Molecular mechanisms of the high pressure inactivation of beer spoiling Lactobacillus plantarum

The application of high pressure (HP) for food preservation requires insight into mechanisms of HP-mediated cell injury and death. The HP-inactivation in model beer of Lactobacillus plantarum TMW1.460, a beer spoiling organism, was investigated at pressures ranging from 200 to 600 MPa. Surviving cells were characterized by determination of (i) cell viability and sublethal injury, (ii) membrane permeability to the fluorescent dyes propidium iodide (PI) and ethidium bromide (EB), (iii) metabolic activity with tetrazolium salts, and (iv) the activity of HorA, an ABC-type multi-drug-resistance transporter conferring resistance to hop compounds. HP-inactivation curves exhibited a lag time, an exponential inactivation phase, and pronounced tailing caused by a barotolerant fraction of the population, about 1 in 106 cells. During exponential inactivation, more than 99,99% of cells were sublethally injured, however, no sublethal injury was detected in the barotolerant fraction of the culture. Sublethally injured cells were metabolically active and loss of metabolic activity corresponded to the decrease of cell viability. Membrane damage measured by PI occurred later than cell death, indicating that dye exclusion may be used as a fail safe method for rapid characterization of HP inactivation. An increase of membrane permeability to EB, and a reduction of HorA activity was observed prior to the loss of cell viability, indicating loss of hop resistance of pressurized cells. Even mild HP treatments thus abolished the ability of cells to survive under adverse conditions. The effects of hop extract and ethanol on growth, high pressure inactivation, and survival of Lactobacillus plantarum were determined in model beer. Corresponding to the beer spoiling ability of this strain, levels of hops and ethanol typical for beer did not inhibit growth. Pressure death time curves determined at 300 MPa were described by an empirical model taking into account the sigmoid shape of survivor curves, sublethal injury, and the presence of baroresistant cells. Ethanol (5 and 10%) enhanced pressure effects on L. plantarum whereas hop extract (50 and 100 ppm) was less effective. In contrast, hop extracts killed pressurized cells during subsequent storage in beer but ethanol did not. The efficiency of high pressure (HP)- treatment as preservation method in foods depends on environmental conditions. The presence of gases may affect the behaviour of micro organisms during HP-treatment. These additional inactivation effects were investigated in model beer (MB) at 200 MPa and 15°C. Oxygen (O2), nitrogen (N2) and carbon dioxide (CO2) were dissolved by two different methods. The effect of CO2 was additional tested at 12 MPa under conditions where a liquid CO2 phase was present. Gases in dissolved state had no additional inactivation effect on L. plantarum. In contrast, the application of liquid CO2 induced a fast inactivation depending on the ratio of the CO2 - aqueous phase interface to the volume of the aqueous phase. A subcritical extraction of L. plantarum cells with CO2 followed by GC-analysis of the extracts demonstrated that fatty acids in form of triglycerides or phospholipids were extracted off the cellular cytoplasmic membrane. The bactericidal effect of liquid and critical CO2 thus appears to involve the extraction of membrane compounds. The effects of pressure on cultures of L. plantarum were characterized by determination of viability and activity of HorA, an ATP-binding cassette multidrug resistance transporter. Changes were determined in the membrane composition of L. plantarum induced by different growth temperatures. Furthermore, the effect of the growth temperature of a culture on the pressure inactivation at 200 MPa was determined. Cells were characterised after pressure treatment by plate counts on selective and non-selective agar, and HorA activity was measured by ethidium bromide efflux. FT-IR and Laurdan fluorescence spectroscopy provided information about the thermodynamic phase state of the cytoplasmic membrane during pressure treatment. A pressure-temperature-diagram for membranes of cells was established. Cells grown at 37°C and pressure treated at 15°C lost greater than 99% of HorA activity and viable cell counts within 36 and 120 min, respectively. The membrane of these cells was in the gel phase region at ambient pressure. In contrast, cells grown at 15°C and pressure treated at 37°C lost greater 99% of HorA activity and viable cell counts within 4 and 8 min, respectively. The membrane of these cells was in the liquid-crystalline phase region at ambient pressure. The kinetic analysis of inactivation of L. plantarum provided further evidence that inactivation of HorA is a crucial step during pressure induced cell death. Comparison of the biological findings and the membrane state during pressure treatment led to the conclusion that the inactivation of cells and membrane enzymes strongly depends on the thermodynamic properties of the membrane. Pressure treatment of cells with a liquid-crystalline membrane at 0.1 MPa resulted in a more rapid HorA inactivation and cell death compared to cells with a gel phase membrane at 0.1 MPa. The adequate application of pulsed electric fields (PEF) for food preservation requires insight into mechanisms of PEF mediated cell injury and death. Lactobacillus plantarum TMW 1.460, a beer spoiling organism, was PEF treated at different field strengths and energy inputs in model beer (MB). The determined threshold values for field strengths and energy inputs were 13 kV cm1 and 64 kJ kg1, respectively. Above these critical values irreversible cell damages occurred indicated by loss of cell counts on MRS. Below critical values metabolic activity and membrane integrity were reduced by 85% without loss of viability. Sublethal injury, defined as difference between cell counts on selective and non-selective media, was not detected with PEF. Addition of nisin and hop extracts, during PEF treatment, exerted different effects. Nisin and PEF reduced cell viability by 1.5 orders of magnitude. Hop extract and PEF reduced cell viability by 1.5 orders of magnitude and induced 99% sublethal injury. The reversibility of membrane damage below critical field strength was measured by propidium iodide (PI) staining. Identical PEF experiments were performed in presence and absence of PI. From the comparison of these results it was concluded, that 30% of membrane damage were resealed after PEF treatment. PEF experiments above critical field strength in hopped MB revealed, that cells were reduced by 5 orders of magnitude. The surviving cells were killed after 14 h of storage in hopped MB.