Multi-pulsed high hydrostatic pressure treatment for inactivation and injury of Escherichia coli

Escherichia coli cells in peptone water were pressurized at 300?MPa at ambient temperature with no holding time (pulse series) and with a total holding duration of 300?s for single- (300?s?×?1 pulse) and multi-pulsed (150?s?×?2 pulses, 100?s?×?3 pulses, 75?s?×?4 pulses, 60?s?×?5 pulses, 50?s?×?6 pulses and 30?s?×?10 pulses) high hydrostatic pressure (HHP) treatments. Multi-pulsed HHP treatment with no holding time indicated that as the pulse number increased the number of inactivated and injured cells also increased. Holding time had significant effect on the inactivation of E. coli. There was low inactivation difference between single- and multi-pulsed HHP treatments with holding time. Escherichia coli cells showed at least 1.6 log₁₀ more reduction on selective medium than the non-selective medium indicating that more than 95?% of the survivors severely injured for both single- and multi-pulsed treatments with holding time. Although the inactivation difference was low between single- and multi-pulsed HHP treatments, storage at 4?°C revealed that there was less recovery from injury for multi-pulsed HHP treatment.     

Extraction methods for lipopolysaccharides from Escherichia coli ATCC 25922 for quantitative analysis by capillary electrophoresis

Lipopolysaccharides (LPS) are major constituents of the cell wall of Gram-negative bacteria. Capillary zone electrophoresis (CZE) was applied to distinguish between lipopolysaccharides extracted from Escherichia coli ATCC 25922 with various techniques. Extraction methods proposed by Westphal and Jann [Methods Carbohydr. Chem. 5 (1965) 83], Galanos et al. [Eur. J. Biochem. 9 (1969) 245], Ni Eidhin and Mouton [FEMS Microbiol. Lett. 110 (1993) 133] and Nichols [Infect. Immun. 62 (1994) 3753] for LPS preparation were evaluated. Electrophoresis buffers with varying pHs were applied to assess the structure stability of the extracted LPS samples. Variations in structural breakdown were apparent demonstrating that different extraction methods removed different LPS molecules. Furthermore, the results obtained proved the CZE useful as an analytical technique for LPS evaluation. The LPS removed with the Nichols extraction procedure presented a unique electrophorogram that could in future be applied in the rapid identification of Gram-negative foodborne pathogens.     

Inactivation of Escherichia coli and Listeria innocua in kiwifruit and pineapple juices by high hydrostatic pressure

Escherichia coli and Listeria innocua in kiwifruit and pineapple juices were exposed to high hydrostatic pressure (HHP) at 300 MPa for 5 min. Both bacteria showed equal resistance to HHP. Using low (0 degrees C) or sub-zero (-10 degrees C) temperatures instead of room temperature (20 degrees C) during pressurization did not change the effectiveness of HHP treatment on both bacteria in studied juices. Pulse pressure treatment (multiple pulses for a total holding time of 5 min at 300 MPa) instead of continuous (single pulse) treatment had no significant (p>0.05) effect on the microbial inactivation in kiwifruit juice; however, in pineapple juice pulse treatment, especially after 5 pulses, increased the inactivation significantly (p<0.05) for both bacteria. Following storage of pressure-treated (350 MPa, 20 degrees C for 60 s x 5 pulses) juices at 4, 20 and 37 degrees C up to 3 weeks, the level of microbial inactivation further increased and no injury recovery of the bacteria were detected. This work has shown that HHP treatment can be used to inactivate E. coli and L. innocua in kiwifruit and pineapple juices at lower pressure values at room temperature than the conditions used in commercial applications (>400 MPa). However, storage period and temperature should carefully be optimized to increase the safety of HHP treated fruit juices.     

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.     

