Inactivation of Escherichia coli and Lactobacillus plantarum in relation to membrane permeabilization due to rapid chilling followed by cold storage.

The relationship between membrane permeabilization and loss of viability by chilling depending on the chilling rate was investigated in two bacterial models: one Gram-positive bacterium, Lactobacillus plantarum, and one Gram-negative bacterium, Escherichia coli. Cells were cold shocked slowly (2 degrees C/min) or rapidly (2,000 degrees C/min) from physiological temperature to 0 degrees C and maintained at this temperature for up to 1 week. Loss of membrane integrity was assessed by the uptake of the fluorescent dye propidium iodide (PI). Cell death was found to be strongly dependent on the rate of temperature downshift to 0 degrees C. Prolonged incubation of cells after the chilling emphasized the effect of treatment on the cells, as the amount of cell death increased with the length of exposure to low temperature, particularly when cells were rapidly chilled. More than 5 and 3-log reductions in cell population were obtained with L. plantarum and E. coli after the rapid cold shock followed by 7-day storage, respectively. A correlation between cell inactivation and membrane permeabilization was demonstrated with both bacterial strains. Thus, loss of membrane integrity due to the chilling treatments was directly involved in the inactivation of vegetative bacterial cells.

A new design intended to relate high pressure treatment to yeast cell mass transfer.

A new optical device has been developed to allow the observation of microorganisms during a high pressure treatment up to 700 MPa. To measure cell volume variation during the high pressure application, an image analysis system was connected with the light microscope. With this device, growth of Saccharomyces cerevisiae was studied at moderate pressure (10 MPa) through the observation of individual cell budding. Cell volume variations were also measured on the yeast Saccharomycopsis fibuligera on fixed cells as well on a population sample and a shrinkage in average cell volume was observed consequently to a pressure increase of 250 MPa. The observed compression rate (25%) under pressure and the partial irreversibility of cell compression (10%) after return to atmospheric pressure lead to the conclusion that a mass transfer between cell and cultivation medium occurred. The causes of this transfer could be explained by a modification of membrane properties, i.e., disruption or increase in permeability.

Influence of thermal and osmotic stresses on the viability of the yeast Saccharomyces cerevisiae.

This work studies the effect of thermal and dehydration kinetics on the viability of Saccharomyces cerevisiae. The influence of the rate of temperature (T) and osmotic pressure (pi) increases are first investigated. Results showed that yeast viability is preserved by slow variations of temperature or osmotic pressure in a precise range of T or pi. The influence of a previous thermal stress on the resistance to a hyperosmotic stress is also studied. Temperatures equal to or lower than 10 degrees C allowed the preservation of viability after an osmotic stress whereas temperatures above 10 degrees C did not preserve yeast survival.

Synergistic action of rapid chilling and nisin on the inactivation of Escherichia coli.

The effect of rapid and slow chilling on survival and nisin sensitivity was investigated in Escherichia coli. Membrane permeabilization induced by cold shock was assessed by uptake of the fluorescent dye 1-N-phenylnapthylamine. Slow chilling (2 degrees C min(-1)) did not induce transient susceptibility to nisin. Combining rapid chilling (2,000 degrees C min(-1)) and nisin causes a dose-dependent reduction in the population of cells in both exponential and stationary growth phases. A reduction of 6 log of exponentially growing cells was achieved with rapid chilling in the presence of 100 IU ml(-1) nisin. Cells were more sensitive if nisin was present during stress. Nevertheless, addition of nisin to cell suspension after the rapid chilling produced up to 5 log of cell inactivation for exponentially growing cells and 1 log for stationary growing cells. This suggests that the rapid chilling strongly damaged the cell membrane by disrupting the outer membrane barrier, allowing the sensitization of E. coli to nisin post-rapid chilling. Measurements of membrane permeabilization showed a good correlation between the membrane alteration and nisin sensitivity. Application involving the simultaneous treatment with nisin and rapid cold shock could thus be of value in controlling Gram negatives, enhancing microbiological safety and stability.

Rates of chilling to 0 degrees C: implications for the survival of microorganisms and relationship with membrane fluidity modifications.

The effects of slow chilling (2 degrees C min(-1)) and rapid chilling (2,000 degrees C min(-1)) were investigated on the survival and membrane fluidity of Escherichia coli, of Bacillus subtilis, and of Saccharomyces cerevisiae. Cell death was found to be dependent on the physiological state of cell cultures and on the rate of temperature downshift. Slow temperature decrease allowed cell stabilization, whereas the rapid chilling induced an immediate loss of viability of up to more than 90 and 70% for the exponentially growing cells of E. coli and B. subtilis, respectively. To relate the results of viability with changes in membrane physical state, membrane anisotropy variation was monitored during thermal stress using the fluorescence probe 1,6-diphenyl-1,3,5-hexatriene. No variation in the membrane fluidity of all the three microorganisms was found after the slow chilling. It is interesting to note that fluorescence measurements showed an irreversible rigidification of the membrane of exponentially growing cells of E. coli and B. subtilis after the instantaneous cold shock, which was not observed with S. cerevisiae. This irreversible effect of the rapid cold shock on the membrane correlated well with high rates of cell inactivation. Thus, membrane alteration seems to be the principal cause of the cold shock injury.

Effects of the kinetics of osmotic pressure variation on yeast viability.

