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Figure 5.11 Modeling of the iso-pKa lines in different buffer systems under different p-T conditions with adiabatic lines (--) and phase transition line from pure water (65).
However, to verify and to evaluate the modeled data, in situ measurements for the dissociation equilibrium shift have to be developed. Up to now, such in situ method is not available and no appropriate online measurement device exists. Applying the these models, sensitive reactions such as cell inactivation or enzyme reactions can be better anticipated in planning experimental designs. For skim milk, Schraml (70) measured the pH shift up to 88 C (compare Fig. 5.10). The dissociation of water and elimination of phosphate ions by crystallization of calcium phosphate decreased the pH value in this case (71). The crystallization led to a dissociation equilibrium shift of phosphoric acid, whereby rearrangement of the equilibrium, phosphoric acid dissociated and more hydroxonium ions (H3O+) were generated. Small changes of the pH value could affect different reactions or inactivations in food matrices. Dannenberg (72) found, that the so-called fouling of skim milk on hot surfaces is drastically increased, when the pH value was decreased from 6.6 to 6.5 6.4. A possible explanation could be increased protein aggregation, because of decreased electrostatic repulsion, when proteins approach the isoelectric point (71). The isoelectric point is the pH value at which a particular molecule or surface carries no net electrical charge. The complex matrix relations in skim milk during thermal processing provide ideas about how many relevant mechanisms might occur under high-pressure, high-temperature processing, but limited data exist.
MICROBIAL EFFECTS
5.4.1 Vegetative Cells The German Research Foundation (DFG) Senate Commission for Food Safety (SKLM) (29) suggests that vegetative cells of bacteria relevant for
MICROBIAL EFFECTS
foodstuffs are destroyed by hydrostatic pressures ranging from 150 to 800 MPa. They derive evidence for this postulation from numerous investigations including pathogenic microorganisms. The kinetics of inactivation show a steady decrease, but at higher inactions a leveling-off at longer treatment times ( tailing ) was observed. There are different theories in the literature to explain this tailing phenomenon. In any case the pressure-induced inaction of vegetative cells strongly depends on the food matrix (73 78). The main lethal effect of pressure treatment on cells is the pressure-induced breakdown of membrane permeability. Hence, the difference between the internal and external pH value could collapse or approach zero, resulting in loss of cell viability (79). The internal pH is critical for the control of many cellular processes, such as ATP synthesis, RNA and protein synthesis, DNA replication, and cell growth, and it plays an important role in secondary transport of several compounds (80). It has a particularly inhibitory effect on membrane ATPase, a very important enzyme in the acid base physiology of cells (81). 5.4.2 Bacterial Spores
Bacterial endospores, as compared with vegetative cells, display a considerably higher resistance to high pressure, as described by the DFG-SKLM (29). Highly resistant spores of Clostridium and Bacillus species are key bacteria for the safety or the spoilage of low-acid (heat-treated) preserved food. The neurotoxin A from Clostridium botulinum has a lethal dose LD50 for humans of 1 ng kg 1. Strains of this species can tolerate extremely high pressures over 1 GPa at room temperature. Using high-pressure thermal sterilization as combined technique it is possible to inactivate such food-relevant bacterial endospores. The required inactivation temperature and/or time can be lowered by combination with high pressure (65, 74, 82 85). Under certain combinations of pressure and temperature these highly resistant spores can be protected or stabilized (26, 65, 86). The main important issue is the pressure-induced spore germination (2, 3, 18, 26, 65). Spore germination and subsequent growth can cause food spoilage and potential toxin formation, which may ultimately lead to foodborne disease. The neurotoxin A from C. botulinum has a lethal dose LD50 for humans of 1 ng kg 1 (87). Infant botulism is the most common type of botulism and may occur in children in the rst year of life, when C. botulinum spores populate the intestines, germinate, and produce toxins. The toxin can lead to the feared paralysis of respiratory muscles, which ultimately leads to death (88). Hence, probably stabilized dormant C. botulinum spores are also a risk factor. Another worse scenario is pulmonary anthrax, because of the germination of Bacillus anthracis in lung macrophage (89). The loss of resistance properties is the crucial step during spore germination, which is of highest importance for sterilization techniques and research. The germination of spores has been studied and reviewed extensively recently