Effect of skimmed milk and its fractions on the inactivation of Escherichia coli K12 by high hydrostatic pressure treatment

We investigated the effects of skimmed milk and its protein fractions (casein, whey, globulin, and albumin) on the injury and inactivation of Escherichia coli K-12 by high hydrostatic pressure (HHP) treatment. The protective effect of skimmed milk on HHP-mediated inactivation and injury of E. coli increased with increases in the skimmed milk concentration. However, protein fractions derived from skimmed milk did not exhibit this protective effect. Microscopy analysis by DAPI/PI staining indicated that some cells were localized in the solid portion of skimmed milk, and some of these cells were alive. The coagulated fraction derived from the autoclaved whey fraction also showed a significant protective effect. We speculate that the solid portion in skimmed milk could provide the protective effect to bacterial cells.     

Effect of negative air ions on Escherichia coli ATCC 25922 inoculated onto mung bean seed and apple fruit

The effect of negative air ions on the reduction of Escherichia coli ATCC 25922 inoculated onto mung bean sprout seed and whole or fresh-cut apple fruit was studied. Mung bean seeds, whole Gala apples, and Gala apple slices were inoculated with E. coli ATCC 25922 before being exposed to negative air ions for up to 18 h at room temperature (-23 degrees C). Results revealed a less than 0.5-log reduction of E. coli on mung bean seed even after 18 h of exposure. The reduction of E. coli on the surface of whole apples increased with increasing exposure time from 0.5 to 3 h, but the maximum reduction was less than 1 log CFU/g. Increasing exposure time from 3 to 18 h did not lead to increased treatment efficacy. No reduction of E. coli was observed on apple slices after 3 h of treatment. When the negative air ion system was applied with acetic acid vapor, no additive or synergistic effect of negative ions on the reduction of E. coli was found. These results suggest that negative air ions have a very limited effect on the population of E. coli on mung bean seed and apples.     

Inactivation of pathogenic Escherichia coli in skimmed milk using hhigh hydrostatic pressure

The pressure resistance of a range of pathogenic Escherichia coli strains was determined in skimmed milk. Pressure resistances varied widely among the 12 strains tested, with the two most resistant being an enterotoxigenic E. coli (NCTC 11601) and an enteroinvasive E. coli (NCTC 9706). A pressure treatment of 500 MPa for 40 min gave only a 4-log reduction of these two most resistant strains, but after a treatment of 600 MPa for 30 min, no survivors of either strain could be detected (>7 log reduction). The effect of the length of time in the stationary phase on the pressure resistances of these two strains was determined. Pressure resistance (600 MPa for 10 min at 20°C) was determined after 12 h incubation at 37°C in tryptone soya broth with 0.6% yeast extract added (TSBYE) (early stationary phase) and at 24-h intervals after this. The pressure resistances of these strains did not change significantly for the first 5 days after the onset of the stationary phase, after which time their resistances decreased. When cells were pressure-treated 7 days after they went into stationary phase, no survivors could be detected.     

Fate of Escherichia coli strains inoculated in model cheese elaborated with or without starter and treated by high hydrostatic pressure

The aim of this research was to study high hydrostatic pressure inactivation of two strains of Escherichia coli (E. coli O59:H21 [CECT 405] and E. coli O157:H7 [CECT 5947]) inoculated in washed-curd model cheese elaborated with and without starter and the ability of these strains for survival, recovery, and growth. Samples were treated at 300, 400, and 500 MPa for 10 min at 20 degrees C and analyzed after the treatment and after 1, 7, and 15 days of storage at 8 degrees C to study the behavior of Escherichia populations. Cheeses elaborated with starter showed the maximum lethality at 400 and 500 MPa, and no significant differences in the baroresistant behavior of either strains were detected, except for E. coli O157:H7 at 400 MPa in cell counts obtained with thin agar layer method medium, where the decrease value was significantly lower. In cheese elaborated without starter, the highest decrease value was observed at 500 MPa, except for E. coli O59:H21 in cell counts obtained with selective culture medium, where the highest decrease value was also found at 400 MPa. The ability to repair and grow was not observed in model cheese elaborated with starter, as cell counts of treated samples decreased after 15 days of storage at 8 degrees C. By contrast, in cheese elaborated without starter, all pressurized samples showed the trend to repair and grow during the storage period in both strains. These results suggest that the presence of starter and low pH values are the main factors that control the ability of Escherichia strains inoculated in this type of cheese and treated by high hydrostatic pressure to recover and grow.     

