Inactivation of Geobacillus stearothermophilus Spores by High-Pressure Carbon Dioxide Treatment

High-pressure CO₂ treatment has been studied as a promising method for inactivating bacterial spores. In the present study, we compared this method with other sterilization techniques, including heat and pressure treatment. Spores of Bacillus coagulans, Bacillus subtilis, Bacillus cereus, Bacillus licheniformis, and Geobacillus stearothermophilus were subjected to CO₂ treatment at 30 MPa and 35°C, to high-hydrostatic-pressure treatment at 200 MPa and 65°C, or to heat treatment at 0.1 MPa and 85°C. All of the bacterial spores except the G. stearothermophilus spores were easily inactivated by the heat treatment. The highly heat- and pressure-resistant spores of G. stearothermophilus were not the most resistant to CO₂ treatment. We also investigated the influence of temperature on CO₂ inactivation of G. stearothermophilus. Treatment with CO₂ and 30 MPa of pressure at 95°C for 120 min resulted in 5-log-order spore inactivation, whereas heat treatment at 95°C for 120 min and high-hydrostatic-pressure treatment at 30 MPa and 95°C for 120 min had little effect. The activation energy required for CO₂ treatment of G. stearothermophilus spores was lower than the activation energy for heat or pressure treatment. Although heat was not necessary for inactivationby CO₂ treatment of G. stearothermophilus spores, CO₂ treatment at 95°C was more effective than treatment at 95°C alone.     

High Pressure Enhances the Growth Rate of the Thermophilic Archaebacterium Methanococcus thermolithotrophicus without Extending Its Temperature Range

Temperature and hydrostatic pressure are essential in determining the assemblage of species in their specific biotopes. To evaluate the effect of high pressure on the range of viability of thermophiles, the pressure and temperature dependence of the growth of the methanogenic archaebacterium Methanococcus thermolithotrophicus was investigated. High pressure up to 50 MPa enhanced the growth rate without extending the temperature range of viability. The optimum temperature remained unaltered (65°C). Beyond 50 MPa, cell lysis predominated over cell proliferation. Destabilization was also observed at temperatures below and above the optimum growth temperature (<60°C, ≥70°C) and at low substrate concentrations.     

High-Pressure-Mediated Survival of Clostridium botulinum and Bacillus amyloliquefaciens Endospores at High Temperature

Endospores of proteolytic type B Clostridium botulinum TMW 2.357 and Bacillus amyloliquefaciens TMW 2.479 are currently described as the most high-pressure-resistant bacterial spores relevant to food intoxication and spoilage in combined pressure-temperature applications. The effects of combined pressure (0.1 to 1,400 MPa) and temperature (70 to 120°C) treatments were determined for these spores. A process employing isothermal holding times was established to distinguish pressure from temperature effects. An increase in pressure (600 to 1,400 MPa) and an increase in temperature (90 to 110°C) accelerated the inactivation of C. botulinum spores. However, incubation at 100°C, 110°C, or 120°C with ambient pressure resulted in faster spore reduction than treatment with 600 or 800 MPa at the same temperature. This pressure-mediated spore protection was also observed at 120°C and 800, 1,000, or 1,200 MPa with the more heat-tolerant B. amyloliquefaciens TMW 2.479 spores. Inactivation curves for both strains showed a pronounced pressure-dependent tailing, which indicates that a small fraction of the spore populations survives conditions of up to 120°C and 1.4 GPa in isothermal treatments. Because of this tailing and the fact that pressure-temperature combinations stabilizing bacterial endospores vary from strain to strain, food safety must be ensured in case-by-case studies demonstrating inactivation or nongrowth of C. botulinum with realistic contamination rates in the respective pressurized food and equipment.     

Combined Effects of High Hydrostatic Pressure and Temperature for Inactivation of Bacillus anthracis Spores

Spores of Bacillus anthracis are known to be extremely resistant to heat treatment, irradiation, desiccation, and disinfectants. To determine inactivation kinetics of spores by high pressure, B. anthracis spores of a Sterne strain-derived mutant deficient in the production of the toxin components (strain RP42) were exposed to pressures ranging from 280 to 500 MPa for 10 min to 6 h, combined with temperatures ranging from 20 to 75°C. The combination of heat and pressure resulted in complete destruction of B. anthracis spores, with a D value (exposure time for 90% inactivation of the spore population) of approximately 4 min after pressurization at 500 MPa and 75°C, compared to 160 min at 500 MPa and 20°C and 348 min at atmospheric pressure (0.1 MPa) and 75°C. The use of high pressure for spore inactivation represents a considerable improvement over other available methods of spore inactivation and could be of interest for antigenic spore preparation.     

