Certain dense gases, including CO(2) and N(2)O, are known to deactivate food pathogens safely. Complete deactivation requires disabling intracellular metabolic pathways by extracellular processing that causes membrane disruption, irreversible denaturation of proteins, or extraction of cell contents. At present, neither the precise physical and metabolic mechanisms nor their kinetics behind dense gas pasteurization are known. The mechanisms depend strongly on both the organism and the environment and may combine membrane disruption, membrane permeabilization, and altering pH. Herein we elucidate the mechanisms of dense gas inactivation with the aid of a novel approach for measuring intracellular pH (pH(i)) under high gas pressure. Using a pH-sensitive GFP-variant of S. cerevisiae as the probe, we demonstrate that membrane permeabilization by a non-acidic gas, N(2)O, contributes to inactivation but at a rate that is relatively low compared to CO(2). CO(2) not only permeabilizes the membrane but also brings about a rapid drop in pH(i), leading to greater deactivation. Mechanistic understanding is vital to develop safe and effective dense gas technologies for food treatment. Knowledge of pH(i) is also important in other cellular processes, including enzyme activity, gene transcription, and protein synthesis. The GFP technique has been demonstrated to be versatile even under pressure.
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