The aim of this thesis was to study the effect of temperature-co-solvent or combined pressure-temperature-co-solvent induced inactivation behaviour of bacteria. Therefore, the focus was set on the fermentative organism Lactococcus lactis ssp. cremoris MG 1363, which should be characterised on its physiological behaviour under extreme conditions. In addition, previously measured data sets were used (Molina-Höppner, 2002) to the pressure-temperature-co-solvent dependent inactivation kinetics that were determined in preparation of this work. Data Mining tools as well as differential equation tools have been used for this bacteria to extract as much information as possible out of the generated data and to describe its behaviour mathematically. The second part of the thesis represents a study about the detection of stress induced enzymes and their influence on the inactivation behaviour of contaminating Escherichia coli TMW 2.497. The prolongation of temperature induced inactivation of L. lactis by the use of protective additives was verified by quantifying viable and stress resistant cell counts after the process as well as by the use of FT-IR spectroscopy during the temperature treatment. Therefore, different buffer systems namely milk bufffer, milk buffer 1.5M sucrose or milk buffer 4M NaCl and a temperature range between 40°C and 75°C was used to detect the protective effect of both additives. NaCl protects L. lactis more effective than sucrose. The inactivation kinetics followed the characteristics of sigmoid asymetric shapes with either with or without an initial shoulder effects. Nevertheless, a complete inactivation of the population was reached by the use of 65°C in each buffer. The protective effect was detected and assigned to the reduced aw in case of NaCl as well as the preferential hydration effect of proteins in carbohydrate solutions. These findings as well as the generated data were used to detect correlations between those physiological states as well as data describing protein unfolding behaviour under extreme temperature conditions. Therefore, FT-IR study was done using D2O and D2O-1.5M sucrose. A powerful tool was found to describe the cumulative behaviour of all proteins in L. lactis and their conformational changes due to the process. The protective effect was proved by an D/H exchange as well as an advanced change from strucured to unstructered protein behaviour in the presence of D2O. Sucrose hampered both reactions which were detected by amide I’ and amide II and amide II’ behaviour. The effects of combined pressure-temperature and the above named buffer systems on the behaviour of L. lactis were characterised by the use of lethal and sublethal measurands, namely viable cell counts, stress resistant cell counts, membrane integrity, metabolic activity and LmrP-activity. The pressure-temperature kinetics differ from those obtained by the use of temperature alone. The inactivation kinetics followed the characteristics of sigmoid asymetric shapes with an initial shoulder and an inactivation phase followed by tailing. L. lactis exhibited maximum resistance to pressure when treated in milk buffer at a temperature that was approximately 6°C below the growth temperature. This temperature of maximum resistance was determined with all of the indicators of lethal or sublethal injury. Therefore, a complete inactivation of the population was only reached, if a temperature of higher or equal than 40°C was used. Temperatures below 30°C inhibit the inactivation of the complete population resulting in a resistant fraction of about 105 bacteria. In the presence of 1.5M sucrose, the population was completely protected using temperatures below 20°C. At each pressure level and temperatures greater than 40°C, the population was sublethally injured after short pressurisation in the presence of this additive. The loss of viability just took place, if a pressure equal to 500 MPa or greater and temperatures equal or greater than 45°C were used. In case of 4M NaCl, L. lactis lost its ability to recover on the selective medium if temperatures below 20°C were used. Otherwise, the population was completely protected at elevated temperatures up to 400 MPa. In case of a pressure level greater than 400 MPa, the inactivation of L. lactis took place at each temperature, and it was strengthened by increasing temperatures. The correlations detected in this work by PCA between viable cell counts and metabolic activity and LmrP and stress resistant cell counts were used in different ways. First, a multi-layer Fuzzy Logic model was generated describing the pressure-temperature effects as well as the effect of 21 different varieties of milk buffer on L. lactis. Therefore, the autonomous output variables viable cell counts and LmrP activity have been defined. The remaining quantities were calculated mainly based on the autonomous output variables. Due to the fact, that the model accurately predicts all five physiological states, the detected findings by PCA were correct. These findings enable a more rapid data aquisition and additionally reduces computation time and computer capacities concerning industrial modelling. For example, the determination of viable and stress resistant cell counts provided information on the metabolic activity of the cells as well as the activity of MDR-transport enzymes involved in the resistance to bactericidal agents. Likewise, the determination of the metabolic activity with tetrazolium salts can be used as a rapid screening of lethal pressure effects at various combinations of pressure, temperature, and co-solvents. Second, inactivation data was recorded for the physiological states viable- and stress resistant cell counts in the presence of milk buffer, milk buffer 1.5M sucrose, or 4M NaCl in a 3.3-litre-medium-sized-high-pressure vessel at two different locations. Generated data out of the 10 ml vessel were used to establish a model using the Logistic equation. The time dependent first order differential equation of the Logistic function has then been used to describe the inactivation behaviour of L. lactis. Therefore, the focus was based on process induced spatiotemporal heterogeneties and their influence on the inactivation behaviour of L. lactis. The model was validated by microbiological data. For the first time, the process heterogeneity of the mass conversion in an industrial-sized high-pressure autoclave could be confirmed both experimentally and numerically. Furthermore, the combination of this state-of-the-art modelling techniques and numerical simulations enabled highly precise prediction of the mass conversion process, i.e. inactivation process of microorganisms or enzymes. Overall, an excellent regression of R2=0.95 or higher were achieved in this work. The second part was focused on the behaviour of Escherichia coli and its ability to maintain a high intracellular pH to prevent damage to intracellular macromolecules, and to provide favourable conditions for intracellular enzymes. Three transport systems are known to contribute to the extrusion of protons in E. coli. Among these, the glutamate decarboxylation pathway is the most favourable because it stabilizes the internal pH without the expense of ATP, and neutralizes the external medium at low pH-values. The availability of glutamate strongly improved the survival of E. coli during post-pressure acid challenge. This improved acid tolerance correlated well to an improved ability of E. coli to restore a high intracellular pH after pressure treatment. Apparently, the enzymes required for the glutamate acid survival pathways remain functional even after pressure treatments that inactivate those enzymes, which are required to establish a transmembrane pH gradient with glucose as sole energy source. The utilisation of glutamate was enhanced in pressure treated cells compared to untreated cells, indicating induction or activation of the glutamate acid survival pathway. Adaptation of E. coli to mild acid conditions further improved the survival of E. coli to post-pressure acid exposure.
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The aim of this thesis was to study the effect of temperature-co-solvent or combined pressure-temperature-co-solvent induced inactivation behaviour of bacteria. Therefore, the focus was set on the fermentative organism Lactococcus lactis ssp. cremoris MG 1363, which should be characterised on its physiological behaviour under extreme conditions. In addition, previously measured data sets were used (Molina-Höppner, 2002) to the pressure-temperature-co-solvent dependent inactivation kinetics that...
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