From Waste to Bioactive Ingredient: Integrated Extraction, Identification, and Validation of Novel Antioxidant...

The valorization of poultry bone by-products into high-value bioactive ingredients aligns with the principles of a sustainable circular bioeconomy. This study established an integrated process for the production, identification, and validation of bioactive antioxidant peptides from Xuefeng black-bone chicken bones (BCB). Alcalase was selected as the optimal protease due to its superior performance in both the degree of hydrolysis and antioxidant activity under the optimized conditions. Using response surface methodology (RSM), the optimal hydrolysis conditions were determined as 50 °C, pH 10.18, and 4.2 h, resulting in a hydrolysate with a hydrolysis degree of 25.10% and ABTS radical scavenging activity of 84.36%. Upon ultrafiltration, the <3 kDa fraction demonstrated a significantly higher antioxidant capacity than the crude hydrolysate. Further purification through gel filtration chromatography yielded the F3 sub-fraction (predominantly <1 kDa peptides), which exhibited the most potent activity across all four antioxidant assays conducted (ABTS, DPPH, hydroxyl radical scavenging, and reducing power). A liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of F3 led to the identification of 21 peptide sequences. An in silico screening based on bioactivity and toxicity predictions pinpointed three promising candidates: DYPF, WDY, and FGYK. These peptides were chemically synthesized and validated to possess significant in vitro radical scavenging activities against both DPPH and hydroxyl radicals. Molecular docking simulations revealed that all three peptides could spontaneously bind to the Keap1 protein with a high affinity (binding energy < -7.0 kcal/mol), primarily through hydrogen bonds and hydrophobic interactions, suggesting a possible molecular mechanism that may involve the Keap1-Nrf2-ARE antioxidant pathway. This computational insight provides a testable hypothesis for their bioactivity, the verification of which is contingent upon future studies demonstrating their cellular delivery and intracellular action. This work not only provides a sustainable strategy for BCB utilization but also identifies potent antioxidant peptides with potential applications in functional foods and nutraceuticals.