Previous Article | Next Article 
Applied and Environmental Microbiology, September 2000, p. 3966-3973, Vol. 66, No. 9
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Effects of High Pressure on Survival and Metabolic
Activity of Lactobacillus plantarum TMW1.460
Helge M.
Ulmer,
Michael G.
Gänzle,* and
Rudi F.
Vogel
Technische Universität München,
Lehrstuhl für Technische Mikrobiologie, D-85350 Freising, Germany
Received 20 March 2000/Accepted 30 June 2000
 |
ABSTRACT |
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 ATP binding cassette-type multidrug resistance transporter conferring resistance to
hop compounds. HP inactivation curves exhibited a shoulder, 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 uptake
occurred later than cell death, indicating that dye exclusion may be
used as a fail-safe method for preliminary characterization of HP
inactivation. An increase of membrane permeability to EB and a
reduction of HorA activity were 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.
| |
INTRODUCTION |
Treatment of food with a high
pressure (HP) of 200 to 800 MPa is a novel process in food technology
employed to change functional food properties, to selectively affect
the activity of food enzymes, to improve food texture, and to eliminate
microorganisms. The application of hydrostatic pressures is especially
promising to achieve preservation of minimally processed foods, as a
pressure treatment does not compromise the sensorial quality of food to the same extent as do thermal treatments with a comparable bactericidal effect. Nevertheless, it is advantageous to achieve food preservation by mild-pressure treatment in order to minimize quality deterioration and to reduce equipment and energy costs. Information on the mechanisms of HP-mediated inactivation of microorganisms will facilitate the
deliberate choice of the parameters pressurization temperature, pH,
pressure level, and holding time and allow the use of synergistic interactions between HP and other preservative principles. Proteins and
membranes are considered to be the primary target for the pressure-induced inactivation of bacteria. Pressures of 150 to 250 MPa
have been shown to induce dissociation of ribosomes in Escherichia coli (25). Wouters et al.
(46) found no morphological changes in the cytoplasmic
membrane upon lethal pressure treatment of Lactobacillus
plantarum; however, the membrane permeability was increased, and
the efflux of protons as well as the ability to maintain a 
pH across
the membrane was impaired. Pressure-induced leakage of sodium and
calcium ions was observed for Saccharomyces cerevisiae
(27). Effects of HP treatment on membrane potential and
membrane-bound transport systems may result from phase transitions in
the membrane (23). Adaptation of barophilic deep-sea
bacteria to HP involved a shift of membrane lipid composition from
saturated to unsaturated fatty acids (47). Both yeasts and
bacteria have been found to exhibit a maximum of barotolerance at
ambient temperature (12, 38). ter Steeg et al.
(42) observed an increased efficacy of HP treatment if the
pressurization temperature was reduced or if the incubation temperature
of the preculture was increased, i.e., under conditions where the
liquid crystalline state of the cytoplasmic membrane during growth of
the organisms is altered to a more rigid, semicrystalline state during pressurization.
Comparison of cell counts of pressurized samples on selective and
nonselective media shows that a large proportion of a given population
is sublethally injured prior to cell death, i.e., pressure-treated cells fail to survive and multiply in harsh environments tolerated by
untreated cells. The validity of this approach for gram-negative organisms was shown by use of selective media to probe the permeability barrier of the outer membrane with bile salts (14, 18, 19, 26). Many foods must be considered selective media for
microorganisms where growth or survival requires specific resistance
mechanisms, e.g., acid tolerance, osmotolerance, or resistance to
inhibitory compounds. Sublethal injury of HP-treated cells may
therefore indicate the inability to survive during food storage;
however, mechanisms accounting for this effect have so far not been elucidated.
As a model system to study the kinetics and mechanisms of HP
inactivation of lactic acid bacteria (LAB), we choose a beer-spoiling organism, L. plantarum TMW1.460. Beer is a highly selective
medium for growth of microorganisms because of the content of hop
bitter compounds, its low pH, and the high content of ethanol and
carbon dioxide. The mechanisms that allow beer-spoiling bacteria to
overcome these hurdles have been characterized in recent years. The
major bactericidal components in beer are hop bitter
compounds
colupulone, humulone, trans-isohumulone, and
trans-humulinic acidwhich dissipate the transmembrane pH
gradient (35, 36). Beer-spoiling LAB have been shown to
possess a plasmid-encoded hop resistance mechanism, HorA (32,
33). HorA mediates ATP-dependent transport of hop bitter
compounds and has high homology to other bacterial ATP-binding cassette-type multidrug transporters as well as mammalian multidrug resistance proteins (33, 44). Sami et al. (31)
screened 95 lactobacilli for the presence of the gene coding for the
hop efflux pump, horA, and found that this resistance
mechanism is a prerequisite for their growth in beer.
