Cristina Arroyo

Thermobacteriological characterization of Enterobacter sakazakii

In the present study the influence of various environmental and physiological factors on the heat resistance of Enterobacter sakazakii (Cronobacter) have been investigated. Our results demonstrated that the heat resistance of E. sakazakii depended on the strain studied, the growth conditions - phase and temperature - the characteristics of treatment medium and the recovery conditions. The strain STCC 858 (ATCC type strain 29544) showed maximum heat resistance among the strains tested and it was selected for the further study. Stationary-phase cells grown between 20 and 37 degrees C (mean D(60)=0.9 min) resulted to be more resistant than cells grown at 10 degrees C (D(60)=0.2 min). Resistance decreased when the treatment medium pH was lower than pH 6.0, and it increased with decreasing water activity of the treatment medium, with a 32-fold increase in resistance when lowering water activity to 0.96. z value at pH 4.0 (z=4.79 degrees C) was significantly higher than at pH 7.0 (z=4.06 degrees C), although E. sakazakii cells were approximately 10 times more heat resistant at pH 7.0 than at pH 4.0 within the range of temperatures tested. Contrary to pH, the magnitude of the influence of a(w) on heat resistance did not significantly change with treatment temperature. The proportion of sublethally damaged cells was similar regardless of the treatment medium pH, but it decreased when lowering the water activity. Nevertheless, increasing treatment temperature would not result in a decreased proportion of sublethally injured E. sakazakii cells within the surviving population. Thus, the design of a theoretical combined process that could take advantage of the occurrence of sublethally injured cells would be similarly effective at low and high temperatures. E. sakazakii proved to be more heat resistant in four different liquid food matrixes than in buffers at the same pH, and this disagreement was especially higher in orange juice, which resulted to be the product that induced a greater protective effect in E. sakazakii cells against heat.

Pulsed electric fields cause sublethal injuries in the outer membrane of Enterobacter sakazakii facilitating the antimicrobial activity of citral

Aims:  The objective was to evaluate the relation of sublethal injury in the outer membrane of Enterobacter sakazakii to the inactivating effect of the combination of pulsed electric fields (PEF) treatments and citral.

Inactivation of Cronobacter sakazakii by ultrasonic waves under pressure in buffer and foods

The objective of this research was to characterize the resistance of Cronobacter sakazakii to ultrasonic waves under pressure (manosonication, MS). The D(MS) value (decimal reduction time value) of C. sakazakii in standard conditions (35°C, 117 μm, 200 kPa, citrate-phosphate buffer pH 7.0) was 0.41 min. This value was higher than that of Yersinia enterocolitica (D(MS)=0.19 min) and lower than those of Salmonella enterica serovar Enteritidis (D(MS)=0.61 min), Listeria monocytogenes (D(MS)=0.86 min), and Enterococcus faecium (D(MS)=1.2 min). Strain studied (ATCC 29544, NCTC 8155, 9238, and 9529), growth temperature (10, 20, 30, and 37°C), and pH of the treatment media (4.0, 5.0, 6.0, and 7.0) did not significantly change C. sakazakii MS resistance. Conversely, entry into stationary growth phase, decreasing water activity of the treatment media (0.98, 0.96, and 0.94), and treatment in food products (apple and orange juices, chicken and vegetable soups, and rehydrated powdered milk) resulted in up to a 1.6-, 3.9-, and 2.5-fold maximum change in D(MS) values, respectively. Whereas an exponential relationship between the amplitude of ultrasonic waves and D(MS) values was found, the relationship between static pressure and D(MS) values was better described by a quadratic equation. The energy transferred into the medium determined the lethality of the ultrasonic waves regardless of the combination of pressure (0, 50, 100, 200 and 300 kPa) and amplitude (34, 62, 90, 117 and 145 μm) applied. There was an exponential relationship between D(MS) values and the power input: an increase of 134 W increased the inactivation rate ten times regardless of the treatment medium. No C. sakazakii cells with sublethally injured cytoplasmic membrane or with sublethal oxidative damage occurred after MS treatments, but the results indicated that damage to the outer membrane preceded microbial death.

Inactivation of Cronobacter sakazakii by manothermosonication in buffer and milk.

