In vitro digestion and fermentation characteristics of ethanol-extracted low molecular weight ginseng polysaccharide (AGP): Interactions with gut microbiota, metabolite regulation, and immunomodulatory effects.

The impact of extraction methods on the structural and functional properties of ginseng polysaccharides (GPs) remains poorly understood, and their immunomodulatory mechanisms are not fully elucidated. To address this gap, seven extraction techniques (hot water, acid, alkaline, hot alkaline, enzyme-assisted, ultrasound-assisted, and 60% ethanol extraction) were systematically compared for the first time. The 60% ethanol-extracted polysaccharide (AGP, 4.56 kDa) exhibited superior antioxidant activity and direct macrophage immunomodulation, and was selected for further investigation. Using an integrated in vitro digestion-fermentation model, AGP demonstrated marked resistance to upper gastrointestinal hydrolysis, but was readily fermented by gut microbiota. Fermentation induced significant structural remodeling to produce AGP48 (2.62 kDa), which exhibited markedly enhanced immunomodulatory activity in RAW264.7 macrophages, as evidenced by increased proliferation, phagocytosis, and up-regulation of NO, IL-6, IL-1β, and TNF-α. Integrative 16S rDNA sequencing and untargeted metabolomics revealed that AGP fermentation enriched beneficial Bifidobacterium, suppressed pathogenic Escherichia-Shigella, and promoted immunomodulatory metabolites including SCFAs and indolelactic acid. These microbial and metabolic shifts were mechanistically linked to enhanced macrophage activity, as evidenced by functional assays, collectively establishing a "gut microbiota-metabolite-immunity" axis. This study provides the first evidence that a LMw ginseng polysaccharide, identified through systematic extraction screening, exerts immunoenhancing effects via a gut microbiota-metabolite-immunity axis, with fermentation-induced structural remodeling as a critical driver of bioactivity. These findings provide a theoretical basis for developing AGP as a functional food ingredient targeting colonic health.