Abstract

Gracilaria bailinae polysaccharides have limited applications due to their high viscosity, large molecular weight, and poor solubility. Probiotic fermentation emerges as an efficient strategy to enhance the bioactivity of plant polysaccharides. This study aimed to prepare fermented water-soluble crude polysaccharide (GBV) from G. bailinae via fermentation with Bacillus velezensis, using water-extracted polysaccharide (GBP) as a control. The physicochemical properties, immunomodulatory activity, and trained immunity effects of GBV were systematically investigated. A significant decrease in molecular weight, accompanied by increased sulfate and uronic acid content, was observed in G. bailinae polysaccharides following fermentation. Modifications to the monosaccharide composition ratio and microstructure also occurred, though the basic polysaccharide structure remained unaffected. In vitro experiments revealed that both GBV and GBP were non-toxic to RAW264.7 macrophages and promoted cell proliferation, phagocytosis, and the secretion of nitric oxide (NO) and pro-inflammatory cytokines (IL-1β, IL-6, TNF-α). Notably, GBV exhibited superior immunostimulatory activity at low concentrations (6.25-50 μg/mL) compared to GBP. Furthermore, at a concentration of 12.5 μg/mL, GBV was found to trigger trained immunity in RAW264.7 cells, as demonstrated by elevated levels of cytokines and NO following LPS restimulation. Untargeted metabolomics analysis indicated that the trained immunity effect was associated with metabolic reprogramming of purine, pyrimidine, and arginine-polyamine pathways, with significant downregulation of spermine potentially amplifying the M1 inflammatory phenotype. These findings demonstrate that B. velezensis fermentation improves the immunomodulatory activity of G. bailinae polysaccharides, and GBV holds great potential as a natural immune activator.