Real time intracellular pH dynamics in Listeria innocua under CO 2 and N 2O pressure

Abstract

This article investigates the inactivation mechanism of high-pressure food treatment, considered as alternative to conventional biocidal processes. We aimed to determine intracellular pH decrease under CO 2 and N 2O pressure, so far postulated as one of the main causes of inactivation. Working with a lab-scale bioreactor in mild conditions – 25 °C and pressures up to 8 MPa – we monitored – for the first time during pressurization – cytoplasmic pH variations of Listeria innocua labeled with pH-sensitive fluorophores based on fluorescein. We show that carbonic acid, due to solubilization of CO 2 into the aqueous phase, causes a rapid pH drop in the cytosol, reaching pH 4.8 at 1 MPa and falls below the detection limit of the indicator fluorophore of pH 4.0. This correlates with a reduced viability (below 90%) in all the pressure ranges investigated. Contrarily, treatment under N 2O pressure reduces cell viability without significant pH-drop neither of intra- nor extra-cellular liquid at any pressure investigated. The pH value remains between 7 and 6 while an inactivation of more than 80% is achieved at 8 MPa. Our data clearly demonstrate that, as a critical pressure is achieved, microbial inactivation is mainly due to pressure-induced membrane permeation – stimulated by non-acidifying fluids as well, rather then cytoplasmic acidification, as widely argued so far. A definitive understanding of the microbial inactivation mechanism due to CO 2/N 2O under pressure has been advanced significantly.