Biochemical and Micrographic Evidence of Escherichia coli Membrane Damage during Incubation in Egg White under Bactericidal Conditions

Bacterial membranes are often thought to be the main targets of the antimicrobial activity of egg white. In order to test this hypothesis, the state of the membranes of Escherichia coli K-12 cells during either bactericidal (45 degrees C) or bacteriostatic (30 degrees C) incubation in egg white at natural alkaline pH was studied by biochemical methods. Namely, the permeability of the outer membrane was evaluated through its ability to incorporate a hydrophobic fluorescent probe (1-N-phenylnaphthylamine), and the permeability of the cytoplasmic membrane was evaluated through the release of a specific intracellular enzyme (beta-galactosidase). The bacteria were observed by atomic force microscopy in order to support the biochemical results. At 45 degrees C, the outer membrane of E. coli K-12 incorporated the hydrophobic probe, suggesting that it was disrupted. In addition, the cytoplasmic beta-galactosidase was released at this temperature. The atomic force microscopy analysis revealed the formation of spheroplasts, which provided further evidence of the cell wall disruption and a progressive release of cellular contents. At 30 degrees C, biochemical and micrographic experiments confirmed that membrane integrity was preserved. These techniques provide a useful approach for studying the mechanisms of bacterial cell death in egg white.