Scalable Jet-O-Mized Eggshell Membrane Processing for Bioactives Promoting Gut Health

This study introduces a novel, simplified, and scalable two-step process for generating bioactive eggshell membrane (ESM) formulations by combining jet-O-mizer ultra-fine milling of ESM (yielding JEM biomaterial) with KOH-mediated hydrolysis, achieving ~50% solubilization of proteins and peptides and enabling the first evaluation of ESM-derived bioactives for gut health applications. The soluble protein fraction (SJ) was separated from the whole hydrolysate (WJ), and subjected to simulated gastrointestinal digestion to assess stability and bioavailability. The antioxidant capacities of the JEM-derived material showed a significant 15-fold increase compared to soluble non-hydrolyzed JEM (NJEM). SJ inhibited E. coli bacterial growth by 50% within 24 h, compared to the untreated bacterial culture. The formulations demonstrated superior anti-inflammatory properties with lipopolysaccharide (LPS)-induced RAW macrophages, resulting in a 80% reduction in NO production compared to untreated cells. Proteomics analysis of SJ revealed key anti-inflammatory (YBX1, YWHAE) and antimicrobial (OCX36, OC-17, TENP, and histones) effectors whose coordinated activities could modulate gut microbial composition. The permeability of the intestinal barrier model Caco-2 monolayer was not significantly affected by treatment with any JEM-derived formulation, thereby predicting maintenance of intestinal integrity. This study provides safe, novel ESM derivatives with high bioavailability and multifunctional bioactivities, including antibacterial, antioxidant, and anti-inflammatory effects, positioning them as promising candidates for dietary supplements to promote gut health.