High hydrostatic pressure treatment impairs AcrAB-TolC pump resulting in differential loss of deoxycholate tolerance in Escherichia coli

We reported previously that high hydrostatic pressure-injured stationary phase cells of Escherichia coli K-12 lost their intrinsic deoxycholate tolerance. The AcrAB-TolC multi-drug resistance pump driven by proton motive force has been argued to be responsible for the tolerance to deoxycholate. In this report, we tested the sensitivity of the AcrAB-TolC (three components) pump to high hydrostatic pressure treatment (HPT). E. coli K-12 treated with HPT became sensitive to AcrAB-TolC-specific drugs such as ethidium bromide, but not to tetracycline which is pumped out by a one-component transporter, Tet. Only E. coli K-12 overproducing both AcrAB and TolC exhibited restored tolerance to deoxycholate after HPT but not E. coli overproducing either TolC or AcrAB. These observations strongly suggest that three-component pumps such as AcrAB-TolC are more susceptible to HPT than one-component pumps such as Tet, resulting in the differential loss of deoxycholate tolerance in high hydrostatic pressure-injured E. coli cells.     

Modelling the inactivation of Escherichia coli ATCC 25922 using pulsed electric field

Microbial tests were conducted to determine the effect of pulsed electric field treatment on microbial inactivation of gram negative Escherichia coli ATCC 25922 suspended in simulated milk ultra filtrate (SMUF). Kinetic analysis of microbial inactivation due to combined pulsed electric field (PEF) and thermal treatments of E. coli was investigated. A generalized correlation for the inactivation rate constant as a function of both electric field intensity and treatment temperature was derived. Comparison between experimental and theoretical variation of E. coli concentration with time after PEF treatment in a complete recirculation mode was conducted using the inactivation kinetics developed from the single pass measurements.

Industrial relevance

PEF technology has a tremendous potential to replace thermal pasteurisation for products which are sensitive to temperature. In this work a pulsed electric field process was mathematically modelled and a generalized correlation for the inactivation rate constant as a function of electric field intensity and treatment temperature was derived. The correlation is needed for the design of industrial PEF systems.     

Potential application of high hydrostatic pressure to eliminate Escherichia coli O157:H7 on alfalfa sprouted seeds

Sprouts eaten raw are increasingly being perceived as hazardous foods as they have been implicated in Escherichia coli O157:H7 outbreaks where the seeds were found to be the likely source of contamination. The objective of our study was to evaluate the potential of using high hydrostatic pressure (HHP) technology for alfalfa seed decontamination. Alfalfa seeds inoculated with a cocktail of five strains of E. coli O157:H7 were subjected to pressures of 500 and 600 MPa for 2 min at 20 degrees C in a dry or wet (immersed in water) state. Immersing seeds in water during pressurization considerably enhanced inactivation of E. coli O157:H7 achieving reductions of 3.5 log and 5.7 log at 500 and 600 MPa, respectively. When dry seeds were pressurized, both pressure levels reduced the counts by <0.7 log. To test the efficacy of HHP to completely decontaminate seeds whilst meeting the FDA requirement of 5 log reductions, seeds inoculated with a ~5 log CFU/g of E. coli O157:H7 were pressure-treated at 600 and 650 MPa at 20 degrees C for holding times of 2 to 20 min. A >5 log reduction in the population was achieved when 600 MPa was applied for durations of > or =6 min although survivors were still detected by enrichment. When the pressure was stepped up to 650 MPa, the threshold time required to achieve complete elimination was 15 min. Un-inoculated seeds pressure-treated at 650 MPa for 15 min at 20 degrees C successfully sprouted achieving a germination rate identical to untreated seeds after eight days of sprouting. These results therefore demonstrate the promising application of HHP on alfalfa seeds to eliminate the risk of E. coli O157:H7 infections associated with consumption of raw alfalfa sprouts.     

