liquid
4.2 Quality and safety aspects of high pressure - low temperature processes
4.2.3 Impact of high pressure-low temperature processing on microorganisms
other methods must be explored for accurate investigation. Furthermore, the infective potential has to be considered in further studies. Nevertheless, the data obtained here are in agreement with a study on parasites (Trichinella spiralis) in pork meat (Nöckler et al., 2001). According to the authors the larvae were affected at 150 MPa and completely inactivated at pressures of 200 MPa in a temperature range of 5 to 25 °C and pressure holding times equal or higher than 10 min.
The experimental and calculated kinetic curves from the inactivation experiments with the microbes suspended in Ringer solution are presented in Figure 4.44. Since the initial bacterial counts were slightly different in each case, the final level of reductions were attained when indicated relative to N0 were divergent.
-8 -6 -4 -2 0
log [N/No]
-8 -6 -4 -2 0
log [N/No]
-8 -6 -4 -2 0
log [N/No]
-8 -6 -4 -2 0
log [N/No]
0 10 20 30 40 50 60
Time [min]
0 10 20 30 40 50 60
Time [min]
0 10 20 30 40 50 60
Time [min]
0 10 20 30 40 50 60
Time [min]
-30°C
-10°C 0°C
-20°C
-8 -6 -4 -2 0
log [N/No]
-8 -6 -4 -2 0
log [N/No]
-8 -6 -4 -2 0
log [N/No]
0 10 20 30 40 50 60
Time [min]
0 10 20 30 40 50 60
Time [min]
0 10 20 30 40 50 60
Time [min]
200 MPa Model (200MPa) 250MPa Model (250MPa) 300MPa Model (300MPa)
+5 °C
+40°C
+10°C
Figure 4.44: Inactivation kinetics of Listeria innocua in Ringer solution at subzero and elevated temperatures at different pressure levels. The ratio N/No is plotted logarithmically versus time. Lines obtained by fitting the model (eqn. 3.19) to the experimental data (symbols). The model parameters are given in Table 4.20.
According to the experimental data from the kinetic studies, it was shown that the inactivation curves exhibited a non-linear behaviour. From the results it can be seen that an increase of the pressure level applied led to higher inactivation rates. A pressure treatment at 200 MPa, for 60 min at 0 °C resulted in a 6 log cycle reduction of Listeria innocua, but the same reduction was obtained after 10 min when applying 300 MPa at 0 °C. However the inactivation curves showed no pronounced shoulder formation and the tailing effect was mainly noticeable at low pressures (200 MPa). It seems that lower temperatures shifted the inactivation rate to higher levels with a maximum at 0 °C. An irregularity of the temperature and pressure effectiveness was seen at –30 °C for 250 and 300 MPa, resulting in the assumption that the physical state of water (liquid or solid) may influence the inactivation kinetics under the tested conditions.
4.2.3.2 Bacteria inactivation in frozen solutions
Since formation of ice is known to reduce the water activity and lower water activity is known to induce protective effects on microorganisms (Smelt et al., 2002), it could be expected that a change of the physical state of water may result in changes of pressure sensitivity of Listeria innocua.
Consequently, a set of experiments was carried out starting from the frozen or the liquid state of the medium, but applying comparable process parameters along the phase boundary. Figure 4.45 (a, b) shows the comparison of the different inactivation kinetics. A reduction of about 6 log cycles was obtained after pressure treatment for 10 min at 300 MPa in both cases (frozen and liquid state).
Contrary to expectations, the inactivation rate was slightly enhanced in the frozen state at 200 MPa and 30 min, but the final reduction values are similar for the frozen and the liquid matrices. As a result, it can be stated that the hydrostatic principle is not affected by the formation of ice, but it is likely that the observations may differ with size of the sample, initial working temperature and the media in which the microorganisms are suspended.
-8 -6 -4 -2 0
log [N/No]
-8 -6 -4 -2 0
log [N/No]
0 10 20 30 40 50 60
Time [min]
0 10 20 30 40 50 60
Time [min]
300MPa 200MPa 250MPa
300MPa 200MPa 250MPa
A: Initial liquid state
-20 °C
B: Initial frozen state
-20 °C
Figure 4.45: Effect of the initial physical state of water (liquid, solid) on the high pressure inactivation kinetics of Listeria innocua suspended in Ringer solution.
4.2.3.3 Influences of p,T-combinations on the rate constant
Using the kinetic data from the regression analysis (Table 4.20) a pTk-diagram was constructed to show the influence of pressure and temperature on the rate constant k’. The generated data are shown in Figure 4.46.
