Elke Y. Wuytack

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 β-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.

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.

Comparative Study of Pressure- and Nutrient-Induced Germination of Bacillus subtilis Spores

ABSTRACT Germination experiments with specific germination mutants ofBacillus subtilis, including a newly isolated mutant affected in pressure-induced germination, suggest that a pressure of 100 MPa triggers the germination cascades that are induced by the nutrient germinant alanine (Ala) and by a mixture of asparagine, glucose, fructose, and potassium ions (AGFK), by activating the receptors for alanine and asparagine, GerA and GerB, respectively. As opposed to germination at 100 MPa, germination at 600 MPa apparently shortcuts at least part of the Ala- and AGFK-induced germination pathways. Inhibitors of nutrient-induced germination (HgCl2and Nα-P-tosyl-l-arginine methyl ester) also inhibit pressure-induced germination at 600 MPa, suggesting that germination at 600 MPa involves activation of a true physiological germination pathway and is therefore not merely a physico-chemical process in which water is forced into the spore protoplast.

A study on the effects of high pressure and heat on Bacillus subtilis spores at low pH.

Bacillus subtilis spore suspensions were subjected to pressure treatments at 100 and 600 MPa at 40 degrees C and over a pH range from 3 to 8. Inactivation of spores under these conditions was maximally 80% and was not increased at low pH. However, higher levels of inactivation were obtained when spores were first pressure treated at neutral pH and then exposed for 1 h to low pH. This large difference in inactivation could be explained by the finding that pressure-induced spore germination, which is known to occur at neutral pH, was inhibited at low pH (< 5). Pressure treatment at low pH made spores more sensitive to heat inactivation, suggesting that demineralized H-spores had been formed. Changes in spore core hydration and pH upon exposure of spores at low pH were studied in a more direct way using green fluorescent protein expressed in recombinant B. subtilis as a reporter protein, and it was confirmed that pressure and heat increase spore permeability for protons. Based on these results, the potential of low temperature, high pressure processes for spore inactivation in acid products is discussed.

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.

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 °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 °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 ≥45 °C, corresponding to an outlet sample temperature >65 °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.

Expression of a P-type Ca2+-transport ATPase in Bacillus subtilis during sporulation

The open reading frame designated yloB in the genomic sequence of Bacillus subtilis encodes a putative protein that is most similar to the typically eukaryotic type IIA family of P-type ion-motive ATPases, including the endo(sarco)plasmic reticulum (SERCA) and PMR1 Ca(2+)-transporters, located respectively in the SERCA and the Golgi apparatus. The overall amino acid sequence is more similar to that of the Pmr1s than to the SERCAs, whereas the inverse is seen for the 10 amino acids that form the two Ca(2+)-binding sites in SERCA. Sporulating but not vegetative B. subtilis cells express the predicted protein, as shown by Western blotting and by the formation of a Ca(2+)-dependent phosphorylated intermediate. Half-maximal activation of phosphointermediate formation occurred at 2.5 microM Ca(2+). Insertion mutation of the yloB gene did not affect the growth of vegetative cells, did not prevent the formation of viable spores, and did not significantly affect 45Ca accumulation during sporulation. However, spores from knockouts were less resistant to heat and showed a slower rate of germination. It is concluded that the P-type Ca(2+)-transport ATPase from B. subtilis is not essential for survival, but assists in the formation of resistant spores. The evolutionary relationship of the transporter to the eukaryotic P-type Ca(2+)-transport ATPases is discussed.