Metabolic, enzymatic, and probiotic profiling of Lactococcus lactis LBM15: inhibition of Staphylococcus aureus biofilm and virulence genes, and GPR-based prediction

Abstract

This study characterized a Lactococcus lactis strain (LBM15) isolated from traditional yogurt and evaluated its probiotic, antimicrobial, and anti-biofilm attributes, along with its physiological robustness and safety-associated characteristics. The isolate was identified by 16S rRNA sequencing (99% similarity) and exhibited a carbohydrate utilization pattern typical of dairy L. lactis , including efficient fermentation of common hexoses and β-glucosides. Enzymatic profiling revealed high phosphatase and peptidase activities, supporting its metabolic adaptation to milk environments. The strain demonstrated notable tolerance under simulated gastrointestinal and environmental stress conditions and maintained substantial growth at pH 4.0–5.5, values decreasing only slightly from 8.60 ± 0.11 Log CFU/mL to 6.80 ± 0.06 Log CFU/mL after 3 h. It exhibited strong surface hydrophobicity (59.5 ± 3.0% for chloroform) and auto-aggregation (73.0 ± 2.5% in 24 h), both promoting adequate adhesion to Caco-2 cells and competitive exclusion of Staphylococcus aureus . Cell-free supernatants (CFS) showed broad antibacterial activity, most notably against S. aureus and L. monocytogenes , with minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of 15 and 30 mg/mL, respectively. qRT-PCR demonstrated significant downregulation of S. aureus virulence genes ( mecA , agr , icaA , spa , and hla ), consistent with the observed reduction in biofilm biomass. The strain also exhibited strong antioxidant potential, moderate cytotoxicity against MCF-7, HeLa, and HT-29 cancer cells, and substantial cholesterol assimilation (54.50 ± 2.80% in 0.2% bile concentration). HPLC confirmed the production of lactic (11.30 ± 0.19 mg/mL), acetic (4.49 ± 0.10 mg/mL), and formic (2.00 ± 0.05 mg/mL) acids. Also, in this study, a Gaussian Process Regression (GPR) model was used to predict acidity and bile salt. The model delivered excellent predictive accuracy, evidenced by low Mean Absolute Percentage Error (MAPE) values (1.39% for bile, 2.54% for acidity) and high R 2 scores (0.98 and 0.94, respectively), with predictions closely mirroring empirical observations. Collectively, these findings indicate that L. lactis LBM15 exhibits promising in vitro probiotic-associated, anti-pathogenic, and antioxidant properties. Its combined probiotic and anti-biofilm activities, supported by data-driven modeling, suggest that it exhibits promising in vitro probiotic-associated properties for food biotechnology applications.