Effects of Hydrostatic Pressure on Growth and Luminescence of a Moderately-Piezophilic Luminous Bacteria Photobacterium phosphoreum ANT-2200

Bacterial bioluminescence is commonly found in the deep sea and depends on environmental conditions. Photobacterium phosphoreum ANT-2200 has been isolated from the NW Mediterranean Sea at 2200-m depth (in situ temperature of 13°C) close to the ANTARES neutrino telescope. The effects of hydrostatic pressure on its growth and luminescence have been investigated under controlled laboratory conditions, using a specifically developed high-pressure bioluminescence system. The growth rate and the maximum population density of the strain were determined at different temperatures (from 4 to 37°C) and pressures (from 0.1 to 40 MPa), using the logistic model to define these two growth parameters. Indeed, using the growth rate only, no optimal temperature and pressure could be determined. However, when both growth rate and maximum population density were jointly taken into account, a cross coefficient was calculated. By this way, the optimum growth conditions for P. phosphoreum ANT-2200 were found to be 30°C and, 10 MPa defining this strain as mesophile and moderately piezophile. Moreover, the ratio of unsaturated vs. saturated cellular fatty acids was found higher at 22 MPa, in agreement with previously described piezophile strains. P. phosphoreum ANT-2200 also appeared to respond to high pressure by forming cell aggregates. Its maximum population density was 1.2 times higher, with a similar growth rate, than at 0.1 MPa. Strain ANT-2200 grown at 22 MPa produced 3 times more bioluminescence. The proposed approach, mimicking, as close as possible, the in situ conditions, could help studying deep-sea bacterial bioluminescence and validating hypotheses concerning its role into the carbon cycle in the deep ocean.     

Asexual reproduction and vegetative growth of Bionectria ochroleuca in response to temperature and photoperiod

Growth and reproduction are two essential life‐history traits for fungi. Understanding life‐history strategies provides insight into the environmental adaption of species. Here, we investigated the colonial morphology, vegetative growth, and asexual reproduction of the ascomycete fungus Bionectria ochroleuca in response to a variety of environmental conditions. We demonstrated that the increased temperature from 15 to 25°C induced mycelial growth and conidiation in B. ochroleuca. We also found that the optimal temperatures for mycelial growth and conidial formation in this fungus species were 25 and 30°C, respectively. However, as the temperature increased from 25 to 30°C, mycelial growth was suppressed, but the total number of conidia was significantly increased. The shift in light–dark cycles dramatically changed the morphological features of the colonies and affected both vegetative growth and asexual reproduction. Under incubation environments of alternating light and dark (16:8 and 8:16 light:dark cycles), conidiophores and conidia in the colonies formed dense‐sparse rings and displayed synchronous wave structures. When the light duration was prolonged in the sequence of 0, 8, 16, and 24 hr per day, mycelial growth was suppressed, but conidiation was promoted. Together, our results indicate that temperature and light period may trigger a trade‐off between vegetative growth and asexual reproduction in B. ochroleuca.     

Pressure Inactivation of Bacillus Endospores

The inactivation of bacterial endospores by hydrostatic pressure requires the combined application of heat and pressure. We have determined the resistance of spores of 14 food isolates and 5 laboratory strains of Bacillus subtilis, B. amyloliquefaciens, and B. licheniformis to treatments with pressure and temperature (200 to 800 MPa and 60 to 80°C) in mashed carrots. A large variation in the pressure resistance of spores was observed, and their reduction by treatments with 800 MPa and 70°C for 4 min ranged from more than 6 log units to no reduction. The sporulation conditions further influenced their pressure resistance. The loss of dipicolinic acid (DPA) from spores that varied in their pressure resistance was determined, and spore sublethal injury was assessed by determination of the detection times for individual spores. Treatment of spores with pressure and temperature resulted in DPA-free, phase-bright spores. These spores were sensitive to moderate heat and exhibited strongly increased detection times as judged by the time required for single spores to grow to visible turbidity of the growth medium. The role of DPA in heat and pressure resistance was further substantiated by the use of the DPA-deficient mutant strain B. subtilis CIP 76.26. Taken together, these results indicate that inactivation of spores by combined pressure and temperature processing is achieved by a two-stage mechanism that does not involve germination. At a pressure between 600 and 800 MPa and a temperature greater than 60°C, DPA is released predominantly by a physicochemical rather than a physiological process, and the DPA-free spores are inactivated by moderate heat independent of the pressure level. Relevant target organisms for pressure and temperature treatment of foods are proposed, namely, strains of B. amyloliquefaciens, which form highly pressure-resistant spores.     

