures from the broth dilution wells were used to determine the MFC, which was defined as the lowest concentration of PMs that prevented the growth of C. albicans on SDA. MFC was also defined as the lowest concentration of an antifungal agent that reduced the initial inoculum by at least 99.99%. All experiments were repeated at least three times.

2.3.2. Determination of the Biofilm Destruction Effect of PMs

A suspension of C. albicans with a concentration of 1.5 × 106 CFU/mL was prepared, and 200 μL was added to each well of a flat‐bottomed 96‐well microplate. The microplate was incubated at 37°C for 24 h to form a C. albicans biofilm. After the incubation period, the suspension was aspirated, and the wells were washed three times with sterile PBS at pH 7.4. Subsequently, 100 μL of each PM at a concentration of 2 × MIC was added to each well, and the microplate was incubated at 37°C for 24 h. After incubation, the C. albicans biofilm was scraped from the bottom of the microplate and serially diluted for plating to determine CFU/mL. All experiments were performed in triplicate (Esfandiary et al. 2024).

2.4. Microscopic Visualization of Biofilm

According to a previous study, tooth slabs were prepared for microbial biofilm formation (Banakar et al. 2023). C. albicans biofilms were formed on the tooth slabs by incubating the slabs in a C. albicans suspension at 1.5 × 106 CFU/mL for 48 h at 37°C. The slabs were then washed three times with sterile PBS and treated with each PM at a concentration of 2 × MIC for 24 h at 37°C (Banakar et al. 2023). After completing the incubation period, the biofilm on the teeth was fixed with 2.5% glutaraldehyde. The samples were subsequently dehydrated using ascending concentrations of ethanol (50%, 60%, 70%, 80%, 90%, and two rounds of 100%) and coated with a thin layer of palladium‐gold for visualization (Banakar et al. 2023; Jeyarajan et al. 2025).

2.5. Evaluation of Metabolic Activity Using the XTT Reduction Assay

To measure the metabolic activity of C. albicans biofilms and avoid effects related to cell death caused by the MIC, biofilms were assessed using the XTT reduction assay kit (2,3‐bis(2‐methoxy‐4‐nitro‐5‐sulfophenyl)‐5‐[(phenylamino)carbonyl]‐2H‐tetrazolium hydroxide) (XTT assay kit; Applied Science Roche, Indianapolis, IN, USA) according to the manufacturer's instructions. Initially, 100 μL of a C. albicans suspension at a concentration of 1.5 × 106 CFU/mL was added to a flat‐bottomed 96‐well microplate and incubated at 37°C for 24 h. The cells were then treated with 1/2 × MIC of each PM for 24 h at 37°C. This concentration was selected to study the effects of PMs on metabolic activity while avoiding effects related to cell death caused by the MIC and higher concentrations. After incubation, 50 μL of the XTT solution was added to each well, and the microplate was incubated in the dark at 37°C for 2 h. Absorbance was measured at 490 nm using a microplate reader (Cosio et al. 2025).

2.6. Statistical Analysis

Each experiment was performed in triplicate (n = 3), and results from the experimental groups are expressed as percentages relative to the control group. Although normality was assessed using the Shapiro‐Wilk test prior to parametric analyses, it is acknowledged that this test has limited statistical power at n = 3; results should therefore be interpreted with appropriate caution. Pairwise comparisons of CFU/mL values between each PM treatment group (LGG or LR) and the untreated control were performed using a two‐tailed Student's t‐test. Comparisons of metabolic activity (XTT absorbance values) across all three groups (LGG PMs, LR PMs, and control) were performed using one‐way analysis of variance (ANOVA) followed by Dunnett's post hoc test. All results are presented as mean ± standard deviation, and a p‐value of less than 0.05 was considered statistically significant.

3. Results

3.1. MIC and MFC of the Postbiotic Mediators

To determine the lowest concentration of PMs that inhibited the growth of C. albicans, serial dilutions of PMs were prepared and tested. The MIC for both LGG‐ and LR‐derived PMs was 25% (v/v), as no visible turbidity was observed at higher concentrations. No growth of C. albicans was observed on SDA at concentrations above 25% (v/v), confirming the MFC value. The MFC values were equal to the MIC (25% v/v) for both PMs, indicating fungicidal activity.

3.2. Biofilm Destruction of C. albicans by Postbiotic Mediators

Colony‐forming unit (CFU/mL) enumeration was used to evaluate the biofilm‐disruption effect of the PMs on C. albicans biofilm (Figure 1). The mean CFU/mL values were (3.2 ± 0.25) × 107, (2.64 ± 0.19) × 107, and (3.01 ± 0.28) × 107 for the control, LGG PM, and LR PM groups, respectively. Both PMs yielded lower CFU counts than the control; however, the reduction was statistically significant only in the LGG group (p = 0.0038) and not in the LR group (p = 0.1537).

