Jet-O-mized Eggshell Membrane Powder: A Novel Hydrolysis Strategy to Unlock Gut-Promoting Bioactivities

Abstract

In developed countries like Canada, significant amounts of eggshell (ES) and eggshell membrane (ESM) waste are generated by egg-breaking plants serving the food and biopharmaceutical industries. ESM is composed of 90% proteins with diverse bioactivities, including antibacterial, immunomodulatory, anti-inflammatory, and antioxidant properties. As a supplement, ESM shows promise in promoting gut health by modulating the microbiome through its bioactive effects. However, advanced solubilization strategies for creating ESM-based functional platforms remain underexplored. This study aimed to develop novel ESM formulations for gut health. Size-reduced ESM particles (JEM) were prepared via jet-O-mizing and hydrolyzed under optimized alkaline conditions using dilute potassium hydroxide, yielding a 50% solubilization of bioactive proteins and peptides. The hydrolyzed JEM suspension (WJ) was centrifuged to obtain the soluble fraction (SJ). Both WJ and SJ were further processed through simulated gastrointestinal digestion (SGID) to produce hydrolyzed metabolites (SJ-G, WJ-G, SJ-GI, and WJ-GI). JEM formulations demonstrated a 15-fold increase in Trolox-equivalent antioxidant capacity (TEAC) compared to non-hydrolyzed JEM (NJEM), with 10 mg/mL SJ showing 667.3 ± 25 µM Trolox versus 43.7 ± 7.1 for NJEM. SJ also exhibited bacteriostatic effects, inhibiting E. coli growth by 50 % over 24 hours compared to untreated culture. Additionally, SJ and WJ-G reduced Lipopolysaccharideinduced nitric oxide production by up to 80% in RAW 264.7 macrophages. The impact on intestinal barrier function was evaluated using Caco-2 cells, showing no significant changes in cell permeability. These findings highlight the development of bioactive ESM formulations with antibacterial, antioxidant, and anti-inflammatory properties, positioning them as promising dietary supplements for gut health.