Besides the investigation of critical process parameters the present study focused on the comparative evaluation of the impact of high pressure – low temperature processes on plant and animal tissues. The high pressure treatment of potato, as an example for plant based tissues, at 250 MPa and subzero temperatures (–28 °C) without phase transition resulted in a low deterioration of the cellular membranes. The state of the membrane was stabilised by high pressure when long holding times (more than 24 h) at subzero temperatures were applied, probably due to the (cold) inactivation of lytic enzymes. Freezing to ice polymorphs of a higher density than liquid water (especially ice III) resulted in a lower membrane damage than conventional freezing. Pressure-shift freezing and freezing to ice III resulted in an excellent preservation of textural characteristics. Short phase transitions (due to a large supercooling) as well as favourable volume changes improved the quality of biological samples. As a result, the phase change with the lowest extent of cellular
damage seems to be freezing to ice III at a pressure of about 360 MPa, close to the triple point liquid/ ice III/ ice V (specific volume change about –3 %) supporting the assumption presented above. This study points out the dependence of freezing damage on volume changes and freezing rates. Crystal transformations between ice I and other ice types demand further examination since these processes can dominate the quality of frozen biomaterials and define the maximum pressure for pressure assisted/induced thawing (around 300 MPa) as indicated by the results.
However, also the direction of the phase change must be considered since solid-solid transformation from ice I to ice III completely destroyed the original tissue matrix while the opposite direction of the solid-solid transformation had significantly minor effects on the biological material. An improvement and better control of high pressure supported phase transitions also demands detailed information on the nucleation mechanisms, since the type of the ice polymorph and supercooling influences the quality of frozen and thawed biological materials decisively. Influences of high pressure during the investigated phase transition processes on the membranes, texture and colour cannot be excluded completely. Especially the influence of enzymes on biomaterials during subsequent processing steps (after high pressure treatments) may not be neglected. More studies on enzymes, especially in their natural and partially permeabilised matrix, have to be carried out in the future to quantify these effects. However, in the case of phase transitions under pressure it is likely that the influence of the phase transition exceeds the influence of high pressure at this pressure level, regarding the quality of the material.
The evaluation of the effect of pressure-assisted thawing on an animal tissue was carried of using fish fillets as samples. It has been shown that by applying high pressure at 200 MPa the required phase transition time can be reduced by approximately 50 % compared to thawing at atmospheric pressure. As expected, the pressure dependent depression of the melting point was comparable to that of the potato taking into consideration the slightly lower melting point of -2.0 °C at ambient pressure. The sensory assessment of raw fillet by QIM revealed that the high pressure thawed samples were at least comparable to those thawed at ambient pressure. However, the demerits of cooked samples after high pressure treatment were higher compared to conventionally thawed ones particularly in taste and texture. Whereas the thaw drip was markedly reduced during high pressure thawing, the water binding ability measured later seems to be reduced compared to conventional thawing. The colour is influenced by high pressure thawing at 200 MPa which is related to an increase in lightness and consequently has to be considered when applying higher pressure levels.
To retain the fresh character of a high pressure thawed fish product, pressure levels below 200 MPa seem to be favourable. The texture parameter hardness increased as a consequence of high pressure thawing, which can be an advantage when post rigor frozen fish fillets have to be processed. The microbial status of thawed fillets was improved when high pressure was used for thawing and it was shown that fish parasites (nematodes) were significantly affected by a high pressure treatment of 200 MPa increasing the product safety. The DSC measurements show that high pressure thawing at
200 MPa is connected with a remarkable denaturation of muscle proteins. This is assumed to be the reason for some quality deterioration observed of both the raw and cooked fillets.
Depending on the required characteristics of processed fish, the process parameters must be selected carefully (Murakami et al., 1992; Amanatidou et al., 2000). Therefore, further studies at different pressure levels are necessary. For designing new fish products an application of pressure levels above 200 MPa seems to be indispensable in order to form certain properties (e.g. modified gels) and to ensure pasteurisation of unwanted microbes or parasites. To obtain comparable properties of fresh fish pressure levels below 200 MPa are suggested depending on pressure holding time and working temperature. However, the various effects of high pressure on the different fish species and the impact of post thawing processing steps must be taken into consideration in each attempt to take advantage of high pressure technology and to improve the final quality of the product. However, the results obtained in this study clearly indicate the different effects of high pressure – low temperature on the quality of animal tissues when compared to plant tissue.
The potential ability of high pressure – low temperature processes to inactivate microorganisms at subzero temperatures was investigated on Listeria innocua which served as a non-pathogenic indicator for L. monocytogenes, in Ringer solution and babyfood and compared to inactivation kinetics at elevated temperatures. The inactivation kinetics was modelled using an empirical formula as described by Ananta et al. (2001) fitting the data obtained in ringer solution with good precision. A plot of the reaction rate constant into a pT-diagram clearly indicated the dominant effect of pressure when compared to the temperature effect within the range of investigation.
However, a maximum of the rate constant was observed at low temperatures, i.e. at 0 °C.
The impact of the processing time t on the inactivation of L. innocua can be demonstrated for different log-cycle reductions using the following equation:
) (
log log 1 1
) 1 (
0 0
c k k
N N N
t
c
⋅ +
−
+
−
−
=
−
, (5.1)
where N0 is the initial count set to 109 (CFU ml-1), N is the final reduction (CFU ml-1), c is the reaction order of the reaction and k is the apparent rate constant (s-1). The required processing time to inactivate a certain amount of the bacteria at 0 °C is plotted versus pressure in Figure 5.4.
While nearly complete inactivation (8 log-cycles) requires a treatment time of 3 h at 200 MPa, the same effect is obtained when applying a pressure of 250 MPa for just 1 h. A further shortening of the pressure holding time to about 50% (30 min) is obtained at a pressure level of 300 MPa. This effect of the pressure decreases with lower inactivation rates. However, freezing and thawing times increase with sample size resulting in extended treatment times to ensure complete phase transition also under high hydrostatic pressure (e.g. 60 min to thaw the fish samples at 200 MPa) and
therefore increasing the product safety with respect to Listeria. Increased pressure inactivation of microorganisms at subzero temperatures was also reported by Takahashi (1992) and Hayashi et al.
(1998). The elliptical shape of the phase boundary in the pT-diagram reported for S. cerevisiae was not clearly obtained for Listeria innocua. Nevertheless such a slope indicates that the inactivation seems to be mainly due to protein denaturation than due to damages of the lipid membrane, since phospholipids change their phases mainly along linear phase transition lines in the pT-diagram (Heremans, 2002). Nevertheless, the inactivation rate was not significantly affected by the physical state of the water (solid or liquid) but enhanced when suspending the Listeria in carrot-potato puree with an pH of 5.3 as compared to Ringer solution (pH 6.7). A freezing/thawing cycle at ambient pressure did not remarkable affect the viable count of Listeria innocua.
0 60 120 180
Treatment time [min]
200 225 250 275 300
Pressure [MPa]
log (N/No) = -8 log (N/No) = -6 log (N/No) = -4 log (N/No) = -2 No = 1E+9 CFU/ml T = 0 °C
Figure 5.4: Impact of the pressure level on the required processing time to inactivate Listeria innocua at 0
°C, calculated (eqn. 5.1) for different log-cycle reductions and constant initial counts.