Figure 1.

Figure 1

Open in a new tab

Changes in Candida albicans colony‐forming units (CFU/mL) after treatment with postbiotic mediators. **p < 0.01.

3.3. Scanning Electron Microscope Analysis

Scanning electron microscopy (SEM) analysis revealed reduced microbial density and fewer microcolonies on the surface following treatment with PMs, indicating changes in biofilm architecture (Figure 2). Consistent with the CFU/mL measurements, LGG‐derived PMs exhibited a more pronounced antibiofilm effect than LR‐derived PMs.

Figure 2.

Figure 2

Open in a new tab

Scanning electron microscopy images of Candida albicans biofilms treated with postbiotic mediators (Scale bar = 10 μm; Mag = 1.00 KX).

3.4. Inhibitory Effect of PMs on C. albicans Metabolic Activity

To measure the metabolic activity of C. albicans biofilms, we used the XTT reduction assay (Figure 3). The mean (± SD) absorbance values of the control, LGG PMs, and LR PMs were 1.78 ± 0.09, 0.96 ± 0.06, and 1.12 ± 0.08, respectively. The PMs from both the LGG and LR groups significantly reduced the metabolic activity of C. albicans compared with the control (p = 0.0009 and p = 0.0015, respectively). These findings indicate that PMs reduced C. albicans biofilm metabolic activity under the tested conditions.

Figure 3.

Figure 3

Open in a new tab

Mean absorbance values of Candida albicans metabolic activity based on the XTT reduction assay. *p < 0.01, **p < 0.001. Error bars represent standard deviations.

4. Discussion

Probiotic Lactobacillus species have received substantial attention for their antimicrobial properties (Ji et al. 2023; Nader‐Macías and De Gregorio 2022; Prajapati et al. 2023), and antibacterial and antifungal effects of these bacteria have been reported (Ji et al. 2023; Matsubara et al. 2016; Prajapati et al. 2023). Among the proposed antifungal mechanisms of Lactobacillus species is inhibition of morphogenesis, including suppression of hyphal growth in Candida spp. (Abdel‐Nasser et al. 2023). Lactobacillus species may also weaken the fungal cell wall through the production of hydrolytic enzymes, such as chitinase and β‐glucanase, potentially increasing susceptibility to other antifungal agents (Abdel‐Nasser et al. 2023). In addition, metabolites produced by Lactobacillus species may influence host immune responses and inhibit fungal growth (Abdel‐Nasser et al. 2023; Nader‐Macías and De Gregorio 2022).

PMs comprise bioactive compounds and metabolites produced by probiotic bacteria through metabolic activity or cellular breakdown and have been reported to possess diverse biological properties (Chakraborty et al. 2025; Fang et al. 2025). PMs can contain substances such as bacteriocins, secreted peptides, antifungal proteins, and hydrogen peroxide that may contribute to antifungal activity (Abdel‐Nasser et al. 2023; Chakraborty et al. 2025; Prajapati et al. 2023). Some Lactobacillus‐derived metabolites can damage fungal cell membranes, leading to leakage of cell contents and cell death (Abdel‐Nasser et al. 2023; Chakraborty et al. 2025). For example, hydrogen peroxide can cause oxidative stress in fungal cells. In a study, the ability of LGG to produce and secrete the major secretory peptidoglycan hydrolase Msp1 (major secretory protein 1) was shown to contribute to chitin degradation in C. albicans hyphae (Allonsius et al. 2019). In addition, lactic acid in LGG‐derived preparations affected the glucan coating on the surface of C. albicans hyphae (Ballou et al. 2016). This may make hyphae more accessible to Msp1 activity, thereby contributing to Candida albicans damage (Allonsius et al. 2019; Ferreira et al. 2024; Song and Lee 2017). LR can also produce various organic acids, including acetic, lactic, oxaloacetic, propanoic, (hexa‐ and octa‐)decanoic, and benzoic acids, which have been detected in LR PMs and may contribute to growth restriction or fungal cell deformation (Bakhshi et al. 2021).

