Isolation and functional evaluation of Lacticaseibacillus casei HUMB07381 isolated from traditional khiki cheese as a potential probiotic candidate.

This study presents the isolation and molecular identification of a Lacticaseibacillus casei strain isolated from traditional Khiki cheese, which showed 99% 16 S rRNA gene sequence similarity to Lcb. casei strain HUMB07381. To assess its probiotic potential, a comprehensive evaluation was conducted including morphological, physiological, and biochemical characterization. The analyses covered carbohydrate fermentation patterns (49 substrates), enzymatic activity, survival under acidic conditions (pH 2.5, 4.0, and 5.5), and tolerance to bile salt concentrations (0.3-0.7%). The strain's capacity to inhibit Staphylococcus aureus biofilm formation and gene expression was also investigated. Antimicrobial activity of neutral and acidic cell-free supernatants (nCFS and aCFS) was determined against Escherichia coli, Salmonella typhimurium, Shigella dysenteriae, Bacillus cereus, Listeria monocytogenes, and S. aureus via agar disc diffusion, agar well diffusion, double-layer, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) assays. The isolate was identified as Gram-positive, rod-shaped, catalase-negative, non-spore-forming, non-motile, and gas-negative. It exhibited versatile carbohydrate fermentation, significant enzymatic activity, and survival under simulated gastrointestinal conditions (pH, bile salts, NaCl, and 25-40 °C). Moreover, the strain showed strong cell-surface hydrophobicity, auto-aggregation, co-aggregation, cholesterol reduction, and inhibition of S. aureus adhesion and biofilm formation. It also displayed antimicrobial activity against pathogens, in vitro cytotoxic effects on HT-29, HeLa, and MCF-7 cell lines, and pronounced antioxidant activity. Thus, Lcb. casei HUMB07381 represents a promising candidate for functional probiotic dairy products.