Purpose/Background: This study aimed to isolate, purify, and characterize a novel bacteriocin produced by Lactobacillus casei strain GO3, isolated from goat milk. Bacteriocins are promising natural antimicrobial agents with potential applications in food preservation and therapeutics, addressing growing concerns over antibiotic resistance and synthetic preservatives. Materials and Methods: L. casei GO3 was isolated from goat milk and screened for antimicrobial activity. The strain was identified through 16S rRNA sequencing, and the sequence was deposited in the NCBI database (accession number ON059683). Purification: The bacteriocin was purified using a multi-step process involving 60% ammonium sulfate precipitation, dialysis (1 kDa cutoff), and ion-exchange chromatography (diethyl aminoethyl-cellulose). Antimicrobial activity was assessed against several pathogens ( Bacillus subtilis, Escherichia coli, Salmonella typhi , and Pseudomonas aeruginosa ) using the agar well diffusion method. Stability was evaluated under various conditions, including pH (2–12), temperature (30°C–121°C), enzymes (trypsin, pepsin, and protease K), and chemicals (ethylenediaminetetraacetic acid (EDTA), urea, NaCl, and dimethyl sulfoxide). Results: The purification process yielded a 2.66% recovery rate with a 54.9-fold increase in specific activity (1,220.89 AU/mg). The bacteriocin demonstrated broad-spectrum inhibition (zone diameters >15 mm) and stability across a wide pH range (2–12) and temperatures up to 100°C. However, activity was lost at 121°C and with proteolytic enzymes, confirming its proteinaceous nature. Notably, EDTA enhanced antimicrobial activity, suggesting metal ion dependence. Conclusion: The bacteriocin produced by L. casei GO3 exhibits robust stability and potent antimicrobial efficacy, making it a promising candidate for applications in food preservation and therapeutics. Future research should focus on optimizing production and evaluating safety for industrial use.

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