Valorization of Grifola frondosa processing by‐products: purification, structural characterization, and anti‐ EV71 bioactivity of a novel low‐molecular‐weight polysaccharide‐peptide

BACKGROUND The large volume of supernatant generated during Grifola frondosa extraction is typically discarded, resulting in substantial resource waste and environmental pressure. Such agro‐fungal processing by‐products are rich in potentially bioactive macromolecules, but efficient recycling strategies and biofunctional verification remain limited. Meanwhile, safe natural antiviral ingredients for pediatric enterovirus infection are lacking. This study established a sustainable chromatographic bioprocess to recover a low‐molecular‐weight polysaccharide‐peptide (LMWPP) from G. frondosa waste liquid, with the aims of achieving high‐value circular utilization of fungal processing wastewater and exploring its anti‐EV71 potential. RESULTS Purified LMWPP with an average molecular weight of 1.252 × 10 4 Da possessed pyranose structures with α ‐ and β ‐glycosidic bonds and consisted of multiple monosaccharides and leucine‐dominant amino acids. Recovered LMWPP exerted marked anti‐EV71 effects by mitigating cytotoxicity, inhibiting viral replication, downregulating VP1 mRNA expression, and alleviating cell apoptosis. Transcriptomic analysis further demonstrated its regulatory effects on the complement and coagulation cascades, peroxisome proliferator‐activated receptor (PPAR) signaling, and oxidative phosphorylation. CONCLUSION This integrated separation strategy enables efficient valorization of G. frondosa liquid by‐products and clarifies the structure–activity relationship of LMWPP. The recycled natural LMWPP shows promise as a pediatric antiviral functional food ingredient, providing a feasible industrial approach for high‐value reuse of edible fungal processing waste.