Nitrate Transport and Not Photoinhibition Limits Growth of the Freshwater Cyanobacterium Synechococcus Species PCC 6301 at Low Temperature

The effect of low temperature on cell growth, photosynthesis, photoinhibition, and nitrate assimilation was examined in the cyanobacterium Synechococcus sp. PCC 6301 to determine the factor that limits growth. Synechococcus sp. PCC 6301 grew exponentially between 20°C and 38°C, the growth rate decreased with decreasing temperature, and growth ceased at 15°C. The rate of photosynthetic oxygen evolution decreased more slowly with temperature than the growth rate, and more than 20% of the activity at 38°C remained at 15°C. Oxygen evolution was rapidly inactivated at high light intensity (3 mE m⁻² s⁻¹) at 15°C. Little or no loss of oxygen evolution was observed under the normal light intensity (250 μE m⁻² s⁻¹) for growth at 15°C. The decrease in the rate of nitrate consumption by cells as a function of temperature was similar to the decrease in the growth rate. Cells could not actively take up nitrate or nitrite at 15°C, although nitrate reductase and nitrite reductase were still active. These data demonstrate that growth at low temperature is not limited by a decrease in the rate of photosynthetic electron transport or by photoinhibition, but that inactivation of the nitrate/nitrite transporter limits growth at low temperature.     

Cloning and Expression of Thermostable α-Amylase Gene from Bacillus stearothermophilus in Bacillus stearothermophilus and Bacillus subtilis

The structural gene for a thermostable α-amylase from Bacillus stearothermophilus was cloned in plasmids pTB90 and pTB53. It was expressed in both B. stearothermophilus and Bacillus subtilis. B. stearothermophilus carrying the recombinant plasmid produced about fivefold more α-amylase (20.9 U/mg of dry cells) than did the wild-type strain of B. stearothermophilus. Some properties of the α-amylases that were purified from the transformants of B. stearothermophilus and B. subtilis were examined. No significant differences were observed among the enzyme properties despite the difference in host cells. It was found that the α-amylase, with a molecular weight of 53,000, retained about 60% of its activity even after treatment at 80°C for 60 min.     

Biophysics of the inhibition of the growth of maize roots by lowered temperature

Roots of hydroponically grown maize (Zea mays cv LG11) have a greatly reduced growth rate at 5°C (0.02 millimeters per hour) compared with those at 20°C (1.2 millimeters per hour). Various physical parameters of roots growing at each temperature were compared. Turgor pressure of cells in the elongation zone increased from 0.59 ± 0.05 megapascal at 20°C to 0.82 ± 0.04 megapascal after 70 hours at 5°C; thus, growth rate was not limited by decreased pressure. On cooling, tissue plasticity (measured by Instron/tensiometer) decreased slowly over 70 hours. On rewarming to 20°C from 5°C, growth rate, turgor pressure, and tissue plasticity all returned concertedly to their original values over a period of days. However, immediately following cooling growth rate dropped rapidly from 1.8 to 0.12 millimeters per hour in 30 minutes but turgor pressure and tissue Instron plasticity remained unchanged. A plot of turgor pressure against growth rate indicated that, following cooling from 30 to 15°C, the in vivo wall extensibility of the tissue was reduced by 75%. Yield threshold was unchanged. Cells whose expansion was arrested in the long-term cold treatment do not resume growth. Root growth recovers by the expansion of cells newly produced by the meristem. Cessation of extension growth is an effect on the individual expanding cell. Growth recovery is not a reverse of this effect but requires the generation of fresh cells.     

