Mechanisms of Pulsed-Light Antifungal Activity: Direct Cellular Damage and Stress Adaptation in Alternaria alternata from Chees

Fungal spoilage in cheese production poses a significant risk because of mycotoxin production. As a result, it is crucial to use a structured approach that combines practical measures with strategies that inhibit fungal growth and activity. In this work, we studied the antifungal effect of pulsed light (PL) technology, mainly focusing on Alternaria alternata, a mycotoxin-producing spoilage fungus, frequently present in the semi-hard cheese matrix. Pulsed light was found to be efficient in controlling A. alternata, inhibiting its growth in a dose-dependent manner both on cheese agar medium (CAM) and potato dextrose agar (PDA). The CAM closely mimics the physicochemical properties of real cheese surfaces, making it a relevant model for practical applications. PL treatment caused up to 30 % inhibition of mycelial growth, while spore germination was significantly reduced by up to 79 %. Confocal microscopy revealed that PL induced cell death in A. alternata FCS26, which was marked by cellular and mitochondrial membrane potential loss, suggesting membrane depolarization. Additionally, PL elicited oxidative stress through increased intracellular levels of reactive oxygen and nitrogen species (ROS/RNS) and calcium, along with DNA damage and mitochondrial impairment. Chitin accumulation and lipid droplet reorganization were identified as adaptive responses reflecting cell wall reinforcement and altered lipid metabolism. Overall, this study provides new mechanistic insight into A. alternata inactivation and highlights PL as a promising, non-thermal antifungal intervention for the dairy industry.