Hydrolyzed eggshell membrane-calcium chelation complex for enhanced antimicrobial activity, antioxidant defense, and male reproductive homeostasis function.

Male infertility is strongly associated with oxidative stress-induced damage to sperm structure and function, necessitating bioactive systems capable of regulating redox balance and calcium homeostasis. In this study, a peptide-Ca2+ chelated eggshell membrane system (Eh-ESM-Ca) was developed via enzymatic hydrolysis and compared with adsorption-based unhydrolyzed ESM-Ca (u-ESM-Ca). To the best of our knowledge, this is the first report of a hydrolyzed ESM-Ca chelation complex for reproductive biomedical applications. Spectroscopic analyses (UV-Vis, FTIR, and XPS) supported coordination between Ca2+ ions and peptide functional groups (-COO- and -NH₂), while morphological studies revealed a transition from fibrous u-ESM to a homogeneous peptide-Ca network with improved solubility and bioavailability. Biologically, Eh-ESM-Ca demonstrated superior antioxidant activity with significantly reduced reactive oxygen species (ROS). In vitro sperm studies showed improved viability (~84%), morphology (~87%), and motility, along with reduced DNA damage under oxidative stress compared with u-ESM-Ca. In vivo results further demonstrated systemic safety, with normal liver (AST, ALT) and kidney (urea, creatinine) markers, enhanced catalase activity, reduced lipid peroxidation, and improved reproductive parameters, including testosterone levels and spermatogenesis. The enhanced performance is attributed to the synergistic interaction of bioactive peptides, metabolites, and Ca2+, contributing to redox regulation and reproductive protection. Overall, this study presents a multifunctional, biocompatible platform for reproductive and biomedical applications.