The objective of this study was to characterize the HP-treated cells
not only to obtain information on cell viability but also to determine
sublethal injury. A number of assays recently proposed for the rapid
determination of cell viability (2, 5, 43) were adapted to
L. plantarum TMW1.460 in order to determine whether the loss
of metabolic activity or membrane integrity or the failure to maintain
hop resistance accounts for sublethal injury of pressurized cells.
| |
MATERIALS AND METHODS |
Strains and culture conditions.
Saccharomyces
cerevisiae subsp. uvarum TMW 3.001, a commercially
available brewer's yeast (Technische Universität München, Lehrstuhl Technologie der Brauerei II, Freising, Germany), was cultured
at ambient temperature on malt extract medium (12% [wt/wt] malt
extract [Ireks, Kulmbach, Germany], sterilized at 121°C for 21 min). L. plantarum TMW1.460, an organism previously isolated from spoiled beer, was cultivated using model beer (MB), MRS agar (7), or MRS agar containing 4% NaCl (MRS-NaCl) at 30°C.
Solid media contained 1.5% agar. MB was prepared by inoculating malt extract medium with S. cerevisiae TMW 3.001 to a cell count
of about 5 × 106 cells ml
1. The mash
was fermented for 140 h at 10°C and autoclaved (121°C, 20 min), and the yeast was removed by centrifugation (20 min, 20,000 × g, 0°C). The clear supernatant was
collected, residual ethanol and CO2 were removed in a
rotary evaporator under vacuum, and the weight loss was compensated for
with demineralized water. The pH was adjusted to 4.0, and the medium
was sterilized at 121°C for 21 min.
HP treatment.
An overnight culture of L. plantarum TMW1.460 in MB was subcultured in MB for 16 h with
1% inoculum to late stationary growth phase. Cells were harvested by
centrifugation and resuspended in an equal volume of MB. This cell
suspension was transferred to 2-ml Eppendorf reaction tubes, sealed
with silicon stoppers avoiding enclosure of air, and stored on ice
until pressurization. The HP inactivation kinetics of L. plantarum were investigated in HP autoclaves precooled to 15°C.
Compression and decompression rates were 200 MPa min1.
The temperature increase in the HP autoclaves due to adiabatic compression was less than 4, 8, 10, and 12°C upon compression to 200, 400, 500, and 600 MPa, respectively, and the temperature reached 15°C
after a 5-min pressure holding time or earlier. Upon pressurization,
samples were stored on ice until further analysis as described below.
For each HP inactivation curve, untreated cultures and cultures
sterilized by treatment with 800 MPa for 10 min were used for
preparation of calibration samples containing 100, 50, and 0% viable cells.
Determination of plate counts.
Cell counts were determined
on MRS agar and MRS-NaCl agar for determination of viable and
sublethally injured cells. Appropriate dilutions were plated using a
spiral plater (IUL, Königswinter, Germany), and plates were
incubated at 30°C for 2 days under a controlled atmosphere (76%
N2, 20% CO2, and 4% O2). Cell
counts of overnight cultures of L. plantarum in MB were
(4.3 ± 2.1) × 108 CFU ml1 on
either MRS or MRS-NaCl agar (mean of 24 determinations). The cell
counts on MRS are referred to as viable cells, and the difference between cell counts on MRS and on MRS-NaCl is referred to as
sublethally injured cells.
Determination of metabolic activity.