Postbiotics have been investigated for potential oral‐health applications, including inhibition of microorganisms associated with oral disease (Harat and Pourjafar 2025; Heidari et al. 2025). As noted above, postbiotic compounds may inhibit the growth of fungi and bacteria, including C. albicans (Banakar et al. 2023; Sharifzadeh et al. 2023; Wu et al. 2022). Recent studies have suggested that postbiotics may help maintain microbial balance by modulating the oral microbiota and enhancing antifungal activity, potentially reducing the risk of oral infections and dental caries (Harat and Pourjafar 2025; Heidari et al. 2025; Marunganathan et al. 2025). Various studies have also documented antifungal compounds produced by lactobacilli (Bakhshi et al. 2021; Banakar et al. 2023; Marunganathan et al. 2025; Song and Lee 2017).

The increasing prevalence of dental caries and its negative effects on public health and quality of life, especially among children, highlight the need for novel prevention methods (Ehizogie Paul Adeghe 2024). To address this need, we evaluated the antifungal activity of postbiotic mediators from LGG and LR against C. albicans. The MIC and MFC results showed that PMs derived from both lactobacilli inhibited C. albicans, corroborating previous findings on lactobacilli‐derived compounds (De Gregorio et al. 2019; Ferreira et al. 2024; Rajão et al. 2025). Biofilm maturation is associated with increased resistance to stress and antifungal therapies (Rajão et al. 2025; Zhang et al. 2024). Furthermore, substantial genetic and phenotypic differences exist between biofilm cells and their planktonic counterparts, contributing to increased tolerance and resistance to antimicrobial drugs (Esfandiary et al. 2024; Matsubara et al. 2016; Rajão et al. 2025). Consequently, treatments may be more effective against less organized cellular structures, such as planktonic cells, than against complex biofilms. Therefore, developing antifungal agents capable of targeting planktonic forms and preventing biofilm formation may improve treatment outcomes (Lu et al. 2023; Rajão et al. 2025).

Based on the biofilm assay, the CFU counts for both types of postbiotic mediators were lower than those of the control group. LGG‐derived PMs produced a greater reduction in C. albicans viability (CFU/mL) than LR‐derived PMs. It is worth noting that the absolute reduction in viable counts, while statistically significant for LGG PMs, was relatively modest (approximately a 17.5% decrease relative to control, or ~0.08 log10). Nevertheless, this viability reduction occurred alongside substantial suppression of metabolic activity (see XTT results below), indicating that the PMs exerted biologically relevant effects on biofilm physiology even though they did not achieve rapid, high‐level killing of biofilm‐embedded cells within the 24‐h exposure window. SEM analysis also revealed marked changes in C. albicans biofilm architecture following treatment with PMs derived from both lactobacilli strains. These findings align with previous studies demonstrating inhibitory effects of lactobacilli on C. albicans (Bakhshi et al. 2021; El‐Ashmony et al. (2023); Sharifzadeh et al. 2023; Song and Lee 2017).

The observed disparity in biofilm reduction between LGG PMs and LR‐derived PMs, despite comparable MIC/MFC values, may reflect strain‐specific differences in metabolite composition and biofilm‐targeted mechanisms (MacAlpine et al. 2021; Poon and Hui 2023). While MIC/MFC assays primarily assess fungicidal activity against planktonic cells, LGG PMs may contain metabolites—such as biosurfactants, proteases, or bacteriocins—that could destabilize biofilm architecture by affecting extracellular matrices or hyphal formation, a critical virulence trait of C. albicans (Jang et al. 2019; Parolin et al. 2015). Furthermore, LGG‐derived metabolites may exhibit enhanced penetration into biofilms or alter metabolic pathways involved in biofilm maintenance (Rossoni et al. 2018). Strain‐specific organic acids (e.g., lactic or acetic acid) could also interact with other antifungal components to impair biofilm viability. These possibilities are consistent with prior studies highlighting Lactobacillus strain variability in antimicrobial metabolite profiles and underscore the need for metabolite characterization to optimize postbiotic formulations for biofilm‐related infections (Heidari et al. 2025; Sharifzadeh et al. 2023; Yocheva et al. 2024).

XTT assay results demonstrated that PMs derived from LGG and LR significantly reduced the metabolic activity of C. albicans biofilms under the tested conditions. This finding indicates suppression of biofilm metabolic activity, but the XTT assay alone does not establish the molecular mechanism of action or demonstrate that a specific virulence pathway was inhibited. In particular, hyphal transition, fungal membrane integrity, extracellular‐matrix production, and biofilm‐associated gene expression were not directly measured in the present study. Previous investigations have shown that Lactobacillus‐derived factors can affect Candida filamentation and biofilm‐related phenotypes (Allonsius et al. 2019; Jang et al. 2019; MacAlpine et al. 2021; Matsubara et al. 2016), but whether the LGG and LR PMs tested here act through the same pathways remains to be determined. Accordingly, the present data should be interpreted as evidence of in vitro antifungal and antibiofilm activity that warrants targeted mechanistic confirmation.

