Structural and functional properties of two novel polysaccharides from seed-utilizing pumpkin and in vitro modulation of gut microbiota in constipated patients.

Two novel acidic polysaccharides, designated LR and BL, were isolated from two seed-utilizing pumpkin cultivars via ultrasonic microscopy-assisted acid extraction coupled with column chromatography purification. Chemical and instrumental analyses revealed that LR, with a molecular weight of 35.794 kDa, is composed of Rha (11.70%), Gal (14.10%), Glu (10.30%), and GalA (63.90%), whereas BL contains Rha (23.20%), Gal (19.30%), Glu (41.30%), Man (8.50%), and GalA (7.70%) and has an average molecular weight of 45.160 kDa. The backbone of LR consists of →3)-α-L-Rha-(1 → 4)-α-D-GalAp-(1 → [4)-α-D-GalAp-(1]3 → 6)-α-D-Glcp-(1 → 6)-β-D-Galp-(1→, whereas the backbone of BL primarily comprises →[6)-α-D-Glcp-(1]4 → 6)-α-D-Glcp-(1 → 6)-α-D-Glcp-(1 → 6)-β-D-Galp-(1 → [2)-α-L-Rha-(1]2 → 2)-α-L-Rha-(1 → 4)-α-D-GalAp-(1→. The two polysaccharides exhibit superior antioxidant capacity, inhibitory activity against digestive enzymes, and enhanced elastic and gel-forming properties. Batch fermentation using intestinal fluid samples from constipated patients demonstrated that they have a good potential to improve the imbalance of intestinal microecology by promoting the relative abundance of beneficial bacteria, especially butyrate-producing Clostridium cluster, while reducing the abundance of pathogenic bacteria Escherichia-Shigella. Moreover, a distinguishing characteristic of the BL and LR is their low gas production coupled with a pronounced capacity to generate short-chain fatty acids, particularly pentanoic acid. Therefore, the comparative characterization of the structural and functional properties of the two seed-utilizing pumpkin polysaccharides demonstrates their application potential in the food additive and functional food, while also supporting their future development as prebiotics and targeted delivery agents for gut health improvement.