Inactivation of a Norovirus by High-Pressure Processing

Murine norovirus (strain MNV-1), a propagable norovirus, was evaluated for susceptibility to high-pressure processing. Experiments with virus stocks in Dulbecco's modified Eagle medium demonstrated that at room temperature (20°C) the virus was inactivated over a pressure range of 350 to 450 MPa, with a 5-min, 450-MPa treatment being sufficient to inactivate 6.85 log₁₀ PFU of MNV-1. The inactivation of MNV-1 was enhanced when pressure was applied at an initial temperature of 5°C; a 5-min pressure treatment of 350 MPa at 30°C inactivated 1.15 log₁₀ PFU of virus, while the same treatment at 5°C resulted in a reduction of 5.56 log₁₀ PFU. Evaluation of virus inactivation as a function of treatment times ranging from 0 to 150 s and 0 to 900 s at 5°C and 20°C, respectively, indicated that a decreasing rate of inactivation with time was consistent with Weibull or log-logistic inactivation kinetics. The inactivation of MNV-1 directly within oyster tissues was demonstrated; a 5-min, 400-MPa treatment at 5°C was sufficient to inactivate 4.05 log₁₀ PFU. This work is the first demonstration that norovirus can be inactivated by high pressure and suggests good prospects for inactivation of nonpropagable human norovirus strains in foods.     

Behavior of Bacillus anthracis Strains Sterne and Ames K0610 in Sterile Raw Ground Beef

The behavior of Bacillus anthracis Sterne spores in sterile raw ground beef was measured at storage temperatures of 2 to 70°C, encompassing both bacterial growth and death. B. anthracis Sterne was weakly inactivated (−0.003 to −0.014 log₁₀ CFU/h) at storage temperatures of 2 to 16°C and at temperatures greater than and equal to 45°C. Growth was observed from 17 to 44°C. At these intermediate temperatures, B. anthracis Sterne displayed growth patterns with lag, growth, and stationary phases. The lag phase duration decreased with increasing temperature and ranged from approximately 3 to 53 h. The growth rate increased with increasing temperature from 0.011 to 0.496 log₁₀ CFU/h. Maximum population densities (MPDs) ranged from 5.9 to 7.9 log₁₀ CFU/g. In addition, the fate of B. anthracis Ames K0610 was measured at 10, 15, 25, 30, 35, 40, and 70°C to compare its behavior with that of Sterne. There were no significant differences between the Ames and Sterne strains for both growth rate and lag time. However, the Ames strain displayed an MPD that was 1.0 to 1.6 times higher than that of the Sterne strain at 30, 35, and 40°C. Ames K0610 spores were rapidly inactivated at temperatures greater than or equal to 45°C. The inability of B. anthracis to grow between 2 and 16°C, a relatively low growth rate, and inactivation at elevated temperatures would likely reduce the risk for recommended ground-beef handling and preparation procedures.     

Temperature-Dependent Fecundity and Life Table of the Fennel Aphid Hyadaphis foeniculi (Passerini) (Hemiptera: Aphididae)

Hyadaphis foeniculi (Passerini) (Hemiptera: Aphididae) is a cosmopolitan species and the main pest of fennel in northeastern Brazil. Understanding the relationship between temperature variations and the population growth rates of H. foeniculi is essential to predict the population dynamics of this aphid in the fennel crop. The aim of this study was to measure the effect of constant temperature on the adult prereproductive period and the life table fertility parameters (infinitesimal increase ratio (r_m), gross reproduction rate (GRR), net reproduction rate (R₀), finite increase ratio (λ), generation time (GT), the time required for the population to double in the number of individuals (DT), and the reproduction value (RV_x)) of the fennel pest H. foeniculi. The values of lx (survival of nymphs at age x) increased as the temperature rose from 15 to 28°C and fell at 30°C, whereas mx (number of nymphs produced by each nymph of age x) increased from 15 to 25°C and fell at 28 and 30°C. The net reproduction rates (R₀) of populations of H. foeniculi increased with temperature and ranged from 1.9 at 15°C to 12.23 at 28°C for each generation. The highest population increase occurred with the apterous aphids at 28°C. The rate of population increase per unit time (r_m) (day) ranged from 0.0033 (15°C) to 0.1995 (28°C). The highest values of r_m were recorded at temperatures of 28°C and 30°C. The r_m values were a good fit to the models tested, with R² > 0.91 and R² _adj > 0.88. The models tested (Davidson, Sharpe and DeMichele modified by Schoolfield et al., Logan et al., Lamb, and Briere et al.) were very good fits for the r_m values observed, with R² > 0.91 and R² _adj > 0.88. The only exception was the Davidson model. Of the parameters studied, the reproductive capacity was higher in the apterous aphids, with the unique exception of daily fecundity at 28°C, which was higher in the alate aphids of H. foeniculi. Parameters relating to the age-specific fertility table for H. foeniculi were heavily influenced by temperature, with the highest biotic potential and population growth capacity found at 34°C. Therefore, the results obtained in this study could be of practical significance for predicting outbreaks of fennel aphids and improving the management of this aphid in fennel crops.     