Physiological changes of Escherichia coli O157:H7 and Staphylococcus aureus following exposure to high hydrostatic pressure

Morphological changes and membrane integrity of Escherichia coli O157:H7 and Staphylococcus aureus cells before and after high hydrostatic pressure (HHP) treatments (200–400 MPa) and time (1–5 min), at a constant temperature (40 °C), in peptone water were examined by using scanning electron microscopy (SEM) and fluorescent microscopy, respectively. SEM images showed that unpressurized cells exhibited a smooth surface appearance. E. coli O157:H7 cells exposed to pressure treatments first appeared larger, then with increasing pressure distorted with dimples and pinches. In case of S. aureus, the cells pressurized at low pressure levels did not show any significant change. The surface appearance became rough and cracked when the cells were exposed to higher pressure levels. Images of fluorescent microscopy showed that a small proportion of bacterial cells were not green fluorescent at lower pressure levels. The other part of the cell population was red fluorescent representing dead cells and the number of red fluorescent cells increased with increasing pressure. The cells with a yellowish color showed that varying levels of membrane damage occurred under HHP. The combinations of mild heat, antimicrobial substances and HHP treatment can be used to inactivate food borne pathogens of varying pressure resistance in the food industry for safe processing conditions. However, the resultant damaged cells at different levels should be taken into account during storage to prevent their recovery.     

Application of high hydrostatic pressure to decontaminate green onions from Salmonella and Escherichia coli O157:H7

Consumption of fecally contaminated green onions has been implicated in several major outbreaks of foodborne illness. The objectives of this study were to investigate the survival and growth of Salmonella and Escherichia coli O157:H7 in green onions during storage and to assess the application of high hydrostatic pressure (HHP) to decontaminate green onions from both pathogens. Bacterial strains resistant to nalidixic acid and streptomycin were used to inoculate green onions at low (∼1 log cfu/g) and high (∼2 log cfu/g) inoculum levels which were then kept at 4 or 22 °C for up to 14 days. Both pathogens grew to an average of 5-6 log cfu/g during storage at 22 °C and the bacterial populations were fairly stable during storage at 4 °C. High-pressure processing of inoculated green onions in the un-wetted, wetted (briefly dipped in water) or soaked (immersed in water for 30 min) conditions at 250-500 MPa for 2 min at 20 °C reduced the population of Salmonella and E. coli O157:H7 by 0.6 to >5 log cfu/g, depending on the pressure level and sample wetness state. The extent of pressure inactivation increased in the order of soaked > wetted > un-wetted state. The pressure sensitivity of the pathogens was also higher at elevated treatment temperatures. Overall, after pressure treatment at 400-450 MPa (soaked) or 450-500 MPa (wetted) for a retention time of 2 min at 20-40 °C, wild-type and antibiotic-resistant mutant strains of Salmonella and E. coli O157:H7 inoculated on green onions were undetectable immediately after treatment and throughout the 15-day storage at 4 °C. The pressure treatments also had minimal adverse impact on most sensorial characteristics as well as on the instrumental color of chopped green onions. This study highlights the promising applications of HHP to minimally process green onions in order to alleviate the risks of Salmonella and E. coli O157:H7 infections associated with the consumption of this commodity.     

The Effects of High-Pressure Processing at Low and Subzero Temperatures on Inactivation of Microorganisms in Frozen and Unfrozen Beef Mince Inoculated with Escherichia coli Strain ATCC 25922

Frozen and unfrozen beef mince inoculated with Escherichia coli strain ATCC 25922 were exposed to a pressure of 300 MPa for 5 min at different temperatures (?10, ?5, 0, 10 and 20 °C). A maximum reduction of 1.5 log in total aerobic count (TAC) was obtained in unfrozen samples at ?5 °C, whereas at 20 °C, the reduction was only 0.6 log. Microbial inactivation in beef mince was enhanced by freezing the beef mince prior to pressurization. An average log reduction of 3.0 (SD?=?0.2) in both E. coli and TAC was obtained in frozen beef mince treated at ?5 °C. The highest bacterial reductions were observed in frozen samples. The extent of bacterial injury was substantially less in frozen samples than unfrozen samples, indicating that the damage inflicted on microorganisms in frozen beef mince by high pressure was irreversible. Lightness (L ^*), redness (a ^*) and yellowness (b ^*) values measured in accordance of Commission Internationale de l’Eclairage (CIE) for all the pressure-treated frozen and unfrozen samples differed slightly from unfrozen control samples (average total colour change, ΔE?=?6.1, SD?=?1.1). Water-holding capacity (WHC) measured by “high-pressure expressed moisture”, a new method proposed in this study, showed that freezing the samples prior to pressurization could increase WHC of minced beef. The results suggest that high-pressure processing could be used to make safer traditional raw minced meat products, such as steak tartare and cig kofte, a traditional Turkish dish made with minced beef, bulgur and spices.     

