Structural and Physicochemical Properties of Chlorella pyrenoidosa Neutral/Acidic Polysaccharides and Their Differential Regulatory Effects on Gut Microbiota and Metabolites in In Vitro Fermentation Model

<b>Background/Objectives</b>: <i>Chlorella pyrenoidosa</i> polysaccharides (CPPs) exhibit digestion-resistant properties, with their bioactivity largely driven by gut microbiota metabolism. However, the fermentation characteristics of CPPs within the intestinal tract remain to be fully elucidated. Elucidating the utilization and metabolic processes of CPPs with respect to the gut microbiota aids in understanding the potential mechanisms underlying the biological activity of these polysaccharides. <b>Methods</b>: This work fractionated CPPs into a neutral polysaccharide fraction (CPP-1) and an acidic polysaccharide fraction (CPP-2), followed by the characterization of their structure, physicochemical properties, and in vitro fermentation characteristics. <b>Results</b>: The results demonstrated that both CPP-1 and CPP-2 were non-starch heteropolysaccharides linked primarily by α-glycosidic bonds and lacking a triple helix structure. Both samples exhibited exceptional thermal stability, high water solubility, and low viscosity properties. CPP-2 selectively promoted <i>Enterocloster</i>, whereas CPP-1 significantly enriched <i>Bacteroides</i> and <i>Bifidobacterium</i> in gut microbiota. This differential regulation may be attributable to structural variations between the polysaccharides. Functional predictions indicated that CPP-1 enhances intestinal barrier integrity and immune homeostasis, whereas CPP-2 has anti-inflammatory activity. CPP-1 and CPP-2 interventions significantly upregulated the levels of health-promoting metabolites, including nicotinamide adenine dinucleotide, putrescine, and 3'-adenosine monophosphate. CPP-1 predominantly modulated amino acid metabolic pathways, while CPP-2 could effectively regulate purine, pyrimidine, amino acid, and butanoate metabolic pathways. <b>Conclusions</b>: This work identifies CPPs (CPP-1 and CPP-2) as novel modulators of gut homeostasis and host metabolism through microbiota-metabolite axis remodeling, supporting their prebiotic potential for functional food innovation.