Carbohydrate Level and Growth of Tomato Plants: II. The Effect of Irradiance and Temperature

The growth response of tomato (Lycopersicon esculentum L.) to temperature and irradiance may be related to carbohydrate concentration. Plants in the exponential phase of vegetative growth were grown under temperatures ranging from 9 to 36°C and under low or high irradiances of approximately 110 or 370 microeinsteins per square meter per second photosynthetically active radiation for a 12 hour photoperiod. The relative growth rate, leaf area ratio, net assimilation rate and whole plant carbohydrate levels were measured. At high irradiance, relative growth rate was 43% faster and total nonstructural carbohydrate concentration was 41% greater than at low irradiance. The change in carbohydrate with irradiance could explain the growth response. Plant growth was fastest at 25°C and decreased parabolically at lower and higher temperatures with a half-maximal rate at 13 and 36°C. Total nonstructural carbohydrate decreased between 13 and 23°C and remained constant at higher temperatures. Soluble sugar concentrations varied little with temperature above 13°C except for sucrose, whose level rose above 30°C. The change in carbohydrate with temperature could not explain the growth response. Above 23°C tomato plants appeared to regulate growth rate to maintain a relatively constant nonstructural carbohydrate concentration.     

THE GROWTH AND DURATION OF LIFE OF CELOSIA CRISTATA SEEDLINGS AT DIFFERENT TEMPERATURES

Daily measurements of hypocotyl length were made on Celosia cristata seedlings cultured in darkness under aseptic conditions at six constant temperatures between 14.5° and 40.5°C. At 40.5° roots did not penetrate the agar and only the hypocotyls that were supported by the wall of the test tube could be measured. The growth curves were of the generalized logistic type, but of different degrees of skewness. The degree of symmetry of the growth curves was influenced by temperature. At the lower temperatures the maximal growth rate came relatively late in the grand period of growth; at successively higher temperatures it came progressively earlier. The mean total time rate of growth (millimeter per diem) was found to be a parabolic function of the temperature. The maximum rate of growth was found from the curve to be at 30.48°C. The maximum observed rate of growth, and the maximum yield, were found to be at 30°C. At all temperatures above 14.5° the maximum growth activity fell in the second quarter of the whole growth period. At all temperatures tested other than 30°, and at all parts of the growth cycle, the growth yield as measured by height of hypocotyl at any given equivalent point was less than at 30°. The total duration of life of the seedlings, and the duration of life after the end of the growth period (intermediate period) were inversely proportional to the mean total growth rate. The observations on Celosia cristata seedlings are thus in accord with the "rate of living" theory of life duration. The optimal temperature for life duration is the minimum temperature, within the range of these observations.     

Modeling the Recovery of Heat-Treated Bacillus licheniformis Ad978 and Bacillus weihenstephanensis KBAB4 Spores at Suboptimal Temperature and pH Using Growth Limits

The apparent heat resistance of spores of Bacillus weihenstephanensis and Bacillus licheniformis was measured and expressed as the time to first decimal reduction (δ value) at a given recovery temperature and pH. Spores of B. weihenstephanensis were produced at 30°C and 12°C, and spores of B. licheniformis were produced at 45°C and 20°C. B. weihenstephanensis spores were then heat treated at 85°C, 90°C, and 95°C, and B. licheniformis spores were heat treated at 95°C, 100°C, and 105°C. Heat-treated spores were grown on nutrient agar at a range of temperatures (4°C to 40°C for B. weihenstephanensis and 15°C to 60°C for B. licheniformis) or a range of pHs (between pH 4.5 and pH 9.5 for both strains). The recovery temperature had a slight effect on the apparent heat resistance, except very near recovery boundaries. In contrast, a decrease in the recovery pH had a progressive impact on apparent heat resistance. A model describing the heat resistance and the ability to recover according to the sporulation temperature, temperature of treatment, and recovery temperature and pH was proposed. This model derived from secondary mathematical models for growth prediction. Previously published cardinal temperature and pH values were used as input parameters. The fitting of the model with apparent heat resistance data obtained for a wide range of spore treatment and recovery conditions was highly satisfactory.     

