Abstract:

【Objective】The effects of high-hydrostatic pressure (HHP) on the membrane damage and oxidative phosphorylation were investigated with Listeria monocytogenes (LM 54004) as the test microorganism in this work. 【Method】The leakage of intracellular metal ions (K+, Mg2+) from LM 54004 subjected to HHP was determined using atomic absorption methods. The cell membrane of LM 54004 was labeled with radioactive probe of lipophilic cation tetraphenylphosphonium chloride ([3H]-TPP+), and the changes of membrane potential for LM 54004 exposed to HHP were measured. The changes of intracellular pH in LM 54004 treated by HHP were detected using carboxyfluorescein succinimidyl ester (cFSE) as fluorescent probe. The effects of HHP on F0F1-ATPase in LM 54004 were monitored using colorimetric methods. 【Result】With increasing pressure at the pressure levels of 150-300 MPa for 10 min, the experimental results showed that survival cells of LM 54004 and the activity of F0F1-ATPase decreased markedly (P＜0.05), intracellular metal ions (K+, Mg2+) were not leaked from LM 54004 cells, and the membrane potential, transmembrane proton concentration gradient and proton-motive force (PMF) did not change basically (P＞0.05). Exposed to 300 MPa for 10 min, although the survival cells on TSAYE medium were below the detection limit and the activity of F0F1-ATPase was significantly decreased to 0% (P＜0.05), integral membrane structure was not damaged and proton motive force (PMF) still could reach maximum as compared to these of untreated cells; the protons expelled from LM 54004 by the proton pump could not be returned to the cells of LM 54004. 【Conclusion】There was a good relationship between the inactivation of LM 54004 and the decrease of F0F1-ATPase activity by HHP. In summary, it can be concluded that the enzymes and electron carriers involved in respiratory chain in cell membrane were more resistant to high pressure than F0F1-ATPase, and the decrease of F0F1-ATPase activity was highly related to cell reduction during HHP, which provided some theoretical evidences for mechanisms of high-hydrostatic pressure sterilization. The major cause of LM 54004 death by HHP was the inactivation of F0F1-ATPase. HHP can be used as an effective tool to explore the development of microbial energy.

Key words: high-hydrostatic pressure (HHP), oxidative phosphorylation, proton motive force (PMF), F1F0-ATPase