Experimental and numerical study of heterogeneous pressure-temperature-induced lethal and sublethal injury of Lactococcus lactis in a medium scale high-pressure autoclave.

The present contribution is dedicated to experimental and theoretical assessment of microbiological process heterogeneities of the high-pressure (HP) inactivation of Lactococcus lactis ssp. cremoris MG 1363. The inactivation kinetics are determined in dependence of pressure, process time, temperature and absence or presence of co-solutes in the buffer system namely 4 M sodium chloride and 1.5 M sucrose. The kinetic analysis is carried out in a 0.1-L autoclave in order to minimise thermal and convective effects. Upon these data, a deterministic inactivation model is formulated with the logistic equation. Its independent variables represent the counts of viable cells (viable but injured) and of the stress-resistant cells (viable and not injured). This model is then coupled to a thermo-fluiddynamical simulation method, high-pressure computer fluid dynamics technique (HP-CFD), which yields spatiotemporal temperature and flow fields occurring during the HP application inside any considered autoclave. Besides the thermo-fluiddynamic quantities, the coupled model predicts also the spatiotemporal distribution of both viable (VC) and stress-resistant cell counts (SRC). In order to assess the process non-uniformity of the microbial inactivation in a 3.3-L autoclave experimentally, microbial samples are placed at two distinct locations and are exposed to various process conditions. It can be shown with both, experimental and theoretical models that thermal heterogeneities induce process non-uniformities of more than one decimal power in the counts of the viable cells at the end of the treatment.

Combined high pressure and temperature induced lethal and sublethal injury of Lactococcus lactis--application of multivariate statistical analysis.

It was the aim of this work to determine the combined effects of pressure, temperature, and co-solutes on Lactococcus lactis, and to detect correlations between culture-dependent and culture-independent methods for assessment of cellular viability and sublethal injury. Therefore, the pressure induced inactivation of L. lactis MG 1363 was investigated in buffer and in buffer with 1.5 M sucrose or 4 M NaCl at a pressure range of 0.1 to 500 MPa and a temperature range of 5 to 50 degrees C. The inactivation was characterised by viable cell counts, stress resistant cell counts, membrane integrity, metabolic activity, and the activity of the multi-drug-resistance transport enzyme LmrP. L. lactis was most resistant to pressure application at 20-30 degrees C. Sucrose protected towards inactivation at any temperature, NaCl provided protection at high temperatures only. By using Principal Component Analysis, correlations were detected between viable cell counts and metabolic activity as well as stress resistant cell counts and LmrP activity. In conclusion, the pressure-inactivation of L. lactis is strongly temperature dependent, baroprotection by sucrose occurs at any temperature but the baroprotective effects of NaCl is temperature dependent. Further on, a combination of two experimental methods fully describe lethal and sublethal injury of pressure treated cells. These simplification of data acquisition and model development facilitates the establishment of pressure processes in food technology.

Differential inactivation of glucose- and glutamate dependent acid resistance of Escherichia coli TMW 2.497 by high-pressure treatments.

The inactivation by 200-400 MPa and post-pressure survival at acid conditions of E. coli TMW 2.497 was characterized by the measurement of intracellular pH (pHin), viable cell counts, glutamate (Glu) and arginine (Arg) consumption, and the influence of mild adaptation to mild acid stress prior to pressure treatment. Glutamate and arginine did not affect viable cell counts or the pHin during pressure application but improved the ability to maintain a high pHin after pressure treatment. In pH 4.0 buffer without arg and glu, a 3 log reduction of cell counts occurred after 24 h of incubation, whereas little or no loss of viability was observed after 24 h incubation in the presence of glu and arg. During post-pressure incubation at pH 4.0, 10 mM glutamate were metabolized but only 2 mM arginine were used, indicating that glutamate rather than arginine was responsible for the protective effect on pHin and survival. In conclusion, the pressure induced, irreversible loss of the transmembrane deltapH correlates to cell death and glu stabilizes the pHin of E. coli during post-pressure incubation.

A fuzzy logic-based model for the multistage high-pressure inactivation of Lactococcus lactis ssp. cremoris MG 1363.

The high-pressure inactivation (200 to 600 MPa) of Lactococcus lactis ssp. cremoris MG 1363 suspended in milk buffer was investigated with both experimental and theoretical methods. The inactivation kinetics were characterised by the determination of the viable cell counts, cell counts of undamaged cells, LmrP activity, membrane integrity, and metabolic activity. Pressures between 200 and 600 MPa were applied, and pressure holding times were varied between 0 and 120 min. Experiments were carried out in milk buffer at pH values ranging between 4.0 and 6.5, and the effect of the addition of molar concentrations of NaCl and sucrose was furthermore determined. The inactivation curves of L. lactis, as characterised by viable cell counts, exhibited typical sigmoid asymmetric shapes. Generally, inactivation of the membrane transport system LmrP was the most sensitive indicator of pressure-induced sublethal injury. Furthermore, the metabolic activity was inactivated concomitant with or prior to the loss of viability. Membrane integrity was lost concomitant with or later than cell death. For example, treatments at 200 MPa for 60 min in milk buffer did not inactivate L. lactis, but fully inactivated LmrP activity and reduced the metabolic activity by 50%. The membrane integrity was unaffected. Thus, the assay systems chosen are suitable to dissect the multistep high-pressure inactivation of L. lactis ssp. cremoris MG 1363. A fuzzy logic model accounting for the specific knowledge on the multistep pressure inactivation and allowing the prediction of the quantities of sublethally damaged cells was formulated. Furthermore, the fuzzy model could be used to accurately predict pressure inactivation of L. lactis using conditions not taken into account in model generation. It consists of 160 rules accounting for several dependent and independent variables. The rules were generated automatically with fuzzy clustering methods and rule-oriented statistical analysis. The set is open for the integration of further knowledge-based rules. A very good overall agreement between measured and predicted values was obtained. Single, deviating results have been identified and can be explained to be measurement errors or model intrinsic deficiencies.