Effects of Solution pH and Ions on Suicidal Germination of Bacillus subtilis Spores Induced by Medium High Temperature-Medium High Hydrostatic Pressure Treatment.

Spores of Bacillus subtilis suspended in water or aqueous solution of NaCl, CaCl2, sodium lactate, or calcium lactate at pH 4 - 7 was subjected to spore inactivation by simultaneous combination of medium high hydrostatic pressure (MHHP; 100 MPa) treatment for germination and medium high temperature (MHT; 65℃) treatment for pasteurization of germinated vegetative cells. The spores at pH 4 in NaCl solution and those at pH 5 and 6 in Na lactate solutions were less killed than in water by MHHP+MHT treatment. Spore inactivation was promoted by calcium ion in NaCl solution at pH 4 and in Na lactate solutions at pH 5 and pH 6, while it was more suppressed at pH 5 and pH 6 in Na lactate solutions than at pH 4 in NaCl solution. The spores treated by MHHP+MHT in NaCl or Na lactate solution at pH 4 were further killed by subsequent MHT treatment.

Characterization of high hydrostatic pressure-injured Bacillus subtilis cells.

High hydrostatic pressure (HHP) affects various cellular processes. Using a sporulation-deficient Bacillus subtilis strain, we characterized the properties of vegetative cells subjected to HHP. When stationary-phase cells were exposed to 250 MPa of HHP for 10 min at 25 °C, approximately 50% of cells were viable, although they exhibited a prolonged growth lag. The HHP-injured cells autolyzed in the presence of NaCl or KCl (at concentrations ≥100 mM). Superoxide dismutase slightly protected the viability of HHP-treated cells, whereas vegetative catalases had no effect. Thus, unlike HHP-injured Escherichia coli, oxidative stress only slightly affected vegetative B. subtilis subjected to HHP.

Injury and recovery of Escherichia coli ATCC25922 cells treated by high hydrostatic pressure at 400-600 MPa.

Escherichia coli cells were inactivated by high hydrostatic pressure (HHP) at 400-600 MPa and their recovery under various conditions was evaluated by colony counting and flow cytometer (FCM) analyses. The lag time in colony formation and an improved recovery of cells under less oxidative conditions (pyruvate addition to the medium and incubation in anaerobic conditions) were observed for HHP treated cells, which indicated that a significant portion of cells were injured and recovered during incubation after HHP treatment. The lag time for colony formation varied, which suggested a wave of resuscitation and recovered cells may multiply before other injured cells complete resuscitation. The recovery process was monitored by FCM: The FCM profile of cells stained using propidium iodide and SYTO9 indicated that while the majority of cells died just after HHP treatment, the staining pattern of possibly injured cells displayed a specific spectrum that gradually became consistent with that of the dead cell population and a living cell population simultaneously appeared. Pyruvate addition to the medium not only enhanced viability of HHP treated cells, but also reduced the lethal effect of HHP. These observations suggested that the degree of damage by HHP may differ cell-by-cell, and oxidative stress may continue after HHP treatment. Pyruvate addition to the recovery medium enhanced viability of E. coli cells inactivated by HHP treatment in tomato juice as well.