Optimization of Medium Composition for High Cell Density Culture of Bifidobacterium longum HSBL001 Using Response Surface Methodology

Abstract

Bifidobacterium longum plays a critical role in the human gut and exhibits diverse probiotic functions. Achieving high-density fermentation of B. longum largely depends on the composition of the culture medium and fermentation conditions. This study aimed to optimize the medium composition and fermentation parameters for B. longum HSBL001, using viable cell counts and optical density at 600 nm (OD600) as indicators. The goal was to improve biomass yield and support the development and industrial application of highly active probiotic preparations. The optimal medium composition and culture conditions were established using a combination of single-factor experiments, the Plackett-Burman design, the steepest ascent method, and the central composite design. The optimized culture medium consisted of yeast extract (19.524 g/L), yeast peptone (25.85 g/L), arginine (0.599 g/L), glucose (27.36 g/L), MnSO₄ (0.09 g/L), MgSO₄ (0.8 g/L), Tween-80 (1 g/L), l-cysteine hydrochloride (0.24 g/L), and methionine (0.15 g/L). The optimal culture conditions included an initial pH of 7.0, 5% inoculum size, and incubation at 37°C, yielding a final viable cell count of 4.20 × 10⁹ colony-forming units (CFU/mL). In a 3 L bioreactor, the viable cell count reached 1.17 × 10¹⁰ CFU/mL, which was 1.786 times higher than that achieved with the modified MRS medium. These findings demonstrate that the optimized medium and fermentation conditions are well-suited for high-density cultivation of B. longum HSBL001 and provide a basis for its industrial application.

Keywords: Bifidobacterium longum; anaerobic fermentation; high cell density culture; medium optimization; response surface methodology.