Physical properties of surimi sausages subjected to high hydrostatic pressure treatment

Food Science and Biotechnology - We aimed to investigate the effect of high pressure treatment (HPT) on the physical properties of surimi sausages. For protein gelation, high hydrostatic pressure...     

Evaluation of high hydrostatic pressure sensitivity of Staphylococcus aureus and Escherichia coli O157:H7 by differential scanning calorimetry

Differential scanning calorimetry (DSC) was used to evaluate the relative high hydrostatic pressure (HHP) resistances of bacterial strains from Staphylococcus aureus and Escherichia coli O157:H7 in vivo. The total apparent enthalpy change and thermal stability were two DSC parameters used to compare bacterial strains of untreated control and pressure-treated bacteria. DSC thermograms indicated that ribosomal denaturation appears to be a major factor in cell death by both thermal and high pressure treatments. However, the analysis of calorimetric data for control samples as well as pressure-treated samples clearly showed that the sensitivities of bacteria to various physical stresses can be different. While S. aureus 765 had a relatively higher resistance to thermal treatment in comparison to S. aureus 485, S. aureus 485 was determined to be more resistant to pressure than S. aureus 765. This information can be utilized in the design of processes specific to targeting certain cellular components by using different physical stresses.     

Sustainable production of pectin from lime peel by high hydrostatic pressure treatment

The application of high hydrostatic pressure technology for enzymatic extraction of pectin was evaluated. Cellulase and xylanase under five different combinations (cellulase/xylanase: 50/0, 50/25, 50/50, 25/50, and 0/50 U/g lime peel) at ambient pressure, 100 and 200 MPa were used to extract pectin from dried lime peel. Extraction yield, galacturonic acid (GalA) content, average molecular weight (M_w,ave), intrinsic viscosity [η]_w, and degree of esterification (DE) were compared to those parameters obtained for pectins extracted using acid and aqueous processes. Pressure level, type and concentration of enzyme significantly (p < 0.05) influenced yield and DE of pectin. Enzyme and high pressure extraction resulted in yields which were significantly (p < 0.05) higher than those using acid and aqueous extraction. Although pressure-induced enzymatic treatment improves pectin yield, it does not have any significant effect on M_w,ave and [η]_w of pectin extracts indicating the potential of high pressure treatment for enzymatic pectin production as a novel and sustainable process.     

Growth of Escherichia coli ATCC 9637 through the uptake of compatible solutes at high osmolarity

The growth rate of Escherichia coli ATCC 9637 was determined in a chemically defined (CD) medium with high osmolarity, 1-1.2 M. The addition of ectoine or glycine betaine to the medium resulted in a significant stimulation of growth rate for this strain. In the presence of ectoine derivatives, hydroxyectoine and homoectoine, cell growth was not stimulated to the same extent as when ectoine was added, but it was improved slightly. The acceleration of growth rate of E. coli ATCC 9637 at elevated osmolarity was ascribed to the accumulation in the cells of ectoine or glycine betaine added to the medium, both of which were proved to be genuine osmolytes in cells. Rapid uptake of ectoine by cells was confirmed when ectoine was available in the CD medium with high osmolarity. Since strain ATCC 9637 did not accumulate ectoine in the absence of an energy source, ectoine uptake might take place not only through cellular sensing of the external high osmolarity but through cellular functioning via energization.