Several limitations should be considered when interpreting these findings. First, only one reference strain of C. albicans (ATCC 10231) was evaluated; therefore, the results cannot be assumed to represent the susceptibility of genetically diverse clinical isolates, antifungal‐resistant strains, or non‐albicans Candida species. Future studies should test a broader panel of clinical isolates, including azole‐resistant strains. Second, the PMs were evaluated as crude cell‐free preparations and were not chemically characterized. Consequently, the concentrations and relative contributions of organic acids, peptides, proteins, bacteriocin‐like compounds, or other metabolites remain unknown. Metabolomic and targeted analytical approaches, including LC‐MS/MS, should be used to identify and quantify the components associated with antifungal and antibiofilm activity. Third, the study did not directly investigate mechanisms of action. Future work should evaluate hyphal transition, membrane permeability or integrity, extracellular‐matrix changes, and expression of genes involved in adhesion, filamentation, biofilm maturation, and stress responses. Fourth, all experiments were performed in vitro and therefore did not reproduce salivary flow, host immune responses, tissue interactions, or polymicrobial oral biofilms. These factors should be incorporated into more physiologically relevant models before efficacy is extrapolated to the oral cavity.

Fifth, cytotoxicity and biocompatibility were not assessed in oral epithelial cells, gingival fibroblasts, or other mammalian cell models; therefore, no conclusion regarding clinical safety can be drawn from the present data. Sixth, the study examined short‐term activity after 24‐h PM exposure and did not evaluate storage stability, persistence of antifungal activity over time, or sustained activity after incorporation into a clinically relevant formulation. Seventh, fixed concentrations were selected for the individual assays (2×MIC for mature‐biofilm disruption and 1/2 × MIC for the XTT assay), but a systematic concentration‐response analysis, exposure‐time optimization, and comparison of delivery approaches were not performed. Eighth, the PMs were not tested in combination with conventional antifungal agents; therefore, potential additive, synergistic, or antagonistic interactions remain unknown. These issues should be addressed in future dose‐optimization, formulation, stability, combination‐treatment, and safety studies.

Additional methodological limitations include the absence of a standard antifungal comparator (e.g., fluconazole) in the biofilm and metabolic‐activity assays, which limits direct benchmarking of PM efficacy. The pH of the PM preparations was adjusted to physiological pH (7.2–7.4), a step that may reduce the activity of pH‐sensitive organic acids such as lactic and acetic acid. Future studies should include uninoculated, pH‐adjusted MRS broth as a vehicle control and should evaluate PM activity across a defined pH range to clarify the contribution of organic acids. In addition, quantitative biofilm assays were performed in polystyrene 96‐well plates, whereas SEM analysis used tooth slabs, introducing a substrate variable that may affect biofilm architecture and limit direct cross‐method comparison. Standardization of the biofilm substrate across complementary assays would strengthen future studies.

5. Conclusions

The present in vitro findings demonstrate that LGG‐ and LR‐derived PMs suppress the metabolic activity of C. albicans biofilms, whereas a statistically significant reduction in viable biofilm counts was detected only with LGG PMs. SEM observations were consistent with altered biofilm architecture, particularly after LGG PM treatment. Under the conditions tested, LGG PMs showed a greater antibiofilm effect; however, these experiments do not establish clinical efficacy, safety, or a specific molecular mechanism.

Further research should evaluate diverse clinical and antifungal‐resistant Candida isolates; chemically characterize active metabolites using LC‐MS/MS or complementary metabolomic approaches; investigate hyphal transition, membrane integrity, biofilm‐associated gene expression, and other relevant mechanisms; and assess cytotoxicity in oral epithelial and fibroblast models. In vivo studies should subsequently evaluate efficacy and safety under conditions that incorporate salivary flow, host responses, tissue interactions, and polymicrobial communities. Dose‐response relationships, exposure time, formulation and delivery strategies, storage stability, sustained activity, and combinations with conventional antifungal agents should also be investigated before therapeutic or preventive applications are considered.

Author Contributions

Fatemeh Amirinia: investigation, data curation, writing – original draft. Morteza Banakar: conceptualization, methodology, supervision, funding acquisition, writing – review and editing. Mojgan Alaeddini: conceptualization and methodology. Shahroo Etemad‐Moghadam: methodology, conceptualization, writing – review and editing. Maryam Pourhajibagher: investigation and formal analysis. Maryam Noori: investigation and data curation.

