Interaction mechanisms of core dominant fungi during fermentation of Massa Medicata Fermentata: volatile compounds of Saccharomycopsis fibuligera inhibit the growth of Rhizopus oryzae

Massa Medicata Fermentata (MMF) is a traditional fermented medicine whose quality is governed by complex microbial interactions. This study investigated the antagonistic effect of the core yeast Saccharomycopsis fibuligera against the filamentous fungus Rhizopus oryzae and its underlying mechanisms. In vitro co-culture assays demonstrated that S. fibuligera suppressed R. oryzae growth and amylase activity in a cell-density-dependent manner, with significant inhibition observed at cell densities of ≥1 × 10⁷ cells·mL⁻¹. The antagonism was driven by two putative pathways: direct spatial/nutritional competition and indirect suppression mediated by yeast-secreted volatile organic compounds (VOCs). HS-SPME-GC-MS analysis identified six major VOCs, primarily fatty acid ethyl esters and 2-phenylethanol, with the latter being the most abundant (0.5664 ± 0.0422 µg·g⁻¹ medium). Single-compound antifungal assays confirmed that isoamyl acetate, ethyl hexanoate, and 2-phenylethanol completely inhibited the growth of R. oryzae at elevated doses. Mechanistic investigations via SEM, PI staining, and biochemical assays revealed that these VOCs induce cell death by disrupting mycelial morphology, compromising membrane integrity, inhibiting ergosterol biosynthesis, and triggering lipid peroxidation. These findings elucidate a key antagonistic interaction between core functional fungi during MMF fermentation, providing a theoretical basis for targeted fermentation regulation and industrial standardization of this traditional herbal medicine. IMPORTANCE By revealing the cell-density-dependent dual-mode antagonism between S. fibuligera and R. oryzae , this study deepens our understanding of microbial interactions in traditional fermentation. The discovery of specific inhibitory VOCs and their membrane-disrupting mechanisms provides a theoretical basis for targeted process regulation. These insights are crucial for achieving precise fermentation control, which is essential for improving product consistency, reducing batch-to-batch variability, and advancing the traditional fermentation standardization of herbal medicines.