Inactivation of Escherichia coli by high-pressure homogenisation is influenced by fluid viscosity but not by water activity and product composition.

The inactivation of Escherichia coli MG1655 by high-pressure homogenisation (HPH) at pressures ranging from 100 to 300 MPa was studied in buffered suspensions adjusted to different relative viscosities (1.0, 1.3, 1.7, 2.7 and 4.9) with polyethylene glycol 6000 (PEG 6000). The water activity of these suspensions was not significantly affected by this high molecular weight solute. Bacterial inactivation was found to decrease with increasing viscosity of the suspensions, an effect that was more pronounced at higher pressures. To study the effect of water activity, series of E. coli suspensions having a different water activity (0.953-1.000) but the same relative viscosity (1.3, 1.7, 2.7 and 4.9) were made using PEG of different molecular weights (400, 600, 1000 and 6000), and subjected to HPH treatment. The results indicated that water activity does not influence inactivation. Finally, inactivation of E. coli MG1655 by HPH in skim milk, soy milk and strawberry-raspberry milk drink was found to be the same as in PEG containing buffer of the corresponding viscosity. These results identify fluid viscosity as a major environmental parameter affecting bacterial inactivation by HPH, as opposed to water activity and product composition, and should contribute to the development of HPH applications for the purpose of bacterial inactivation.

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.

Moderate temperatures affect Escherichia coli inactivation by high-pressure homogenization only through fluid viscosity.

The inactivation of suspensions of Escherichia coli MG1655 by high-pressure homogenization was studied over a wide range of pressures (100-300 MPa) and initial temperatures of the samples (5-50 degrees C). Bacterial inactivation was positively correlated with the applied pressure and with the initial temperature. When samples were adjusted to different concentrations of poly(ethylene glycol) to have the same viscosity at different temperatures below 45 degrees C and then homogenized at these temperatures, no difference in inactivation was observed. These observations strongly suggest, for the first time, that the influence of temperature on bacterial inactivation by high-pressure homogenization is only through its effect on fluid viscosity. At initial temperatures > or =45 degrees C, corresponding to an outlet sample temperature >65 degrees C, the level of inactivation was higher than what would be predicted on the basis of the reduced viscosity at these temperatures, suggesting that under these conditions heat starts to contribute to cellular inactivation in addition to the mechanical effects that are predominant at lower temperatures. Second-order polynomial models were proposed to describe the impact of a high-pressure homogenization treatment of E. coli MG1655 as a function of pressure and temperature or as a function of pressure and viscosity. The pressure-viscosity inactivation model provided a better quality of fit of the experimental data and furthermore is more comprehensive and versatile than the pressure-temperature model because in addition to viscosity it implicitly incorporates temperature as a variable.

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.

Na+-mediated piezoprotection in Rhodotorula rubra.

Sodium concentrations as low as 2 mM exerted a significant protective effect on the high-pressure inactivation (160-210 MPa) of Rhodotorula rubra at pH 6.5, but not on two other yeasts tested (Schizosaccharomyces pombe and Saccharomyces cerevisiae). A piezoprotective effect of similar magnitude was observed with Li+ (2 and 10 mM), and at elevated pH (8.0-9.0), but no effect was seen with K+, Ca2+, Mg2+, Mn2+, or NH4(+). Intracellular Na+ levels in cells exposed to low concentrations of Na+ or to pH 8.0-9.0 provided evidence for the involvement of a plasma membrane Na+/H+ antiporter and a correlation between intracellular Na+ levels and pressure resistance. The results support the hypothesis that moderate high pressure causes indirect cell death in R. rubra by inducing cytosolic acidification.

Modelling inactivation of Staphylococcus aureus and Yersinia enterocolitica by high-pressure homogenisation at different temperatures.

A detailed study of the inactivation of Staphylococcus aureus and Yersinia enterocolitica by high-pressure homogenisation was performed at, respectively, 25 and 35 different combinations of process temperature and process pressure covering a range of 5-50 degrees C and 100-300 MPa. It appeared that in the entire studied pressure-temperature domain, S. aureus was more resistant to high-pressure homogenisation than Y. enterocolitica. Furthermore, the effect of the process pressure on the inactivation of S. aureus was considerably smaller than on the inactivation of Y. enterocolitica. Also, temperature between 5 and 40 degrees C did not affect inactivation of S. aureus by high-pressure homogenisation, while Y. enterocolitica inactivation was affected by temperature over a much wider range. Different mathematical models were compared to describe the inactivation of both bacteria under the experimental conditions applied. Such pressure-temperature inactivation models form the engineering basis for design, evaluation and optimisation of high-pressure homogenisation processes as a new preservation technique.

Decontamination of seeds for seed sprout production by high hydrostatic pressure.

