Abstract

Chondroitin sulfate (CS) is a glycosaminoglycan that plays essential roles in physiological functions. However, the relationship between CS and the intestines of midlife women remains unclear. This study investigated the degradation and utilization of low-molecular-weight CS (LMWCS) and high-molecular-weight CS (HMWCS) using an in vitro fermentation model with fecal samples from four midlife women. The result showed that 51% of LMWCS and 41% of HMWCS were degraded within 48 h, with interindividual variation. Both CS forms were ultimately degraded to an unsaturated disaccharide (udp2), while LMWCS was metabolized more efficiently. This result in higher production of short-chain fatty acids, particularly acetate and propionate. Fermentation of both CS forms increased Bacteroides abundance, with LMWCS more effectively promoting beneficial bacteria and reducing Klebsiella . Bacteroides ovatus AP1 was identified as a key CS-degrading species, efficiently utilizing both LMWCS (87%) and HMWCS (58%), producing udp2 and propionate. Whole-genome sequencing of B. ovatus AP1 revealed 649 carbohydrate-active enzyme genes, including multiple polysaccharide lyases involved in CS depolymerization. qPCR confirmed the upregulation of CS-metabolism-related genes during fermentation. These findings highlight the potential of LMWCS as a prebiotic for modulating gut microbiota and provide insights into the metabolism of CS by the gut microbiota.