Identifying the Components in Eggshell Membranes Responsible for Reducing the Heat Resistance of Bacterial Pathogens

Abstract

Enzymatically and biologically active protein fractions from hen eggshell membrane (ESM) and egg white (HEW) were isolated to evaluate their specific biological role in providing defense against bacterial pathogens. Numerous attempts to purify active protein/enzyme fractions from the ESM proved somewhat limited; however, isoelectric focusing and ion exchange chromatography provided a technical means whereby relatively pure samples were obtained. HEW β-N-acetylglucosaminidase (β-NAGase) was isolated using a two-step chromatographic procedure, during which relatively pure fractions of ovotransferrin were also obtained. These fractions were used assist in identifying ESM components that reduce the heat resistance of bacterial pathogens and to define the possible mechanism of action. Ovotransferrin, lysozyme and β-NAGase were identified as primary contributors to ESM antimicrobial activity. Cellular loss of Ca²⁻, Mg²⁻, Na⁻ K⁻ from S. Typhimurium cells, after treatment with ESM components was observed. Transmission electron microscopy indicated the integrity of the outer membrane was compromised when ovotransferrin, lysozyme and β-NAGase were applied to the cells. Thus, we hypothesized that ESM-bound ovotransferrin functions as an ion chelator and disrupts lipopolysaccharide stability, resulting in a compromised lipopolysaccharide and increased susceptibility to heat, enzymes and possibly other stressors.

Differences in enzymatic and biological activity of ESM as a function of layer breeds, age and ESM stabilization methods were determined. ESM biological activity remains fairly constant across different layer and breeds and throughout the laying cycle. Significant reductions in biological activity was not observed in refrigerated (4°C), frozen (-20°C) or freeze-dried ESM, however significant reductions in enzymatic and biological activities were observed for air and heat dried ESM. Refrigerated and frozen ESM also exhibited gradual loss of enzymatic activity during a 6 month storage study, where as air, heat and freeze-dried ESM showed no significant loss of enzyme activity after processing treatments.