Methodological estimation of pore size in hen eggshell membranes

Eggshell membranes (ESM) are proteinaceous biobarriers critical to avian embryonic development and microbial exclusion. Despite their importance, quantitative assessments of ESM pore dimensions remain limited. This study aimed to determine the effective pore size of chicken ESM and to validate methodological approaches for its estimation. Two orthogonal strategies were employed to evaluate ESM pore architecture. First, pressure-driven pure-water permeation was performed within a low transmembrane pressure range (0.01–0.05 MPa). The resulting flux–pressure data were analyzed using the Guéout–Bjerrum–Manegold relation to approximate average pore dimensions. Second, size-selective filtration experiments were conducted using colloidal silica and polystyrene latex particles ranging from 14 to 204 nm. Rejection rates were plotted against particle size and interpreted through the Ferry–Renkin model to derive effective pore estimates. Comparison of the two methods demonstrated strong consistency, supporting their mutual applicability. While numerical outcomes suggested pore dimensions on the order of ~ 100–200 nm, the emphasis of this work lies in validating complementary approaches. Together, these techniques provide a robust framework for probing semipermeable biological barriers and open avenues for applying ESM, an abundant biowaste, in bioinspired membrane technologies and biosafety studies.