The inactivation of Cronobacter sakazakii by heat and ultrasound treatments under pressure at different temperatures [manosonication (MS) and manothermosonication (MTS)] was studied in citrate-phosphate pH 7.0 buffer and rehydrated powdered milk. The inactivation rate was an exponential function of the treatment time for MS/MTS treatments (35-68 °C; 200 kPa of pressure; 117 μm of amplitude of ultrasonic waves) in both media, and for thermal treatments alone when buffer was used as heating media. Survival curves of C. sakazakii during heating in milk had a concave downward profile. Up to 50 °C, the lethality of ultrasound under pressure treatments was independent of the treatment temperature in both media. At temperatures greater than 64 °C in buffer and 68 °C in milk, the inactivating effect of MTS was equivalent to that of the thermal treatments alone at the same temperature. Between 50 and 64 ºC for buffer and 50 and 68 °C for milk, the lethality of MTS was the result of a synergistic effect, where the total lethal effect was higher than the lethal effect of heat added to that of ultrasound under pressure at room temperature. The maximum synergism was found at 60 °C in buffer and at 56 °C in milk. A heat treatment of 12 min (60 °C) or 4 min of an ultrasound under pressure at room temperature treatment (35 °C; 200 kPa; 117 μm) would be necessary to guarantee the death of 99.99% of C. sakazakii cells suspended in milk. The same level of C. sakazakii inactivation can be achieved with 1.8 min of a MTS treatment (60 °C; 200 kPa; 117 μm). Damaged cells were detected after heat treatments and after ultrasound under pressure treatments at lethal but not at non-lethal temperatures.

Effect of Pulsed Electric Field treatments at various stages during conditioning on quality attributes of beef longissimus thoracis et lumborum muscle

Beef longissimus thoracis et lumborum (LTL) muscle was used to evaluate the effect of PEF treatments (1.4 kV/cm, 10 Hz, 20 μs, 300 and 600 pulses) on meat quality attributes (weight loss, colour, cook loss and texture) and its evolution at various stages during ageing (2, 10, 18 and 26 days post-mortem). The length of meat ageing before and after PEF application exerted no influence on weight loss, colour and cook loss. Results also demonstrated that PEF treatments applied at different times post-mortem (2, 10, 18 and 26 days) showed a tendency towards reducing toughness of beef samples but that the application of PEF did not affect the tenderization process provided by ageing itself. 60% of the sensory panellists scored PEF treated samples as tender (≥6.0 points out of 9.0) whereas only 27.5% did so for untreated samples.

Development of resistance in Cronobacter sakazakii ATCC 29544 to thermal and nonthermal processes after exposure to stressing environmental conditions

Aims:  The objective was to study the response of Cronobacter sakazakii ATCC 29544 cells to heat, pulsed electric fields (PEF), ultrasound under pressure (Manosonication, MS) and ultraviolet light (UV‐C) treatments after exposure to different sublethal stresses that may be encountered in food‐processing environments.

An assessment of the impact of pulsed electric fields processing factors on oxidation, color, texture, and sensory attributes of turkey breast meat.

Pulsed electric fields (PEF) is a novel nonthermal technology that has the potential to cause physical disruption to muscle tissue which in turn could alter the sensorial aspects of meat in both a positive (e.g., enhanced tenderization) and a negative way (e.g., off-flavor development). If there is a risk of off-flavor development it should be identified prior to embarking on an extensive investigation on PEF in meat tenderization and turkey meat was chosen for this purpose as it is particularly prone to oxidation. The objective of this study was to investigate the effect of various PEF treatments on the quality attributes of turkey breast meat. Turkey breast meat obtained 1 d postslaughter was treated in a batch PEF chamber with increasing electric field strength up to 3 kV/cm and analyzed for lipid oxidation by thiobarbituric acid reactive substances assay (TBARS) with up to 5 d storage at 4°C in aerobic conditions. In a separate experiment, turkey breast meat samples were exposed to PEF under various combinations of pulse number, frequency, and voltage. Following PEF treatments weight loss, cook loss, lipid oxidation, texture, and color were assessed by instrumental methods. A sensory analysis was also performed to determine consumer acceptability for color, texture, and odor of the samples. Lipid oxidation in all PEF-treated samples progressed at the same rate with storage as the untreated samples and was not found to be significantly different to the control. Under the conditions examined PEF treatments did not induce differences in instrumentally measured weight loss, cook loss, lipid oxidation, texture, and color (raw and cooked) either on fresh or frozen samples. However, the sensory evaluation suggested that panelists could detect slight differences between the PEF-treated samples and the controls in terms of texture and odor.

