Abstract
Natural antioxidants derived from agricultural by-products have great promise and ecological advantages in the treatment of oxidative stress-related diseases. The eggshell membrane (ESM) from hatched eggs, i.e., the hatched ESM, is a globally abundant agricultural byproduct, and its high-value utilization has been rarely studied compared to the well-studied ESM from fresh eggs. In this research, we systematically characterized the hatched ESM as a novel source of antioxidant hydrolysates and explored their potential role in H2O2-induced human chondrocytes. The results showed that the hatched ESM is a protein-rich fibrous mesh material with a significantly different structure and composition from those of fresh ESM. Enzymatic hydrolysis of hatched ESM can produce antioxidant hydrolysates rich in low molecular weight (MW) peptides, which mainly derived from the Lysyl oxidase homolog by Nano-LC-MS/MS analysis. The peptide fraction with MW < 3 kDa (HEMH-I) exhibited the highest DPPH radical scavenging, Fe2+-chelating, and Fe3+-reducing abilities. In H2O2-induced human SW1353 chondrocytes, HEMH-I treatment significantly increased the cell viability and ameliorated oxidative stress, inflammatory response, and cartilage matrix degradation by reducing the level of ROS, matrix metalloprotease 3 (MMP3), MMP13, and IL-6, and by promoting the expression of SOD and type II collagen, potentially through activating the cellular Keap1/Nrf2/HO-1 pathway. This study provides a theoretical basis for the value-added application of hatched ESM waste to produce antioxidant hydrolysates and indicates their potential as functional food and pharmaceuticals.