High-density cultivation of Lacticaseibacillus paracasei GY-1 in a novel medium produces a structurally distinct immunostimulatory exopolysaccharide.
Lactic acid bacteria are important probiotics and producers of valuable released exopolysaccharides (r-EPS). However, achieving high cell density cultivation (HCDC) and high r-EPS yields in a simple batch culture remains challenging, partly due to the lack of an effective medium. To address this gap, we developed a novel multifunctional Spirulina platensis (SP) broth, designated MSP, for cultivating Lacticaseibacillus paracasei GY-1 by replacing all nitrogen sources (peptone, beef extract, and yeast extract) in De Man, Rogosa, and Sharpe (MRS) broth with 15.48 g/L SP hydrolysates, supplemented with 0.5 g/L corn steep liquor and 1 g/L trehalose. MSP achieved an unprecedented HCDC of 1.72 × 1011 viable cells/mL (70-fold higher than MRS) in 20 h, thereby increasing total r-EPS yields by 4.16 times. Purification identified two MSP-specific fractions: r-EPS1 and r-EPS2. In vitro assays demonstrated that r-EPS2 significantly promoted RAW264.7 cell proliferation and cytokine secretion (IL-1β, TNF-α, IL-6, and IL-10) compared to r-EPS1 and lipopolysaccharide. Structural characterization identified r-EPS2 as a novel polysaccharide (7.66 × 105 Da) composed of 98.90% glucose, with a backbone of (1 → 4)-α-D-Glcp and branches of (1 → 4,6)-α-D-Glcp and (1 → 3,4)-α-D-Glcp. Collectively, MSP is the first versatile medium that enables HCDC, enhances r-EPS yield, and produces structurally distinct r-EPS with potent immunostimulatory activity in a single batch culture, highlighting its potential for probiotic and r-EPS applications.