Strategic multistage optimization enhances bile salt hydrolase production in <i>Heyndrickxia coagulans</i> ATCC 7050 and <i>Lactiplantibacillus plantarum</i> ATCC 10012

Bile salt hydrolase (BSH), a key probiotic enzyme with antimicrobial activity, has gained considerable attention for its cholesterol-lowering potential. To enhance its commercial applicability, this study optimized media composition and fermentation conditions to maximize BSH production from Heyndrickxia coagulans ATCC 7050 and Lactiplantibacillus plantarum ATCC 10012. Initial screening of carbon sources using one-factor-at-a-time (OFAT) approach identified dextrose as the most suitable substrate for both organisms. Subsequently, Plackett–Burman design revealed beef extract, polysorbate 80, and K2HPO4 as key factors for H. coagulans ATCC 7050, while peptone, beef extract, and K2HPO4 influenced BSH production in L. plantarum ATCC 10012. These variables were optimized using response surface methodology and numerical optimization, followed by OFAT-based refinement of physical fermentation parameters. Under optimized conditions, maximum BSH yields of 190.66 ± 9.35 U/mL and 253.61 ± 12.47 U/mL were obtained at 16 hours using a 3% inoculum for H. coagulans ATCC 7050 (pH 6.5, 40 °C) and L. plantarum ATCC 10012 (pH 6.0, 37 °C), respectively. Growth curve analysis indicated a reduced lag phase and earlier onset of exponential and stationary phases, with peak BSH production in the stationary phase. Overall, optimization resulted in 3.54-fold and 6.12-fold increases in BSH production, demonstrating an effective strategy for enhancing BSH yield.