Mechanisms of Inactivation of Dry Escherichia coli by High-Pressure Carbon Dioxide

Abstract

High-pressure carbon dioxide processing is a promising technology for nonthermal food preservation. However, few studies have determined the lethality of high-pressure CO₂ on dry bacterial cells, and the mechanism of inactivation remains unknown. This study explored the mechanisms of inactivation by using Escherichia coli AW1.7 and mutant strains differing in heat and acid resistance, in membrane composition based on disruption of the locus of heat resistance, and in genes coding for glutamate decarboxylases and cyclopropane fatty acid synthase. The levels of lethality of treatments with liquid, gaseous, and supercritical CO₂ were compared. The cell counts of E. coli AW1.7 and mutants with a water activity (a_W) of 1.0 were reduced by more than 3 log₁₀ (CFU/ml) after supercritical CO₂ treatment at 35°C for 15 min; increasing the pressure generally enhanced inactivation, except for E. coli AW1.7 ΔgadABE. coli AW1.7 Δcfa was more susceptible than E. coli AW1.7 after treatment at 10 and 40 MPa; other mutations did not affect survival. Dry cells of E. coli were resistant to treatments with supercritical and liquid CO₂ at any temperature. Treatments with gaseous CO₂ at 65°C were more bactericidal than those with supercritical CO₂ or treatments at 65°C only. Remarkably, E. coli AW1.7 was more susceptible than E. coli AW1.7 Δcfa when subjected to the gaseous CO₂ treatment. This study identified CO₂-induced membrane fluidization and permeabilization as causes of supercritical mediated microbial inactivation, and diffusivity was a dominant factor for gaseous CO₂IMPORTANCE The safety of dry foods is of increasing concern for public health. Desiccated microorganisms, including pathogens, remain viable over long periods of storage and generally tolerate environmental insults that are lethal to the same organisms at high water activity. This study explored the use of high-pressure carbon dioxide to determine its lethality for dried Escherichia coli and to provide insight into the mechanisms of inactivation. The lethality of high-pressure CO₂ and the mechanisms of CO₂-mediated inactivation of dry E. coli depended on the physical state of CO₂ Liquid and supercritical CO₂ were ineffective in reducing the cell counts of dry E. coli isolates, and the effectiveness of gaseous CO₂ was related to the diffusivity of CO₂ Results provide a novel and alternative method for the food industry to enhance the safety of low a_W products.

Keywords: CO2 diffusivity; cyclopropane fatty acid synthase; gaseous CO2; membrane fluidity and permeability; supercritical CO2; water activity.