Abstract
Background:
Kojic acid (KA) has recently garnered significant attention due to its broad-spectrum biological properties, including both antibacterial and antifungal activities, in animal, plant, and human applications. However, its clinical application remains constrained by poor physicochemical stability and susceptibility to oxidation. Nanoformulation strategies have emerged as promising approaches to overcome these issues. Natural kojic acid nanoencapsulation exhibited broad-spectrum antibacterial activity by disrupting microbial membranes, inducing oxidative stress, and interfering with metal-dependent enzymatic pathways. Furthermore, it exhibited excellent antifungal efficacy through improved cellular uptake and sustained release mechanisms. Simultaneously, its metal-chelating properties inhibit essential enzymes, such as chitin synthase and tyrosinase, which are vital for fungal cell wall formation.

Aim:
The present study aims to investigate the antibacterial and antifungal activities of natural KANEC against twelve pathogenic bacterial and fungal strains of animal, plant, and human origin.

Methods:
40 fungal isolates recovered from diverse food sources were tested for kojic acid production. The antibacterial and antifungal effects of KANEC against 12 pathogenic bacterial and fungal strains of animal, plant, and human origin were assessed at concentrations of 10, 50, 100, and 200 µg/mL using the well-diffusion method.

Results:
The results indicated that the minimum inhibitory concentration (MIC) values for bacteria ranged from 20 to 60 µg/ml. Natural KANEC was more effective against Gram-negative bacteria than Gram-positive bacteria, likely due to electrostatic interactions between the positively charged KANEC and negatively charged lipopolysaccharides (LPS) in the outer membrane of Gram-negative bacteria, leading to enhanced membrane disruption and increased cell permeability. For fungi, MIC values ranged from 20 to 40 µg/ml.

Conclusion:
Natural KANEC shows strong potential for use in veterinary medicine, plant, and human health as a natural alternative for combating drug-resistant pathogens. Nonetheless, further in vivo studies and clinical validation are essential to confirm its safety and translational potential. The evaluated kojic acid nanoparticle–orange peel oil nanoemulsion exhibited a novel green nanoformulation and promising antimicrobial and antibiofilm activities against various veterinary pathogens. These findings support further investigation of this nanoformulation for biomedical applications. To avoid misunderstanding, we have clarified in the Materials and Methods section that the nanoformulation evaluated in this study was previously developed, and the present work focused on assessing its biological activities. The evaluated nanoformulation exhibited significant antimicrobial activity, suggesting its potential as an alternative antimicrobial agent.

Key words: Antibacterial; Antifungal; Kojic acid; Nanoencapsulation.