-30 -20 -10 0 10 20 30 40
Temperature [°C]
200 225 250 275 300
Pressure [MPa]
0.000404
0.000544 0.000684
0.000824 0.000964
0.001104
0.001244
0.001384 0.001525
0.001665 0.001805
0.0019450.002085
solid state of water k-values
Figure 4.46: Effect of solid-liquid transition on the rate constant of L. innocua inactivation. The lines indicate p,T-combinations with constant inactivation rates.
As expected from the inactivation kinetics (Figure 4.44) the pressure level (within the range of consideration) affected the rate constant more than the temperature, leading to a more vertical slope of the curves in the pTk-plot. At 0 °C there is a significant change of the slope of the rate constant indicating accelerated inactivation at all pressure levels between 200 and 300 MPa. By implementing the phase boundary of pure water into the pTk-diagram of the L. innocua inactivation in Ringer solution, an effect of the physical state of water can be assumed to describe the divergent slope of the k-values near the phase transition line. However, this effect (more pronounced at pressures above 250 MPa) mainly derived form the experimental data in which the inactivation kinetics show irregular behaviour at –30 °C (Figure 4.44) and the subsequent estimation of the rate constant based on these data. The results of the experimental sets carried out to evaluate the protective effect of the solid state of water on Listeria innocua didn’t support the assumption suggested above. Further investigations are required to provide more details on the effect of the solid state of water on the mechanical force transmission of high hydrostatic pressure and other mechanisms probably affecting the inactivation kinetics of microorganisms.
4.2.3.4 Pressure resistance of Listeria innocua in food matrix
The comparative evaluation of the effect of the matrix on the inactivation kinetics is shown in Figure 4.47 Though similar final levels of reductions were observed for both Ringer solution and baby food for the same temperature and pressure conditions, it was easier to inactivate the microorganisms suspended in baby food which had an initial concentration of ~107 CFU/ml as against ~109 CFU/ml in Ringer solution. The reason for this could be because of the pH of baby food (5.3) being lesser than that of Ringer solution (pH 6.75). However at low pressure (200 MPa) the inactivation curves showed pronounced shoulder formation at 10 °C and 0 °C when L. innocua was inoculated in baby food, as against Ringer solution. An explanation for this could be that the initial concentration suspended in the Ringer solution was two log cycles higher than that for baby food as mentioned before. The reduction in the former was therefore drastic in the first few minutes of treatment and then slowed down. It was also possible that a certain portion of the microorganisms were inactivated during the pressure build-up time itself, the effect of which was not taken into account in the present study.
-10 -8 -6 -4 -2 0
log [N/No]
-10 -8 -6 -4 -2 0
log [N/No]
-10 -8 -6 -4 -2 0
log [N/No]
-10 -8 -6 -4 -2 0
-10 -8 -6 -4 -2 0
-10 -8 -6 -4 -2 0
0 20 40 60 80 100 120
0 20 40 60 80 100 120
0 20 40 60 80 100 120 Time [min]
0 20 40 60 80 100 120
0 20 40 60 80 100 120
0 20 40 60 80 100 120 Time [min]
200 MPa 250 MPa 300 MPa
200 MPa 250 MPa 300 MPa
0 °C
10 °C
0 °C
-10 °C -10 °C
10 °C Ringer
solution
Ringer solution
Ringer solution
Baby food
Baby food
Baby food
Figure 4.47: Comparison of inactivation kinetics of Listeria innocua in Ringer solution and baby food (carrot-potato puree) at 3 different pressures and temperatures. The ratio N/N0 is plotted logarithmically versus time.
At 200 MPa and 10°C, low inactivation rates was observed even after 30 min whereas a combination of 300 MPa and –10 °C produced complete inactivation within 10 minutes. The inactivation at 0 and –10°C was in both cases greater than for that for elevated temperature and the highest inactivation rates was obtained at 0 °C for Ringer solution and –10 °C for baby food. Such a behaviour of the dependence of the inactivation on temperature corresponds with the dependence of protein denaturation with temperature (Heremans and Smeller, 1998) which goes to suggest that denaturation of proteins in the cell membranes of the microorganisms could be one of the major mechanisms causing inactivation. It can thus be expected that the high pressure supported phase transition processes which are normally associated with sub-zero temperatures can also benefit from the considerable amount of inactivation attained during the process. For example, thawing of a frozen product at 250 MPa for 40 min can cause complete microbial inactivation of the tested microorganism, Listeria innocua besides shortening the thawing time.