Environmental factors influencing the inactivation of Cronobacter sakazakii by high hydrostatic pressure

The effect of High Hydrostatic Pressure (HHP) on the survival of Cronobacter sakazakii was investigated. Deviations from linearity were found on the survival curves and the Mafart equation accurately described the kinetics of inactivation. Comparisons between strains and treatments were made based on the time needed for a 5-log(10) reduction in viable count. The ability of C. sakazakii to tolerate high pressure was strain-dependent with a 26-fold difference in resistance among four strains tested. Pressure resistance was greatest in the stationary growth phase and at the highest growth temperatures tested (30 and 37 °C). Cells treated in neutral pH buffer were 5-fold more resistant than those treated at pH 4.0, and 8-fold more sensitive than those treated in buffer with sucrose added (a(w)=0.98). Pressure resistance data obtained in buffer at the appropriate pH adequately estimated the resistance of C. sakazakii in chicken and vegetables soups. In contrast, a significant protective effect against high pressure was conferred by rehydrated powdered milk. As expected, treatment efficacy improved as pressure increased. z values of 112, 136 and 156 MPa were obtained for pH 4.0, pH 7.0 and a(w)=0.98 buffers, respectively. Cells with sublethal injury to their outer and cytoplasmic membranes were detected after HHP under all the conditions tested. The lower resistance of C. sakazakii cells when treated in media of pH 4.0 seemed to be due to a decreased barostability of the bacterial envelopes. Conversely, the higher resistance displayed in media of reduced water activity may relate to a higher stability of bacterial envelopes.

UV-C Inactivation of Cronobacter sakazakii

The purpose of this research was to study the effect of different factors on the resistance of Cronobacter sakazakii NCTC 9238 to UV-C light (which includes germicidal ultraviolet light at 254 nm) and to determine whether a combined treatment with heat would produce a synergistic effect for its inactivation. Growth temperature between 10°C and 37°C did not change the UV-C resistance of C. sakazakii. On the contrary, cells in the logarithmic phase of growth were more sensitive to UV-C light than in the stationary phase. The lethality of UV-C was independent of pH (between 3.0 and 7.0) and a(w) (between 0.94 and > 0.99) of the treatment media, but it exponentially decreased with the absorption coefficient (α). When applying a UV-C treatment of 27.1 J/mL at 25°C to C. sakazakii suspended in vegetable soup and apple juice, 1 and 2 log10 cycles of inactivation were barely achieved, respectively. However, the bactericidal effect of UV-C light increased with temperature. The lethality of the combined process was the result of a synergistic effect that was maximum at 52.5°C for apple juice and 55°C for vegetable soup. In conclusion, these results indicate that UV-C efficacy may be influenced by microbial growth conditions and food characteristics, and that its combination with heat may act synergistically against C. sakazakii.

Bacterial maximum non-inhibitory and minimum inhibitory concentrations of different water activity depressing solutes

The NaCl MNICs (maximum non-inhibitory concentrations) and MICs (minimum inhibitory concentrations) for growth of various strains of six bacterial species were determined and then compared with those obtained for seven other solutes. The influence of prior growth conditions on the MNICs and MICs was also evaluated. No significant changes on the MNICs and MICs were found among the strains studied within each species. Among all factors investigated, only growth phase -for Gram-negatives- and growth at high NaCl concentrations led to a change in the NaCl MNICs. Species could be classified depending on its NaCl MNICs and MICs (in decreasing order) as follows: Staphylococcus aureus, Listeria monocytogenes, Cronobacter sakazakii, Enterococcus faecium, Escherichia coli and Salmonella Typhimurium. Similar results were obtained for KCl, LiCl, and sodium acetate, but not for the remaining solutes investigated (sucrose, glycerol, MgCl2 and CaCl2). Results obtained indicate that, in general, Gram-negatives showed lower MNICs and MICs than Gram-positives for all the solutes, S. aureus being the most solute tolerant microorganism. When compared on a molar basis, glycerol showed the highest MNICs and MICs for all the microorganisms -except for S. aureus- and LiCl the lowest ones. NaCl MNICs and MICs were not significantly different from those of KCl when compared on a molar basis. Therefore, the inhibitory action of NaCl could not be linked to the specific action of Na(+). Results also showed that the Na(+) tolerance of some species was Cl(-) dependent whereas for others it was not, and that factors others than aw-decrease contribute to the inhibitory action of LiCl, CaCl2 and MgCl2.