Abstract
Eggshell membrane (ESM) is a food-processing poultry by-product containing over 500 proteins and a valuable source of bioactive peptides that remain minimally exploited for functional foods and nutraceuticals. The current study developed a distinct valorization and fractionation strategy using clean, size-selected particalized ESM (PEM) as a defined feedstock to generate peptide-containing fractions with improved antioxidant and potential intestinal barrier-supportive activities. PEM batches (<53 μm) were subjected to alkaline hydrolysis using potassium hydroxide (KOH), followed by neutralization, fractionation, simulated gastrointestinal digestion, and desalting. Bioactivities of different fractions were evaluated using Trolox equivalent antioxidant capacity (TEAC), nitric oxide (NO) production in LPS-stimulated RAW264.7 macrophages, and transepithelial electrical resistance (TEER) in differentiated Caco-2 intestinal epithelial monolayers. KOH hydrolyzed the crosslinked ESM protein meshwork, with increased recovery of soluble proteins and peptides, achieving ~78% solubilization. The soluble PEM hydrolysate fraction (PS) exhibited the greatest reduction in LPS-induced NO production, whereas its intestinal digestion product (PSI) showed 65-fold greater TEAC compared to unprocessed PEM. Several PEM-derived fractions reduced LPS-induced NO production and improved TEER responses. These findings demonstrate that alkaline hydrolysis and gastrointestinal digestion markedly alter the bioactivity of ESM-derived fractions, supporting further investigation of these fractions as a multifunctional ingredient for potential food and nutraceutical applications.