Ethics Statement

This study was conducted with the approval and supervision of the National Institute for Medical Research Ethics Committee (IR.NIMAD.REC.1400.148). All procedures were carried out following the relevant guidelines and regulations.

Consent

The authors have nothing to report.

Conflicts of Interest

The authors declare no conflicts of interest.

AI Tool Disclosure

In producing this manuscript, the authors used grammar and language assistance tools, including Grammarly and a large language model (Claude, Anthropic), to improve clarity and readability. Following use of these tools, all content was critically reviewed, revised as necessary, and approved by the authors, who take full responsibility for the accuracy, integrity, and originality of the published work.

Acknowledgments

This study was supported by grants from the National Institute of Medical Sciences Research, Tehran, Iran (Grant number: 4001382). The funding bodies did not participate in its design, collection, analysis, data interpretation, or manuscript writing.

Contributor Information

Morteza Banakar, Email: Dr.mbanakar@gmail.com.

Mojgan Alaeddini, Email: malaeddini@yahoo.com.

Data Availability Statement

The data sets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

References

  1. Abdel‐Nasser, M. , Abdel‐Maksoud G., Eid A. M., et al. 2023. “Antifungal Activity of Cell‐Free Filtrate of Probiotic Bacteria Lactobacillus rhamnosus ATCC‐7469 Against Fungal Strains Isolated From a Historical Manuscript.” Microorganisms 11, no. 5: 1104. 10.3390/microorganisms11051104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Abu‐Humaidan, A. H. , Alshdaifat A., Awajan D., et al. 2025. “The Antimicrobial Resistance of Candida: A 5‐Year Retrospective Analysis at a Tertiary Hospital in Jordan.” Journal of Fungi 11, no. 2: 87. 10.3390/jof11020087. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Allonsius, C. N. , Vandenheuvel D., Oerlemans E. F. M., et al. 2019. “Inhibition of Candida albicans Morphogenesis by Chitinase From Lactobacillus rhamnosus GG.” Scientific Reports 9, no. 1: 2900. 10.1038/s41598-019-39625-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bakhshi, M. , Salari S., Almani P. G. N., and Afshari S. A. K.. 2021. “Evaluation of the Antifungal Activity of Lactobacillus reuteri Against Candida Species.” Gene Reports 25: 101369. 10.1016/j.genrep.2021.101369. [DOI] [Google Scholar]
  5. Ballou, E. R. , Avelar G. M., Childers D. S., et al. 2016. “Lactate Signalling Regulates Fungal β‐glucan Masking and Immune Evasion.” Nature Microbiology 2, no. 2: 16238. 10.1038/nmicrobiol.2016.238. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Banakar, M. , Pourhajibagher M., Etemad‐Moghadam S., et al. 2023. “Antimicrobial Effects of Postbiotic Mediators Derived From Lactobacillus rhamnosus GG and Lactobacillus reuteri on Streptococcus mutans .” Frontiers in Bioscience‐Landmark 28, no. 5: 88. 10.31083/j.fbl2805088. [DOI] [PubMed] [Google Scholar]
  7. Cao, J. , Ma Q., Shi J., et al. 2025. “Cariogenic Microbiota and Emerging Antibacterial Materials to Combat Dental Caries: A Literature Review.” Pathogens 14, no. 2: 111. 10.3390/pathogens14020111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chakraborty, T. , Roy S., Sen S. K., et al. 2025. “Postbiotics From Lactobacillus .” In Postbiotics, 51–76. Elsevier. [Google Scholar]
  9. Cilo, B. D. , and Ener B.. 2021. “Comparison of Clinical Laboratory Standards Institute (CLSI) Microdilution Method and VITEK 2 Automated Antifungal Susceptibility System for the Determination of Antifungal Susceptibility of Candida Species.” Cureus 13, no. 12: e20220. 10.7759/cureus.20220. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Clinical and Laboratory Standards Institute . 2008. “Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts (CLSI document M27‐A3).” CLSI.
  11. Clinical and Laboratory Standards Institute . 2017. “Performance Standards for Antifungal Susceptibility Testing of Yeasts (CLSI supplement M60).” CLSI.
