L-cysteine-assisted enzymatic hydrolysis of eggshell membrane generates gastrointestinal-stable peptides with...

This study aimed to develop an efficient strategy for releasing bioactive peptides derived from eggshell membrane (ESM) and to evaluate their gastrointestinal stability and potential anti-inflammatory activity. In this study, an L-cysteine (L-Cys)-assisted enzymatic hydrolysis system was used to disrupt the compact disulfide-crosslinked network of ESM. The hydrolytic effects of different proteases were screened, and the stability, structural changes, and potential mechanisms of the resulting peptides were systematically investigated using free sulfhydryl/disulfide bond analysis, in vitro simulated gastrointestinal digestion, anti-inflammatory activity evaluation in RAW264.7 macrophages, atomic force microscopy (AFM) structural characterization, liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based peptide sequence identification, network pharmacology, and molecular docking analysis. The results showed that Flavourzyme combined with L-Cys exhibited the highest hydrolysis efficiency. The addition of L-Cys significantly increased free sulfhydryl content while decreasing disulfide bond content, indicating an alteration of the thiol-disulfide balance during enzymatic hydrolysis. The resulting hydrolysates significantly inhibited lipopolysaccharid (LPS) induced nitric oxide (NO) production in RAW264.7 macrophages and maintained good stability during simulated gastrointestinal digestion after simulated gastrointestinal digestion, with the degree of hydrolysis and digestibility stabilizing at approximately 28% at the end of the intestinal phase. AFM observations indicated that digestion gradually transformed the peptide system from large aggregated structures into a more uniform nanoscale dispersion. LC-MS/MS identified a total of 238 peptide sequences, from which 20 candidate peptides were screened based on predicted bioactivity and molecular docking performance. Among them, YVHSSFSGFLF, SSEYVHSSFSGFLF, and ARGPAGPQGPR showed stable interactions with inflammation-related targets, including MAPK1, MAPK14, and NFKB1. Cellular validation further demonstrated that these three peptides reduced LPS-induced interleukin-6 (IL-6), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and NO production while enhancing interleukin-10 (IL-10) secretion, with P5 showing the strongest overall anti-inflammatory activity. The study demonstrates that L-Cys-assisted hydrolysis can effectively promote the release of gastrointestinal-stable anti-inflammatory peptides from ESM, providing a theoretical basis for the high-value utilization of eggshell membrane resources and their development as functional food ingredients.