Ultrasonic assisted three-phase partitioning extraction and bioactivities validation of polysaccharides from Flammulina velutipes residues.

Mushroom residues, especially Flammulina velutipes residues (FVR) rich in bioactive polysaccharides, are under exploited due to inefficient extraction methods conflicting with green chemistry. This study developed an ultrasonic-assisted three-phase partitioning (UAE-TPP) approach to extract crude polysaccharides (cTP-FVRP) from FVR, combined with structural and functional characterization. An integrated optimization strategy combining response surface methodology (RSM), artificial neural network (ANN), and genetic algorithm (GA) identified global optimum conditions (18 min, 1:1 crude extract to n-Butanol, 44 % (NH₄)₂SO₄), yielding cTP-FVRP at 3.49 ± 0.05 %-significantly higher than that of UAE method (2.38 ± 0.04 %). Purified TP-FVRP was a 108.13 kDa non-starch polysaccharide with a triple helix, α-linkages, and 9 monosaccharides, stable under physiological conditions. It exhibits potent antioxidant activity, biocompatibility, and immunomodulatory potential. UAE-TPP reduces solvents while preserving polysaccharide integrity. This green technique enables high-value utilization of FVR, aligning with sustainability goals and providing a scalable model for industrial by-product valorization.