The variation rate of the osmotic pressure increase was found to have a great effect on the viability of yeasts subjected to hyperosmotic stress. A low intensity of the increase rate of osmotic pressure could maintain an important viability of the cells (about 90 to 100%) even for very high levels of osmotic pressure (about 10(8) Pa). The viability level was found to be highly dependent on the physiological state of the cells: Variations in the properties of the cell membrane were supposed to be involved in such a dependence.

Coupling effects of osmotic pressure and temperature on the viability of Saccharomyces cerevisiae.

The osmotic tolerance of cells of Saccharomyces cerevisiae as a function of glycerol concentration and temperature has been investigated. Results show that under isothermal conditions (25 degrees C) cells are resistant (94% viability) to hyperosmotic treatment at 49.2 MPa. A thigher osmotic pressure, cell viability decreases to 25% at 99 MPa. Yeast resistance to high osmotic stress (99 Mpa) is enhanced at low temperatures (5-11 degrees C). Therefore, the temperature at which hyperosmotic pressure is achieved greatly affects cell viability. These results suggest that temperature control is a suitable means of enhancing cell survival in response to osmotic dehydration.

The effect of osmotic pressure on the membrane fluidity of Saccharomyces cerevisiae at different physiological temperatures.

Membrane fluidity in whole cells of Saccharomyces cerevisiae W303-1A was estimated from fluorescence polarization measurements using the membrane probe, 1,6-diphenyl-1,3,5-hexatriene, over a wide range of temperatures (6-35 degrees C) and at seven levels of osmotic pressure between 1.38 MPa and 133.1 MPa. An increase in phase transition temperatures was observed with increasing osmotic pressure. At 1.38 MPa, a phase transition temperature of 12 +/- 2 degrees C was observed, which increased to 17 +/- 4 degrees C at 43.7 MPa, 21+/- 7 degrees C at 61.8 MPa, and 24 +/- 9 degrees C at an osmotic pressure of 133.1 MPa. From these results we infer that, with increases in osmotic pressure, the change in phospholipid conformation occurs over a larger temperature range. These results allow the representation of membrane fluidity as a function of temperature and osmotic pressure. Osmotic shocks were applied at two levels of osmotic pressure and at nine temperatures, in order to relate membrane conformation to cell viability.

Passive response of Saccharomyces cerevisiae to osmotic shifts: cell volume variations depending on the physiological state.

The passive osmoregulation phase of S. cerevisiae has been characterized for different physiological states. The relative cell volume decrease of exponential cells was found to be greater than the volume decrease of stationary ones during the hyperosmotic shock. The application of a slow and linear increase in osmotic pressure has allowed the distinguishing of two phases in the passive osmoregulatory response of cells and to calculate the turgor pressure of S. cerevisiae. Cells in the exponential phase have a weak turgor pressure (0.05 MPa) compared to the turgor pressure of stationary phase cells (0.2 MPa). Nevertheless, the turgor pressure and the relative decrease in volume were found to be independent of the size of cells for the same physiological state.

Effects of the kinetics of water potential variation on bacteria viability.

The effect of the kinetics of water potential variation (psi) on the viability of bacteria subjected to hyperosmotic stresses in water-glycerol solution was studied. The three bacteria used were Lactobacillus plantarum L-73, Leuconostoc mesenteroides LM057 and Escherichia coli TG1. These strains were submitted to a final water potential of -107.2 MPa, -170.9 MPa and/or -244.7 MPa. In any case the kinetics of water potential variation was found to have a great effect on the cell viability. The application of slow water potential decreases could maintain an important cell viability (about 80-100%) with regard to the corresponding viability observed after a sudden step change for the same final water potential (15-57%). For each strain tested, an optimal dehydration kinetics was determined which depended on the final water potential. The existence of this optimum could be explained thanks to the opposition of two actions affecting cell viability: a positive action relative to the slowness of the water potential variation and a negative action relative to the residence time of cells in a critical range of water potential.

Saccharomyces cerevisiae viability is strongly dependant on rehydration kinetics and the temperature of dried cells.

The effects of rehydration kinetics and temperature on the viability of Saccharomyces cerevisiae dehydrated by drying were studied. During rehydration, a water activity range of 0.117-0.455 must be crossed slowly in order to maintain cell viability. If this range is crossed rapidly, cell viability can be preserved if rehydration takes place at 50 degrees C. Several hypotheses have been proposed to explain previous results. One hypothesis, which relates cell mortality after rapid rehydration to water flow through the membrane in phase transition, is the more plausible and requires further investigation.

Escherichia coli and Lactobacillus plantarum responses to osmotic stress.

Escherichia coli and Lactobacillus plantarum were subjected to final water potentials of -5.6 MPa and -11.5 MPa with three solutes: glycerol, sorbitol and NaCl. The water potential decrease was realized either rapidly (osmotic shock) or slowly (20 min) and a difference in cell viability between these conditions was only observed when the solute was NaCl. The cell mortality during osmotic shocks induced by NaCl cannot be explained by a critical volume decrease or by the intensity of the water flow across the cell membrane. When the osmotic stress is realized with NaCl as the solute, in a medium in which osmoregulation cannot take place, the application of a slow decrease in water potential resulted in the significant maintenance of cell viability (about 70-90%) with regard to the corresponding viability observed after a sudden step change to same final water potential (14-40%). This viability difference can be explained by the existence of a critical internal free Na+ concentration.