Optimizing lipase production by Bacillus subtilis on cheese whey and evaluating its antimicrobial, antibiofilm, anti virulence and biosafety properties

Abstract

This study optimized lipase production using cheese whey, biofilm inhibition, and antibacterial efficacy of Bacillus subtilis (DSM 1088)derived lipase against Staphylococcus aureus (ATCC 6538). Peak lipase activity, growth rate, and inhibitory potential were observed at 48 h and 30 °C. Using Plackett-Burman and Central Composite Designs (PBD and CCD), whey, peptone, and agitation speed were identified as significant factors, achieving optimal lipase activity of 1314 U/mL and an inhibitory zone diameter (IZD) of 48 mm against S. aureus. Partial purification through ammonium sulfate precipitation and dialysis increased partial purified lipase (PPL) activity by twofold and fivefold, respectively. PPL exhibited effective bactericidal properties with a minimum inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of 1/8 and 1/16, confirming a bactericidal effect (MIC/MBC ratio ≤ 2). Biofilm inhibition assays demonstrated 95% biofilm reduction at 80 µg/mL PPL, with SEM imaging revealing significant biofilm matrix disruption. Time-kill assays showed concentration-dependent bactericidal action, while inhibition of hemolysin and protease activities (25-100%) indicated reduced S. aureus pathogenicity. Cytotoxicity assays on normal liver cells showed an IC50 > 300 µg/mL, indicating low toxicity. GC/MS analysis of oil waste before degradation identified predominantly oleic acid 3-hydroxypropyl ester and octadecane derivatives, while after degradation, it revealed enriched free fatty acids including myristic, palmitic, linoleic, and oleic acids, which could enhance antimicrobial efficacy. Molecular docking suggested that PPL inhibits essential bacterial enzymes (folic acid synthetase, RNA polymerase, DNA gyrase), potentially disrupting DNA synthesis and promoting cell death. These findings highlight B. subtilis-derived lipase as a promising bio-agent for combating biofilm-associated, drug-resistant pathogens with clinical and industrial applications.