IntroductionIn the quest for the development of antibiotic alternatives, this study aimed to systematically evaluate the probiotic potential and mechanism of action of Ligilactobacillus agilis R22, a novel strain isolated from chickens.MethodsThe in vitro probiotic properties of L. agilis R22, including growth ability, gastrointestinal tolerance, and antimicrobial activity, were initially assessed. Genomic and untargeted metabolomic analyses were conducted to explore its genetic and metabolic pr