Cells from a 100-µl
sample were harvested by centrifugation at 0°C and 10,000 × g for 10 min, resuspended in 100 µl of phosphate buffer with
glucose (PBG; 50 mM H2KPO4, 0.1 g of
MgSO4 × 7H2O liter1,
0.05 g of MnSO4 × H2O
liter1 and 4 g of glucose liter1, pH
6.5), and transferred to microtiter plates. To this cell suspension was
added a stock solution of 4 mmol of
2-(-iodophenyl)-3-(p-nitrophenyl)5-phenyltetrazolium chloride (INT; Molecular Probes, Eugene, Oreg.) liter1 in
PBG to a final concentration of 2 mmol of INT liter1. The
kinetics of reduction of the colorless INT to the red formazan dye was
monitored by measuring absorption at 590 nm in a Spectrafluor microtiter plate reader (Tecan, Grödig, Austria) at 1-min
intervals for 60 min at 30°C. The initial rate of INT reduction was
used for calculation of the metabolic activity of the cells. A
calibration curve was established for each inactivation curve using the
calibration samples described above, and the results are reported as
percent metabolic activity.
Determination of HorA activity.
Ethidium bromide (EB) is a
substrate for HorA conferring hop resistance and related multidrug
resistance transport systems of LAB (3, 33). Therefore, an
assay for HorA activity was developed using EB as a substrate. EB stock
solutions were prepared by dissolving 40 µmol of EB
liter1 in PBG and PB0 (50 mM
H2KPO4, 0.1 g of MgSO4
· 7H2O liter1, and 0.05 g of
MnSO4 · H2O liter1, pH
6.5). Cells were harvested from 1-ml samples by centrifugation (10 min
at 15°C, 6,000 × g) and resuspended in 1 ml of PB0.
Each sample was divided into two aliquots. One aliquot received 200 µl of cell suspension and 200 µl of EB stock solution in PB0 to obtain an EB concentration of 20 µmol liter1. The other
aliquot received 200 µl of EB stock solution in PBG to obtain the
same buffer and EB concentrations and additionally 2 g of glucose
liter1 as an energy source. Samples were mixed and
incubated at 30°C for 0, 20, 45, 100, and 140 min (assay validation)
or 1.5 h (characterization of HP-treated samples) in the dark.
After incubation, cells were harvested, resuspended in 200 µl of PB0,
and transferred to black microtiter plates. The fluorescence of this
cell suspension was measured using a Spectrafluor microtiter plate
reader (
Ex = 485 nm, Em = 595 nm). The difference between EB fluorescence of starved cells and that
of energized cells was considered to indicate HorA activity.
Determination of membrane integrity.
The integrity of the
cytoplasmic membrane of HP-treated cells was determined using two
different dye exclusion assays. (i) The BacLight Live/Dead Kit
(Molecular Probes) was used according to the instructions of the
supplier. In short, cells from 100-µl samples were harvested by
centrifugation, resuspended in an equal volume of PB0, and transferred
onto black microtiter plates. To each sample, 100 µl of dye solution
(33.4 µmol of Syto9 liter1 and 200 µmol of propidium
iodide [PI] liter1 in PB0) was added, and the plates
were incubated for 15 min at ambient temperature. The ratio of Syto9
and PI fluorescence was used to calculate the percentage of intact
membranes in the sample. The applicability of the BacLight kit to
L. plantarum was verified with cells killed by severe heat
(80°C for 10 min) or HP treatment (800 MPa, 10 min). A calibration
curve was established for each inactivation curve using the calibration
samples described above, and the results are reported as percent intact
membranes. (ii) The determination of HorA activity described above
required incubation of cells of L. plantarum with EB in the
presence and absence of an energy source, glucose. In the absence of
glucose (i.e., in the absence of a functional EB efflux system), EB
uptake was considered to depend solely on the barrier properties of the
cytoplasmic membrane. The fluorescence intensity of samples stained
with EB in PB0 (see the HorA assay described above) thus provides
further information on the membrane integrity of pressurized cultures of L. plantarum. The samples with known contents of viable
and dead cells were stained with EB in the absence of glucose as
described above, and the resulting calibration curve was used to
calculate the percent intact membranes in the HP-treated samples.
| |
RESULTS |
Assay validation for determination of metabolic activity with
tetrazolium salts.
The use of tetrazolium salts has become a
standard laboratory method to determine metabolic activity and
viability of microorganisms. The assay is based on the ability of
metabolically active cells to reduce INT to insoluble red formazan.
However, the feasibility of this method to characterize metabolic
activity of lactobacilli has so far not been demonstrated, and previous
investigators have used the microscope to determine the deposition of
formazan crystals in individual cells (43, 45). To develop a
miniaturized assay for the rapid analysis of a large amount of samples,
we evaluated whether the initial rate of formazan production by cell
suspensions is a suitable means to estimate their metabolic activity.
