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

The biological activity (D-value determination) of eggshell membrane (ESM) was examined to determine the membrane components and mechanisms responsible for antibacterial activity. Biological and enzymatic activities (i.e., β-N-acetylglucosaminidase [β-NAGase], lysozyme, and ovotransferrin) of ESM denatured with trypsin, lipases, or heat were compared with those of untreated ESM. Trypsin-treated ESM lost all biological activity (D-values at 54°C were 5.12 and 5.38 min for immobilized and solubilized trypsin, respectively) but showed no significant loss of enzymatic activities. Treatments with porcine lipase and a lipase cocktail did not impact biological or enzymatic activities. Heat denaturation of ESM (at 80 and 100°C for 15 min) resulted in significant decreases in biological activity (D-values of 3.99 and 4.43 min, respectively) and loss of β-NAGase activity. Lysozyme and ovotransferrin activities remained but were significantly reduced. Purified ESM and hen egg white components (i.e., β-NAGase, lysozyme, and ovotransferrin) were added to Salmonella Typhimurium suspensions (in 0.1% peptone water) at varying concentrations to evaluate their biological activity. D-values at 54°C were 4.50 and 3.68 min for treatment with lysozyme or β-NAGase alone, respectively, and 2.44 min for ovotransferrin but 1.47 min for a combination of all three components (similar to values for ESM). Exposure of Salmonella Typhimurium cells to a mixture of ovotransferrin, lysozyme, and β-NAGase or ESM resulted in significant increases in extracellular concentrations of Ca²⁺, Mg²⁺, and K⁺. Transmission electron microscopic examination of Salmonella Typhimurium cells treated with a combination of ovotransferrin, lysozyme, and β-NAGase revealed membrane disruption and cell lysis. The findings of this study demonstrate that ovotransferrin, lysozyme, and β-NAGase are the primary components responsible for ESM antibacterial activity. The combination of these proteins and perhaps other ESM components interferes with interactions between bacterial lipopolysaccharides, sensitizing the outer bacterial membrane to the lethal affects of heat and possibly pressure and osmotic stressors.