Probiotic, functional, and safety assessment of Levilactobacillus brevis M3R3 isolated from traditional Khiki cheese.

Traditional Khiki cheese, as a naturally fermented dairy product, represents a valuable source for the isolation of indigenous lactic acid bacteria with probiotic potential. In this study, Levilactobacillus brevis M3R3, isolated from traditional Khiki cheese, was evaluated for its probiotic, functional, and safety properties. The strain was identified based on morphological, physiological, biochemical, API 50 CHL, and API ZYM profiles. Strain M3R3 demonstrated a notable ability to withstand gastrointestinal barriers, maintaining viable counts of 4.847 log CFU/mL after exposure to the highly acidic condition of pH 2.5. In simulated gastric and intestinal fluids, the strain exhibited survival rates of 67.8% and 83.0%, respectively. Its cell-surface characteristics, including hydrophobicity toward chloroform, xylene, and toluene, 78.2% auto-aggregation, and 13.7% adhesion to Caco-2 cells, indicate its potential to interact with the intestinal epithelium and transiently persist in the gastrointestinal tract. Functionally, the cell-free supernatant of this strain showed strong inhibitory activity against Staphylococcus aureus and reduced biofilm formation to 17.8% at 4 × MIC. This effect was associated with downregulation of the icaA and hla genes by 83.2% and 58.4%, respectively. Furthermore, DPPH and ABTS radical-scavenging activities reached 54.05% and 62.75%, while cholesterol removal in the presence of bile reached 39.0%. The absence of hemolysis, DNase activity, biogenic amine production, and β-glucuronidase activity further supports the relative safety of strain M3R3 as a probiotic candidate.