Targeting Alternaria alternata in cheese: Multi-mechanism antifungal activity of essential oils integrating lethal and adaptive stress responses with preliminary olfactory evaluation.

Mold spoilage is a major challenge in semi-hard cheese production, causing economic losses and quality deterioration. This study evaluated the antifungal activity and mechanisms of essential oils (EOs) from Origanum vulgare (cultivar CARVA) and Thymus vulgaris (cultivar VARICO) against A. alternata isolated from Caciocavallo cheese alongside a preliminary assessment of their olfactory impact. A cheese agar medium (CAM) was developed to mimic the cheese matrix. The MICs of O. vulgare and T. vulgaris EOs were 0.94% and 0.12% (v/v), respectively, and both concentrations prevented fungal growth on inoculated cheese slices during refrigerated storage. T. vulgaris EO showed greater antifungal potency, achieving complete inhibition at a lower concentration than O. vulgare EO in both in vitro and in situ assays. Confocal microscopy showed plasma membrane depolarization, mitochondrial dysfunction, ROS/RNS accumulation, nuclear and structural alterations, and ultimately loss of fungal viability. At sublethal exposure, lipid droplet and chitin accumulation indicated adaptive stress responses. Overall, oregano and thyme EOs acted through a multi-target antifungal mechanism involving membrane disruption, oxidative/nitrosative stress, and cellular damage. Furthermore, a preliminary olfactory evaluation conducted alongside the efficacy trials revealed that high EO concentrations (3.75% and 1.88%) imparted intense herbal aromas and produced greater olfactory alteration relative to the untreated control, whereas treatments at the MIC levels produced comparatively limited olfactory changes. These results suggest that, at the concentrations required for antifungal efficacy, the tested EOs have a detectable olfactory impact, warranting further investigation into their potential application as natural preservatives for hard cheeses.