  12. Cosio, T. , Romeo A., Pistoia E. S., et al. 2025. “Retinoids as Alternative Antifungal Agents Against Candida albicans: In Vitro and In Silico Evidence.” Microorganisms 13, no. 2: 237. 10.3390/microorganisms13020237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Du, Q. , Ren B., He J., et al. 2021. “ Candida albicans Promotes Tooth Decay by Inducing Oral Microbial Dysbiosis.” ISME journal 15, no. 3: 894–908. 10.1038/s41396-020-00823-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Ehizogie Paul Adeghe, A. 2024. “The Multifaceted Role of Fluoride in Preventing Early Childhood Caries: A Comprehensive Review.” International Journal of Life Science Research Updates 2, no. 01: 009–017. [Google Scholar]
  15. Eidt, G. , Andrade C. G., Negrini T. C., and Arthur R. A.. 2019. “Role of Candida albicans on Enamel Demineralization and on Acidogenic Potential of Streptococcus mutans in Vitro Biofilms.” Journal of Applied Oral Science 27: e20180593. 10.1590/1678-7757-2018-0593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. El‐Ashmony, M. M. , Raslan F. S., Abdelhamid D. H., Hafez H. M., and A El‐Rahman O. A.. 2023. “Potential Anti‐Fungal Activity of Lactobacilli Probiotic Against Fluconazole Resistant Candida albicans Clinical Isolates.” Azhar International Journal of Pharmaceutical and Medical Sciences 3, no. 1: 120–131. [Google Scholar]
  17. El‐Mashad, N. , Foad M. F., Saudy N., and Salem D. A.. 2012. “Susceptibility Tests of Oropharyngeal Candida albicans From Egyptian Patients to Fluconazole Determined by Three Methods.” Brazilian Journal of Microbiology 43: 266–273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Esfandiary, M. A. , Khosravi A. R., Asadi S., Nikaein D., Hassan J., and Sharifzadeh A.. 2024. “Antimicrobial and Anti‐Biofilm Properties of Oleuropein Against Escherichia coli and Fluconazole‐Resistant Isolates of Candida albicans and Candida glabrata .” BMC Microbiology 24, no. 1: 154. 10.1186/s12866-024-03198-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Ev, L. D. , Damé‐Teixeira N., Do T., Maltz M., and Parolo C. C. F.. 2020. “The Role of Candida albicans in Root Caries Biofilms: An RNA‐seq Analysis.” Journal of Applied Oral Science 28: e20190578. 10.1590/1678-7757-2019-0578. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Fang, H. , Rodrigues e‐Lacerda R., Barra N. G., et al. 2025. “Postbiotic Impact on Host Metabolism and Immunity Provides Therapeutic Potential in Metabolic Disease.” Endocrine Reviews 46, no. 1: 60–79. 10.1210/endrev/bnae025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ferreira, R. L. P. S. , Nova B. G. V., Carmo M. S., and Abreu A. G.. 2024. “Mechanisms of Action of Lactobacillus spp. in the Treatment of Oral Candidiasis.” Brazilian Journal of Biology 84: e282609. 10.1590/1519-6984.282609. [DOI] [PubMed] [Google Scholar]
  22. De Gregorio, P. R. , Silva J. A., Marchesi A., and Nader‐Macías M. E. F.. 2019. “Anti‐Candida Activity of Beneficial Vaginal Lactobacilli in In Vitro Assays and in a Murine Experimental Model.” FEMS Yeast Research 19, no. 2: foz008. 10.1093/femsyr/foz008. [DOI] [PubMed] [Google Scholar]
  23. Harat, S. G. , and Pourjafar H.. 2025. “Health Benefits and Safety of Postbiotics Derived From Different Probiotic Species.” Current Pharmaceutical Design 31, no. 2: 116–127. 10.2174/0113816128321617241009074137. [DOI] [PubMed] [Google Scholar]
  24. Heidari, F. , Heboyan A., Rokaya D., et al. 2025. “Postbiotics and Dental Caries: A Systematic Review.” Clinical and Experimental Dental Research 11, no. 1: e70114. 10.1002/cre2.70114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Hill, C. , Guarner F., Reid G., et al. 2021. “Expert Consensus Document: The International Scientific Association for Probiotics and Prebiotics Consensus Statement on the Scope and Appropriate Use of the Term Postbiotic.” Nature Reviews Gastroenterology & Hepatology 18, no. 9: 649–665. 10.1038/s41575-021-00488-4. [DOI] [PubMed] [Google Scholar]
