Antifungal Efficacy and Chemical Profiles of Salvia, Oregano, and Laurus Essential Oils Against Mold Species Associated with Caciocavallo Cheese Spoilage

Mold contamination is a major cause of spoilage in ripened cheeses. In this study, 171 fungal isolates were recovered from semi-hard Caciocavallo cheese sampled across three seasons. The number of recovered isolates showed a descriptive decrease from summer to winter. Thirty-two representatives were selected for ITS and/or β-tubulin sequencing after morphotype screening. Among these sequenced representatives, Penicillium commune was the most frequent provisional taxonomic designation (8/32; 25.0%), followed by Aspergillus flavus (3/32; 9.4%). Penicillium echinulatum, Fusarium iranicum, Paecilomyces variotii, and Cladosporium-like isolates each accounted for 2/32 (6.3%). ITS-only assignments in taxonomically difficult genera are treated as provisional closest-match designations rather than definitive species identifications. Essential oils (EOs) from three Salvia rosmarinus Spenn. chemotypes (SR-CV2-23, SR-CV3-23, and SR-CV6-23), oregano cv. Carva (Origanum vulgare L. subsp. viridulum (Martrin-Donos) Nyman × O. vulgare L. subsp. hirtum Ietswaart; Ov-C-23), and Laurus nobilis L. (Ln-23) were chemically characterized and evaluated for their antifungal activity against mold species associated with Caciocavallo cheese spoilage. GC-MS showed that the rosemary EOs were rich in camphor, α-pinene, and 1,8-cineole; Ov-C-23 contained 83.95% carvacrol; and Ln-23 contained mainly 1,8-cineole, linalool, and bornyl acetate. All EOs exhibited concentration-dependent antifungal activity, with spore germination generally more sensitive than mycelial growth. At 0.35% (v/v), SR-CV2-23 showed the broadest observed in-vitro activity, whereas Ov-C-23 produced the highest observed inhibition of A. alternata mycelial growth. CFDA/PI confocal microscopy showed the largest relative shift toward reduced metabolic activity and membrane compromise after SR-CV3-23 treatment. In-vitro assays and cheese-slice experiment used different quantitative endpoints and were therefore not directly compared. Ov-C-23 produced the highest observed reduction in visible colonies in the cheese slice model. These EOs may contribute to integrated mold control strategies, but practical application requires further validation under industrial conditions.