Assessment of high pressure induced damage on Lactobacillus rhamnosus GG by flow cytometry

To assess the mode of action of high-pressure treatment on Lactobacillus rhammosus GG (LGG) a flow cytometric analysis was applied. This fluorescence-based approach could give additional insights on process-induced changes of cellular events, which were not explicitly assessable by culture techniques, such as cellular inactivation sites, specific metabolic activities, etc. To achieve this goal, combined staining with carboxyfluorescein diacetate (cFDA) and propidium iodide (PI) was applied. With this staining strategy pressure-induced changes of microbial esterase activity and membrane integrity were monitored. Moreover, the ability of the cells to extrude intracellular accumulated carboxyfluorescein (cF) upon energisation was ascertained as an additional vitality marker. The determination of microbial viability status with help of different physiological and metabolic parameters and their relative changes following pressure treatment is of increasing importance in evaluating sterilisation and/or pasteurisation processes. Comparison of conventional culture techniques with flow cytometric viability assessment (after pressure treatments at 100-600 MPa) revealed the occurrence of certain cell populations, which were stressed and lost their ability to grow on agar, but still showed metabolic activity. This fraction is often described as viable but not culturable cells. The presence of such injured bacteria in food might be critical in terms of their potential activity on excreting toxic or food spoiling metabolites. (C) 2003 Elsevier Ltd. All rights reserved.