  26. Homayouni Rad, A. , Pourjafar H., and Mirzakhani E.. 2023. “A Comprehensive Review of the Application of Probiotics and Postbiotics in Oral Health.” Frontiers in Cellular and Infection Microbiology 13: 1120995. 10.3389/fcimb.2023.1120995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Jang, S. J. , Lee K., Kwon B., You H. J., and Ko G.. 2019. “Vaginal Lactobacilli Inhibit Growth and Hyphae Formation of Candida albicans .” Scientific Reports 9, no. 1: 8121. 10.1038/s41598-019-44579-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Jeyarajan, S. , Kandasamy I., Veerapandian R., et al. 2025. “Antibiofilm Activity of Protamine Against the Vaginal Candidiasis Isolates of Candida albicans, Candida tropicalis and Candida krusei .” Applied Biosciences 4, no. 1: 5. 10.3390/applbiosci4010005. [DOI] [Google Scholar]
  29. Ji, J. , Jin W., Liu S. J., Jiao Z., and Li X.. 2023. “Probiotics, Prebiotics, and Postbiotics in Health and Disease.” MedComm 4, no. 6: e420. 10.1002/mco2.420. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Li, W. , Yu D., Gao S., Lin J., Chen Z., and Zhao W.. 2014. “Role of Candida Albicans‐Secreted Aspartyl Proteinases (Saps) in Severe Early Childhood Caries.” International Journal of Molecular Sciences 15, no. 6: 10766–10779. 10.3390/ijms150610766. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lu, Y. , Lin Y., Li M., and He J.. 2023. “Roles of Streptococcus mutans–Candida albicans Interaction in Early Childhood Caries: A Literature Review.” Frontiers in Cellular and Infection Microbiology 13: 1151532. 10.3389/fcimb.2023.1151532. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. MacAlpine, J. , Daniel‐Ivad M., Liu Z., et al. 2021. “A Small Molecule Produced by Lactobacillus Species Blocks Candida albicans Filamentation by Inhibiting a DYRK1‐Family Kinase.” Nature Communications 12, no. 1: 6151. 10.1038/s41467-021-26390-w. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Marunganathan, V. , Guru A., and Arockiaraj J.. 2025. “Exploring Microbiome‐Based Interventions in Periodontitis: The Role of Prebiotics and Postbiotics in Oral Health Management.” In Metagenomics, 289–299. Elsevier. [Google Scholar]
  34. Matsubara, V. H. , Wang Y., Bandara H. M. H. N., Mayer M. P. A., and Samaranayake L. P.. 2016. “Probiotic Lactobacilli Inhibit Early Stages of Candida albicans Biofilm Development by Reducing Their Growth, Cell Adhesion, and Filamentation.” Applied Microbiology and Biotechnology 100, no. 14: 6415–6426. 10.1007/s00253-016-7527-3. [DOI] [PubMed] [Google Scholar]
  35. Mudenda, S. 2024. “Global Burden of Fungal Infections and Antifungal Resistance From 1961 to 2024: Findings and Future Implications.” Pharmacology & Pharmacy 15, no. 4: 81–112. 10.4236/pp.2024.154006. [DOI] [Google Scholar]
  36. Nader‐Macías, M. E. F. , and De Gregorio P. R.. 2022. “Probiotics and Urogenital Health.” In Probiotics, 355–388. Elsevier. [Google Scholar]
  37. Pallavi, P. , Kumar V., Prakash S., and Raut S.. 2024. “Probiotics: A Novel Natural Therapy for Oral Health.” In Applied Biotechnology and Bioinformatics: Agriculture, Pharmaceutical Research and Environment, edited by Thatoi H., Mohapatra S., Das S. K., and S. K. Pradhan, 157–180. John Wiley & Sons. 10.1002/9781119896869.ch7. [DOI] [Google Scholar]
  38. Parolin, C. , Marangoni A., Laghi L., et al. 2015. “Isolation of Vaginal Lactobacilli and Characterization of Anti‐Candida Activity.” PLoS One 10, no. 6: e0131220. 10.1371/journal.pone.0131220. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Poon, Y. , and Hui M.. 2023. “Inhibitory Effect of Lactobacilli Supernatants on Biofilm and Filamentation of Candida albicans, Candida tropicalis, and Candida parapsilosis .” Frontiers in Microbiology 14: 1105949. 10.3389/fmicb.2023.1105949. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Prajapati, N. , Patel J., Singh S., et al. 2023. “Postbiotic Production: Harnessing the Power of Microbial Metabolites for Health Applications.” Frontiers in Microbiology 14: 1306192. 10.3389/fmicb.2023.1306192. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Rajão, A. , Silva J. P. N., Almeida‐Nunes D. L., Rompante P., Rodrigues C. F., and Andrade J. C.. 2025. “ Limosilactobacillus reuteri AJCR4: A Potential Probiotic in the Fight Against Oral Candida spp. Biofilms.” International Journal of Molecular Sciences 26, no. 2: 638. 10.3390/ijms26020638. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Raposa, J. , and Vazquez J. A.. 2025. “New Pharmacotherapeutic Strategies for Drug‐Resistant Candida Infections: A Review.” Expert Opinion on Pharmacotherapy 26, no. 16: 1713–1723. 10.1080/14656566.2024.2433605. [DOI] [PubMed] [Google Scholar]
