Enhancement of Ozonated oil for veterinary applications

The therapeutic potential of ozonated extra virgin olive oil (EOO) has gained increasing attention due to its antimicrobial, antifungal, antioxidant and regenerative properties. This study investigates the production, characterisation and biological effects of EOO, with particular emphasis on its application in veterinary medicine, in particular in bovine mastitis. EOO was produced by a controlled ozonation process, optimising the conditions to achieve stable ozonide formation while preserving the bioactive components of the oil. Titration and gas chromatography-mass spectrometry (GC-MS) analyses confirmed a direct correlation between ozonation time and ozonide concentration. In view of the increasing prevalence of antibiotic-resistant pathogens in bovine mastitis, the study investigated the antimicrobial and antibiofilm activity of EOO against Gram-positive (Staphylococcus aureus, Streptococcus agalactiae) and Gram-negative (Escherichia coli, Pseudomonas aeruginosa) bacteria. The results showed a dose- and time-dependent bactericidal effect, with S. aureus and S. agalactiae showing significant viability reduction at lower concentrations, while E. coli and P. aeruginosa required higher concentrations due to their outer membrane resistance. In addition, EOO effectively inhibited biofilm formation, particularly for S. aureus, reducing biofilm biomass by up to 70% after 24 hours of treatment. These results support the potential of EOO as an alternative therapeutic approach to mastitis management, reducing the reliance on antibiotics in the dairy industry. In addition to its antibacterial properties, EOO exhibited potent antifungal activity against Candida albicans and Candida glabrata. The study showed a dose-dependent inhibition of fungal viability and biofilm formation, with over 90% C. albicans cell death at a concentration of 3% (v/v) of ozonated oil. Transmission electron microscopy (TEM) revealed structural damage to the fungal cell wall, while reactive oxygen species (ROS) levels increased by 60% within 10 minutes of exposure. Upregulation of autophagy-related genes (atg-7 and atg-13) further suggested a stress-induced cell death response. These results pose EOO as a promising alternative antifungal agent, particularly in biofilm-associated infections. In addition, EOO demonstrated antioxidant and regenerative properties in eukaryotic cells, reinforcing its therapeutic and cosmetic potential. In 3T3 fibroblasts and HaCaT keratinocytes, EOO improved cell viability, mitochondrial function and resistance to oxidative stress. JC-1 and MitoSOX assays indicated improved mitochondrial membrane potential and reduced superoxide production, while catalase activity assays confirmed its ability to alleviate intracellular oxidative stress. Protein 2 carbonylation levels were significantly reduced, further supporting its cytoprotective effects. These findings highlight EOO as a multifunctional bioactive agent with broad-spectrum antimicrobial, antifungal and antioxidant properties, making it a potential candidate for applications in veterinary medicine. Future research should focus on elucidating the molecular mechanisms and evaluating its applicability for sustainable therapeutic solutions not only restricted to bovine mastitis.