Carbon source modulates the antifungal and antibiofilm activities of Lactiplantibacillus plantarum S61 supernatants against Rhodotorula glutinis UMP22

Natural metabolites produced by lactic acid bacteria (LAB) are emerging as sustainable alternatives to synthetic antifungal agents. This study evaluated the antifungal and antibiofilm activities of cell-free supernatants (CFSs) derived from <i>Lactiplantibacillus plantarum</i> S61, cultivated with glucose (CFS-Gl) or arabinose (CFS-Ar), against the spoilage and opportunistic yeast <i>Rhodotorula glutinis</i> UMP22. Antifungal activity was determined by agar diffusion and MIC/MFC assays, while stability was assessed under variable temperature, pH, and enzymatic conditions. Membrane permeability, evaluated through propidium iodide uptake, confirmed that CFSs disrupt fungal cell integrity, indicating a multifactorial mode of action involving organic acids and protease-sensitive peptides. CFS-Ar exhibited significantly higher antifungal potency than CFS-Gl, achieving an inhibition zone of 36.7 ± 0.4 mm. When combined with sub-inhibitory concentrations of cycloheximide (MIC/2 = 15.5 μg/mL; MIC/3 = 10.3 μg/mL), both CFSs produced a synergistic effect, achieving near-complete growth suppression while lowering the required antifungal dose. In addition, both CFSs demonstrated strong preventive and curative antibiofilm activities across a wide range of temperatures (4-37 °C), concentrations, and exposure durations. The persistence of activity under refrigeration highlights their relevance for food preservation. Overall, these findings identify <i>L. plantarum</i> S61 as a promising source of thermostable, pH-tolerant bioactive metabolites with dual antifungal and antibiofilm functions. The observed synergy between CFSs and conventional antifungals offers an innovative, eco-friendly, and resistance-mitigating strategy for controlling spoilage yeasts and emerging fungal contaminants in food and health-related applications.