Next-Generation Antifungal Preservation of Caciocavallo Cheese

This thesis investigated clean-label and non-thermal strategies for controlling fungal spoilage of Caciocavallo cheese while preserving quality of product. The work progressed from fungal ecology and essential-oil screening and 2nd control strategies by using pulsed-light, its validation, cellular mechanisms, gene expression, fungal metabolomics, and cheese-surface metabolomics. The integrated findings establish that fungal spoilage of Caciocavallo cheese represents a surface-driven, multi-species problem requiring targeted hurdle strategies rather than single antimicrobial interventions. Obtained results successfully bridges the gap between fundamental food science and industrial dairy application by evaluating plant derived Essential Oils (EOs) and Pulsed Light (PL) technology as independent, sustainable, surface-targeted hurdle strategies. Intended as waterless alternatives to traditional post-contamination washing protocols, both technologies were systematically assessed against dominant Caciocavallo cheese-spoiling fungal strains (Penicillium commune, and Alternaria alternata) to establish practical processing windows that balance microbial safety with matrix integrity and sensory acceptability. Thymus vulgaris, Origanum vulgare and PL treatments exert distinct, dose-dependent inhibitory effects on cheese borne fungi. EOs rich in carvacrol and thymol compounds significantly reduced mycelial growth, spore germination, and relative viability signals. In the cheese-slice model, a concentration of 0.125% for both oils achieved an optimal compromise between surface fungal suppression and olfactory acceptability. Similarly, PL fluence levels dictated fungal survival, effectively clearing superficial, early-stage fungal conidia. However, PL encountered physical barriers such as surface roughness, cheese opacity, and shadowing effects, which lowered its efficacy against highly pigmented established mycelium. Neither technology achieved complete fungal eradication or inhibition as a standalone treatment under real-world conditions. While both demonstrated robust fungistatic capabilities and not able to completely replace conventional chemical agents such as natamycin. Instead, the findings establish that both strategies should be integrated into multi-hurdle preservation frameworks to optimize performance. Mechanistic investigations clarified that fungal inactivation is achieved not through a single inhibitory pathway, Infact through multi-target cellular damage. EO exposure triggered plasma membrane depolarization, mitochondrial dysfunction, DNA damage, lipid droplet accumulation, and cell-wall chitin remodeling/accumulation. PL induced fungal death resulted from a synergistic combination of photochemical, photothermal, and photophysical mechanisms. PL caused extensive DNA damage and cell membrane disruption, accompanied by fluence-dependent accumulation of reactive oxygen and nitrogen species (ROS/RNS) that overwhelmed fungal antioxidant defenses. Furthermore, untargeted metabolomics confirmed that PL induces complex metabolic reprogramming in A. alternata FCS26, specifically disrupting fungal lipid and membrane homeostasis, sulfur/amino acid pathways, and secondary metabolism pathways. Defining precise treatment windows was essential to prevent detrimental modifications to the Caciocavallo matrix and quality parameters. The application of 0.125% EOs limited physical alterations mostly to the rind, with only minor changes in color and hardness, while leaving under-rind pH, water activity, and internal texture largely unaffected. Higher concentrations, however, were restricted by poor water solubility, high volatility, and intense herbal aromas that compromised the sensory profile. For the light-based treatment, a clear operational optimal treatment window was established at low to moderate intensities (30 PL and 60 PL), which significantly reduced natural contaminants while preserving the cheese’s physical and organoleptic properties which are important for traditional cheeses. Conversely, excessive exposure (120 PL) reached a detrimental threshold, triggering increased lipid oxidation, surface discoloration, increased firmness, and oxidized off-odors, although these sensory defects remained confined mainly to the outer rind and further validation is needed. Advanced integrated profiling revealed that PL operates as a tool for modulating the biochemical dynamics of the cheese rind during refrigerated storage. Untargeted metabolomics showed that while PL exposure restructured surface organic acids, amino acids, peptides, and carbohydrates, the differences between the individual PL doses (30 PL to 120 PL) were less pronounced than the baseline shift from the untreated control cheese. This confirms that optimized, lower-fluence treatments can drive favorable microbial restructuring without causing excessive, uncontrolled biochemical degradation of the dairy matrix. Seasonal sampling recovered 171 fungal isolates belonging to several genera, confirming that Caciocavallo spoilage is associated with a diverse surface mycobiota influenced by environmental conditions rather than a single contaminant. This diversity supported the selection of Penicillium commune FCS25 as a representative spoilage mold and Alternaria alternata FCS26 as an emerging cheese-associated fungus of potential mycotoxicological relevance. These findings established the biological basis for developing and evaluating surface targeted hurdle technologies based on essential oils and pulsed light.