  43. Rawnuck, T. , Reza M. S., Jahan N., Hossain A. I., Fatema B., and Fatteh Ul Islam M.. 2025. “Risk Elements Linked to Oral Candidiasis.” Medicine Today 37, no. 1: 93–98. [Google Scholar]
  44. Regis, W. F. M. , Mustafa Gomes Muniz F. W., Rocha F. R., et al. 2025. “Streptococcus Mutans Serotyping, Collagen‐Binding Genes and Candida albicans in Dentin Carious Lesions: A Molecular Approach.” Clinical Oral Investigations 29, no. 1: 52. 10.1007/s00784-024-06122-y. [DOI] [PubMed] [Google Scholar]
  45. Rodríguez‐Tudela, J. L. , Barchiesi F., Bille J., et al. 2003. “Method for the Determination of Minimum Inhibitory Concentration (MIC) by Broth Dilution of Fermentative Yeasts.” Clinical Microbiology and Infection 9, no. 8: i–viii. 10.1046/j.1469-0691.2003.00789.x. [DOI] [Google Scholar]
  46. Rossoni, R. D. , de Barros P. P., de Alvarenga J. A., et al. 2018. “Antifungal Activity of Clinical Lactobacillus Strains Against Candida albicans Biofilms: Identification of Potential Probiotic Candidates to Prevent Oral Candidiasis.” Biofouling 34, no. 2: 212–225. 10.1080/08927014.2018.1425402. [DOI] [PubMed] [Google Scholar]
  47. Samuel, A. M. , Lammers M. G., Nachreiner J., et al. 2024. “Multiple Lactobacillus Infections Caused by Probiotics at Pediatric and Adult Academic Medical Centers.” WMJ: Official Publication of the State Medical Society of Wisconsin 123, no. 4: 272–277. [PubMed] [Google Scholar]
  48. Seyedjavadi, S. , Hatami F., and Jahanshiri Z.. 2024. “Antifungal Resistance in Candida albicans .” In Candida albicans—Epidemiology and Treatment,edited by Behzadi P., 69–92. IntechOpen. 10.5772/intechopen.1004578. [DOI] [Google Scholar]
  49. Sharifzadeh, S. S. , Lotfali E., Lesan S., and Farrokhnia T.. 2023. “Antifungal Effect of Probiotic Lactobacillus casei on Drug‐Resistant Oral Candida albicans Isolated From Patients With Hematological Malignancy: An In Vitro Study.” Journal of dentistry (Shiraz, Iran) 24, no. 4: 389–394. 10.30476/dentjods.2022.94792.1812. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Song, Y.‐G. , and Lee S.‐H.. 2017. “Inhibitory Effects of Lactobacillus Rhamnosus and Lactobacillus casei on Candida Biofilm of Denture Surface.” Archives of Oral Biology 76: 1–6. 10.1016/j.archoralbio.2016.12.014. [DOI] [PubMed] [Google Scholar]
  51. Wu, Y. , Hu S., Wu C., Gu F., and Yang Y.. 2022. “Probiotics: Potential Novel Therapeutics Against Fungal Infections.” Frontiers in Cellular and Infection Microbiology 11: 793419. 10.3389/fcimb.2021.793419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Yeşilyurt, N. , Yılmaz B., Ağagündüz D., and Capasso R.. 2021. “Involvement of Probiotics and Postbiotics in the Immune System Modulation.” Biologics: Targets & Therapy 1, no. 2: 89–110. 10.3390/biologics1020007. [DOI] [Google Scholar]
  53. Yocheva, L. , Tserovska L., Danguleva‐Cholakova A., et al. 2024. “In Vitro Inhibitory Effects and Co‐Aggregation Activity of Lactobacilli on Candida albicans .” Microbiology Research 15, no. 3: 1576–1589. 10.3390/microbiolres15030105. [DOI] [Google Scholar]
  54. Zhang, Q. , Zhang J., Zhang Y., et al. 2024. “Antifungal and Anti‐Biofilm Activities of Patchouli Alcohol Against Candida albicans .” International Journal of Medical Microbiology 314: 151596. 10.1016/j.ijmm.2024.151596. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The data sets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Articles from MicrobiologyOpen are provided here courtesy of Wiley

RESOURCES