Cold Limonene Vapor and UV Treatment Against <i>Salmonella</i> Enteritidis and <i>Staphylococcus aureus</i> in Quail Eggs: Potential Mode of Action

This study aimed to develop a novel, refrigeration-free preservation method to extend quail egg shelf life and enhance consumer safety. The antimicrobial efficacy of limonene vapor against <i>Salmonella</i> Enteritidis and <i>Staphylococcus aureus</i> was evaluated. Limonene nanoemulsions (0.04%, 0.08%, and 0.12%) were used to generate vapor for coating eggshells. The coating process was performed using an ultrasonic device with a controllable temperature chamber to maintain different temperatures: 4°C (cold), 25°C (medium), and 37°C (high temperature). The antimicrobial efficacy was enhanced by applying UV irradiation for 10 min. The mode of action was analyzed through bacterial morphology, biofilm formation, and cell leakage. Limonene vapor (0.08%) at 4°C with ultraviolet (UV) reduced <i>Salmonella</i> Enteritidis and <i>S. aureus</i> by 6-7 log₁₀ on eggshells, whereas at 25°C and 37°C, the reductions were only 3.5 log₁₀ and 2.8 log₁₀, respectively. <i>Salmonella</i> was not detected, and <i>S. aureus</i> remained within food safety standards when quail eggs were stored at 30 ± 2°C for 10 days, compared with the control, which showed bacterial growth within one day. Possible mechanisms of action suggested that cold-generated limonene formed a thin film on the eggshell surface. This film caused deformation and abnormal morphology in <i>Salmonella</i> Enteritidis cells and induced pore formation in <i>S. aureus</i> cell walls, enhancing bacterial cell injury. UV exposure further exacerbated cellular damage, resulting in a synergistic antimicrobial effect. However, this method did not cause significant membrane damage or lead to substantial leakage of intracellular materials in <i>S. aureus</i>. This method was cost-effective and could be adapted for on-farm application before distribution, ensuring safer eggs for consumers.