Multi-omics characterization of Saccharomyces boulardii in the biocontrol of Aspergillus flavus.

Aspergillus flavus pose a major threat due to aflatoxin contamination to the crops and is commonly controlled through chemical fungicides. However, these treatments raise serious concerns regarding environmental sustainability and human health, underscoring the pressing need for safer and more sustainable alternatives. This study investigates the Saccharomyces boulardii mediated inhibition of Aspergillus flavus using integrated proteomic and metabolomic approach. The activity of S. boulardii extract was assessed by the MTT assay, which demonstrated significant inhibition of A. flavus conidia with an MIC₅₀ of 1.57 mg/mL. Co-cultures of S. boulardii and A. flavus at ratios of 1:1, 10:1, and 20:1 were monitored from day 1 to day 6. A clear inhibition of A. flavus was observed at the 20:1 ratio. To understand the mechanism of inhibition, the proteomic analysis using nLC-ESI-MS/MS at 7 and 24 h (20:1, S. boulardii: A. flavus) identified 2,247 proteins and 2,142, respectively. From 2094 commonly expressed proteins, 782 were differentially expressed proteins (at 2-fold change); including 646 upregulated and 112 were downregulated proteins from S. boulardii, while upregulated and 6 downregulated proteins from A. flavus. The predominance of S. boulardii proteins indicates higher metabolic activity, particularly in pathways related to lipid, amino acids, and nutrient metabolism, whereas downregulation of A. flavus proteins involved in signaling and glucose metabolism suggests impaired energy production and stress response. Using LC/MS, metabolomic analysis of 20:1 co-culture at 24 h showed 16 metabolites linked to antimicrobial and antioxidant activities compared to metabolites from monoculture of A. flavus and S. boulardii. In addition, the co-culture extract exhibited significantly higher antioxidant activity (80%) compared to S. boulardii monoculture (22%). These findings highlight S. boulardii as a biocontrol agent against A. flavus, mediated through growth inhibition and metabolic interference.