Garden cress, sesame, radish, and mustard seeds immersed in water were treated with high pressure (250, 300, 350, and 400 MPa) for 15 min at 20 degrees C. After treatment, percentages of seeds germinating on water agar were recorded for up to 11 days. Of the seeds tested, radish seeds were found to be the most pressure sensitive, with seeds treated at 250 MPa reaching 100% germination 9 days later than untreated control seeds did. Garden cress seeds, on the other hand, were the most pressure resistant, with seeds treated at 250 MPa reaching 100% germination 1 day later than untreated control seeds did. Garden cress sprouts from seeds treated at 250 and 300 MPa also took about 1 day longer to reach average sprout length than sprouts from untreated control seeds did, indicating that sprout growth was not retarded once germination had occurred. Garden cress seeds were inoculated with suspensions of seven different bacteria (10(7) CFU/ml) and processed with high pressure. Treatment at 300 MPa (15 min, 20 degrees C) resulted in 6-log reductions of Salmonella Typhimurium, Escherichia coli MG1655, and Listeria innocua, > 4-log reductions of Shigella flexneri and pressure-resistant E. coli LMM1010, and a 2-log reduction of Staphylococcus aureus. Enterococcus faecalis was virtually not inactivated. For suspensions of the gram-positive bacteria, similar levels of inactivation in water in the absence of garden cress seeds were found, but the inactivation of E. coil LMM1010 and S. flexneri in water in the absence of garden cress seeds was significantly less extensive. These data suggest that garden cress seeds contain a component that acts synergistically with high hydrostatic pressure against gram-negative bacteria.

Comparison of sublethal injury induced in Salmonella enterica serovar Typhimurium by heat and by different nonthermal treatments.

We have studied sublethal injury in Salmonella enterica serovar Typhimurium caused by mild heat and by different emerging nonthermal food preservation treatments, i.e., high-pressure homogenization, high hydrostatic pressure, pulsed white light, and pulsed electric field. Sublethal injury was determined by plating on different selective media, i.e., tryptic soy agar (TSA) plus 3% NaCl, TSA adjusted to pH 5.5, and violet red bile glucose agar. For each inactivation technique, at least five treatments using different doses were applied in order to cover an inactivation range of 0 to 5 log units. For all of the treatments performed with a technique, the logarithm of the viability reductions measured on each of the selective plating media was plotted against the logarithm of the viability reduction on TSA as a nonselective medium, and these points were fined by a straight line. Sublethal injury between different techniques was then compared by the slope and the y intercept of these regression lines. The highest levels of sublethal injury were observed for the heat and high hydrostatic pressure treatments. Sublethal injury after those treatments was observed on all selective plating media. For the heat treatment, but not for the high-pressure treatment, sublethal injury occurred at low doses, which were not yet lethal. The other nonthermal techniques resulted in sublethal injury on only some of the selective plating media, and the levels of injury were much lower. The different manifestations of sublethal injury were attributed to different inactivation mechanisms by each of the techniques, and a mechanistic model is proposed to explain these differences.

Bacterial inactivation by high-pressure homogenisation and high hydrostatic pressure.

The resistance of five gram-positive bacteria, Enterococcus faecalis, Staphylococcus aureus, Lactobacillus plantarum, Listeria innocua and Leuconostoc dextranicum, and six gram-negative bacteria, Salmonella enterica serovar typhimurium, Shigella flexneri, Yersinia enterocolitica, Pseudomonas fluorescens and two strains of Escherichia coli, to high-pressure homogenisation (100-300 MPa) and to high hydrostatic pressure (200-400 MPa) was compared in this study. Within the group of gram-positive bacteria and within the group of gram-negative bacteria, large differences were observed in resistance to high hydrostatic pressure, but not to high-pressure homogenisation. All gram-positive bacteria were more resistant than any of the gram-negative bacteria to high-pressure homogenisation, while in relative to high hydrostatic pressure resistance both groups overlapped. Within the group of gram-negative bacteria, there also existed another order in resistance to high-pressure homogenisation than to high hydrostatic pressure. Further it appears that the mutant E. coli LMM1010, which is resistant to high hydrostatic pressure is not more resistant to high-pressure homogenisation than its parental strain MG1655. The preceding observations indicate a different response of the test bacteria to high-pressure homogenisation compared to high hydrostatic pressure treatment, which suggests that the underlying inactivation mechanisms for both techniques are different. Further, no sublethal injury could be observed upon high-pressure homogenisation of Y. enterocolitica and S. aureus cell population by using low pH (5.5 7), NaCl (0 6%) or SDS (0-100 mg/l) as selective components in the plating medium. Finally, it was observed that successive rounds of high-pressure homogenisation have an additive effect on viability reduction of Y. enterocolitica and S. aureus.

High-Pressure Transient Sensitization of Escherichia coli to Lysozyme and Nisin by Disruption of Outer-Membrane Permeability.

Escherichia coli MG1655 suspensions in 10 mM phosphate buffer (pH 7.0) were subjected to high pressures in the range of 180 to 320 MPa for 15 min. Cell death was evident at 220 MPa and increased exponentially with pressure. Surviving populations were sublethally injured, as demonstrated by their reduced ability to form colonies on violet red bile glucose agar, a selective growth medium containing crystal violet and bile salts. During exposure to high pressure (> 180 MPa), cells were sensitive to lysozyme, nisin, and ethylenediaminetetraacetic acid (EDTA), as was apparent from an increased lethality of pressure in the presence of these agents. Sublethal injury in the surviving population was lower in the presence of nisin and lysozyme, but higher in the presence of EDTA. Combinations of EDTA with nisin or lysozyme present during pressure treatment increased lethality in an additive manner. However, the addition of lysozyme, nisin and/or EDTA to pressurized cell suspensions immediately after pressure treatment did not cause any viable count reduction. Finally, we observed leakage of the periplasmic enzyme ß-lactamase from an ampicillin-resistant recombinant E. coli MG1655 under high pressure. These results suggest that high pressure transiently disrupts the permeability of the E. coli outer membrane for water-soluble proteins.