Screening for Bacillus subtilis mutants deficient in pressure induced spore germination: identification of ykvU as a novel germination gene.

Exposure to high pressure induces germination in spores of Bacillus subtilis. To investigate the mechanisms of this process and to compare the pressure and nutrient induced germination pathways, a random transposon knock-out library of B. subtilis was constructed and screened for clones with a compromised pressure induced germination at 100 MPa. Two mutants were isolated and their transposon insertion was mapped to gerAC and ykvU respectively. While GerAC is required for production of the l-alanine receptor which has been implicated in pressure-induced germination before, YkvU is shown here to be a novel germination determinant in B. subtilis, affecting germination by high (100 MPa) and very high (600 MPa) pressure, by nutrients and by dodecylamine, but not by Ca(2+)-dipicolinic acid.

Inactivation of Escherichia coli by high hydrostatic pressure at different temperatures in buffer and carrot juice.

The inactivation of Escherichia coli MG1655 was studied at 256 different pressure (150-600 MPa)-temperature (5-45 degrees C) combinations under isobaric and isothermal conditions in Hepes-KOH buffer (10 mM, pH 7.0) and in fresh carrot juice. A linear relationship was found between the log10 of inactivation and holding time for all pressure-temperature combinations in carrot juice, with R2-values>or=0.91. Decimal reduction times (D-values), calculated for each pressure-temperature combination, decreased with pressure at constant temperature and with temperature at constant pressure. Further, a linear relationship was found between log10D and pressure and temperature. A first order kinetic model, describing log10D in carrot juice as a function of pressure and temperature was formulated that allows to identify process conditions (pressure, temperature, holding time) resulting in a desired level of inactivation of E. coli. For Hepes-KOH buffer, the Weibull model more accurately described the entire set of inactivation curves of E. coli MG1655 compared to the log-linear or the biphasic model. Several secondary models (first and second order polynomial and Weibull) were evaluated, but all had poor fitting capacities. When the Hepes-KOH dataset was limited to 22 of the 34 pressure-temperature combinations, a first order model was appropriate and enabled us to use the same model structure as for carrot juice, for comparative purposes. The major difference in kinetic behaviour of E. coli in buffer and in carrot juice was that inactivation rate as a function of temperature showed a minimum around 20-30 degrees C in buffer, whereas it increased with temperature over the entire studied temperature range in carrot juice.

Inactivation of Bacillus cereus spores in milk by mild pressure and heat treatments.

The objective of this work was to study the germination and subsequent inactivation of Bacillus cereus spores in milk by mild hydrostatic pressure treatment. In an introductory experiment with strain LMG6910 treated at 40 degrees C for 30 min at 0, 100, 300 and 600 MPa, germination levels were 1.5 to 3 logs higher in milk than in 100 mM potassium phosphate buffer (pH 6.7). The effects of pressure and germination-inducing components present in the milk on spore germination were synergistic. More detailed experiments were conducted in milk at a range of pressures between 100 and 600 MPa at temperatures between 30 and 60 degrees C to identify treatments that allow a 6 log inactivation of B. cereus spores. The mildest treatment resulting in a 6 log germination was 30 min at 200 MPa/40 degrees C. Lower treatment pressures or temperatures resulted in considerably less germination, and higher pressures and temperatures further increased germination, but a small fraction of spores always remained ungerminated. Further, not all germinated spores were inactivated by the pressure treatment, even under the most severe conditions (600 MPa/60 degrees C). Two possible approaches to achieve a 6 log spore inactivation were identified, and validated in three additional B. cereus strains. The first is a single step treatment at 500 MPa/60 degrees C for 30 min, the second is a two-step treatment consisting of pressure treatment for 30 min at 200 MPa/45 degrees C to induce spore germination, followed by mild heat treatment at 60 degrees C for 10 min to kill the germinated spores. Reduction of the pressurization time to 15 min still allows a 5 log inactivation. These results illustrate the potential of high-pressure treatment to inactivate bacterial spores in minimally processed foods.

High sucrose concentration protects E. coli against high pressure inactivation but not against high pressure sensitization to the lactoperoxidase system.

The inactivation of Escherichia coli by high hydrostatic pressure treatment at up to 550 MPa and 20 degrees C was studied in potassium phosphate buffer containing high concentrations of sucrose. E. coli strain MG1655 was pressure-sensitive in the absence of sucrose, but became highly pressure resistant in the presence of 10% to 50% (w/v) sucrose. The pressure resistance of E. coli strain LMM1010, a previously described derivative of MG1655 that is pressure resistant in the absence of sucrose, was further increased in the presence of sucrose, to a similar level as for strain MG1655 in the presence of sucrose. When cell suspensions of either strain were stored after pressure treatment for 24 h at 20 degrees C, a further reduction of the plate counts indicative of pressure induced sublethal injury was observed, that was positively correlated with pressure intensity and negatively with sucrose concentration. Addition of the lactoperoxidase system to the cell suspensions strongly enhanced high pressure inactivation of E. coli at high sucrose concentrations. Using a pressure intensity of only 250 MPa, both E. coli strains were sensitized for the lactoperoxidase system in up to 30% (w/v) sucrose, resulting in at least 10(6)-fold inactivation within 24 h or less after pressure treatment. For comparison, a pressure treatment at 250 MPa in the absence of the lactoperoxidase system did not cause any inactivation of either strain even in the absence of sucrose. At sucrose concentrations above 30% (w/v), no or very little inactivation occurred even in the presence of the lactoperoxidase system.

The lactoperoxidase system increases efficacy of high-pressure inactivation of foodborne bacteria.

The inactivation of eight different bacteria comprising Escherichia coli LMM1010 and MG1655, respectively a pressure-resistant strain and the corresponding wild-type, Salmonella Typhimurium, Pseudomonas fluorescens, Staphylococcus aureus, Enterococcus faecalis, Listeria innocua and Lactobacillus plantarum, by high hydrostatic pressure in skim milk supplemented with the lactoperoxidase-hydrogen peroxide-thiocyanate (LP) system at naturally occurring concentration was studied. In the absence of pressure treatment, the LP system had either no effect, i.e. on S. Typhimurium and E. coli LMM1010, a growth inhibiting effect, i.e. on E. coli MG1655, L. innocua, S. aureus, L. plantarum and E. faecalis, or a bactericidal effect, i.e. on P. fluorescens. The presence of the LP system affected inactivation by high pressure in a cell density-dependent manner. At low cell concentration (10(6) cfu/ml), the LP system strongly increased high-pressure inactivation as measured immediately after pressure treatment of all bacteria except the pressure-resistant E. coli. At high cell density (10(9) cfu/ml), only inactivation of L. innocua, E. faecalis and L. plantarum were enhanced. For both E. coli strains, the fate of the bacteria during 24 h following pressure treatment was also studied. It was found that in the presence of the LP system, considerable further inactivation occurred in the first hours after pressure treatment. The potential of the LP system to improve the bactericidal efficiency of high-pressure treatment for food preservation is discussed.