Effects of Hyperbaric Pressure on a Deep-Sea Archaebacterium in Stainless Steel and Glass-Lined Vessels

The effects of hyperbaric helium pressures on the growth and metabolism of the deep-sea isolate ES4 were investigated. In a stainless steel reactor, cell growth was completely inhibited but metabolic gas production was observed. From 85 to 100°C, CO₂ production proceeded two to three times faster at 500 atm (1 atm = 101.29 kPa) than at 8 atm. At 105°C, no CO₂ was produced until the pressure was increased to 500 atm. Hydrogen and H₂S were also produced biotically but were not quantifiable at pressures above 8 atm because of the high concentration of helium. In a glass-lined vessel, growth occurred but the growth rate was not accelerated by pressure. In most cases at temperatures below 100°C, the growth rate was lower at elevated pressures; at 100°C, the growth rates at 8, 250, and 500 atm were nearly identical. Unlike in the stainless steel vessel, CO₂ production was exponential during growth and continued for only a short time after growth. In addition, relatively little H₂ was produced in the glass-lined vessel, and there was no growth or gas production at 105°C at any pressure. The behavior of ES4 as a function of temperature and pressure was thus very sensitive to the experimental conditions.     

Variation in Resistance to Hydrostatic Pressure among Strains of Food-Borne Pathogens

Among food-borne pathogens, some strains could be resistant to hydrostatic pressure treatment. This information is necessary to establish processing parameters to ensure safety of pressure-pasteurized foods (N. Kalchayanand, A. Sikes, C. P. Dunne, and B. Ray, J. Food Prot. 61:425–431, 1998). We studied variation in pressure resistance among strains of Listeria monocytogenes, Staphylococcus aureus, Escherichia coli O157:H7, and Salmonella species at two temperatures of pressurization. Early-stationary-phase cells in 1% peptone solution were pressurized at 345 MPa either for 5 min at 25°C or for 5, 10, or 15 min at 50°C. The viability loss (in log cycles) following pressurization at 25°C ranged from 0.9 to 3.5 among nine L. monocytogenes strains, 0.7 to 7.8 among seven S. aureus strains, 2.8 to 5.6 among six E. coli O157:H7 strains, and 5.5 to 8.3 among six Salmonella strains. The results show that at 25°C some strains of each species are more resistant to pressure than the others. However, when one resistant and one sensitive strain from each species were pressurized at 345 MPa and 50°C, the population of all except the resistant S. aureus strain was reduced by more than 8 log cycles within 5 min. Viability loss of the resistant S. aureus strain was 6.3 log cycles even after 15 min of pressurization. This shows that strains of food-borne pathogens differ in resistance to hydrostatic pressure (345 MPa) at 25°C, but this difference is greatly reduced at 50°C. Pressurization at 50°C, in place of 25°C, will ensure greater safety of foods.     

TEMPERATURE CHARACTERISTICS FOR THE OXYGEN CONSUMPTION OF GERMINATING SEEDS OF LUPINUS ALBUS AND ZEA MAYS

The rate of oxygen consumption by germinating seeds of Lupinus albus and of Zea mays was studied as a function of temperature (7–26°C.). The Warburg manometer technique was used, with slight modifications. Above and below a critical temperature at 19.5°C. the temperature characteristic for oxygen consumption by Lupinus albus was found to be µ = 11,700± and 16,600 respectively. The same critical temperature was encountered in the case of Zea mays, with temperature characteristics µ = 13,100± above and µ = 21,050 below that temperature.     

Effects of Pressure-Induced Membrane Phase Transitions on Inactivation of HorA, an ATP-Dependent Multidrug Resistance Transporter, in Lactobacillus plantarum

The effects of pressure on cultures of Lactobacillus plantarum were characterized by determination of the viability and activity of HorA, an ATP-binding cassette multidrug resistance transporter. Changes in the membrane composition of L. plantarum induced by different growth temperatures were determined. Furthermore, the effect of the growth temperature of a culture on pressure inactivation at 200 MPa was determined. Cells were characterized by plate counts on selective and nonselective agar after pressure treatment, and HorA activity was measured by ethidium bromide efflux. Fourier transform-infrared spectroscopy and Laurdan fluorescence spectroscopy provided information about the thermodynamic phase state of the cytoplasmic membrane during pressure treatment. A pressure-temperature diagram for cell membranes was established. Cells grown at 37°C and pressure treated at 15°C lost >99% of HorA activity and viable cell counts within 36 and 120 min, respectively. The membranes of these cells were in the gel phase region at ambient pressure. In contrast, cells grown at 15°C and pressure treated at 37°C lost >99% of HorA activity and viable cell counts within 4 and 8 min, respectively. The membranes of these cells were 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 HorA inactivation and cell death more rapid than those of cells with a gel phase membrane at 0.1 MPa.