Mixtures of heat-killed, metabolically inactive cells of L. plantarum with metabolically active cells were incubated with INT,
and the rate of formazan formation was determined. The increase of
absorption at 595 nm is shown in Fig. 1.
A linear rate of INT reduction was observed during the first 20 to 30 min of incubation, and the INT reduction rate was calculated from these
data by linear regression. The INT reduction rate correlated well with
the content of viable cells in the culture (r2 = 0.9985). This assay was therefore used for characterization of
HP-treated cultures, and an r2 of 0.95 or
greater was obtained for all calibration curves determined with HP
experiments (n = 12).
View larger version (18K):
[in this window]
[in a new window]
|
FIG. 1.
Formazan formation by cultures of L. plantarum TMW1.460 containing 100% ( ), 90% ( ), 50% ( ),
10% ( ), and 0% ( ) viable cells. Lines represent curves obtained
by linear regression. OD, optical density.
|
|
Assay validation for determination of HorA activity.
To
demonstrate that L. plantarum TMW1.460, a highly
hop-resistant beer isolate, has a functional HorA efflux system and to develop a HorA activity assay, the hop resistance was evaluated at the
genetic and physiological level. Using horA-targeted primers and PCR conditions as described by Sami et al. (31), we
obtained a PCR product of the expected size, 345 bp (data not shown),
using chromosomal DNA from L. plantarum TMW1.460 as
template. Sequencing of this PCR product revealed identity to the
corresponding horA fragment of Lactobacillus
brevis but for one conservative base pair exchange (references
31 and 32 and data not shown).
This demonstrates that L. plantarum TMW1.460 carries a
horA gene. HorA activity of L. plantarum TMW1.460
was investigated using EB as substrate. Energy-dependent EB efflux by
L. plantarum was assessed by incubation of cells with EB in
the presence or absence of an energy source, glucose, and monitoring of
EB uptake over time. The results are shown in Fig.
2. In the presence of glucose, cells were
able to maintain an internal low EB concentration, whereas starved
cells accumulated EB. This points to the presence of an energy-dependent EB efflux system which was attributed to HorA activity. Based on the kinetics of EB accumulation, an incubation time
of 1.5 h was chosen for determination of HorA activity in pressurized cells.
View larger version (19K):
[in this window]
[in a new window]
|
FIG. 2.
Kinetics of EB diffusion into starved () and
energized () cells of L. plantarum TMW1.460. EB influx
was determined by measuring the fluorescence of cells harvested after
incubation times of 0 to 145 min. Symbols represent means ± standard deviations of two independent experiments.
|
|
For assay calibration, mixtures of viable cells with cells killed by
either HP treatment (800 MPa, 10 min) or heat (80°C,
10 min) were
used. In Fig.
3 is shown the correlation
of EB fluorescence
in the presence and absence of glucose with the
content of viable
cells in the culture. Dead cells exhibited high EB
fluorescence,
and an increasing content of viable cells resulted in a
decrease
of EB fluorescence, as in viable cells EB influx occurs by
diffusion
through the lipid bilayer only. For samples containing viable
cells, EB fluorescence in the presence of glucose was lower than
that
in the absence of glucose, in accordance with HorA activity
of viable,
energized cells. Cultures sterilized either by heat
or by HP treatment
exhibited the same behavior in the assay.
View larger version (20K):
[in this window]
[in a new window]
|
FIG. 3.
Correlation of EB fluorescence of starved (open symbols)
and energized (black symbols) cells with the content of viable cells in
the sample. Samples were prepared by mixing untreated cultures with
cultures sterilized by heat (80°C, 10 min) (circles) or HP (800 MPa,
10 min) (inverted triangles). Symbols represent means ± standard
deviations of three independent experiments.
|
|
It must be emphasized that the killing of cells by severe heat (80°C,
10 min) or HP (800 MPa, 10 min) treatments results in
simultaneous
disruption of membrane integrity, inactivation of
glycolytic enzymes,
and inactivation of HorA activity. However,
during pressurization not
all of these three components required
for EB transport across the
membrane are necessarily inactivated
at the same time. Therefore, the
interpretation of the assay results
with respect to HorA activity
requires additional information
on metabolic activity and membrane
integrity. Comparison of the
EB fluorescence data presented in Fig.
2
and
3 shows that several
mechanisms account for EB influx and efflux
into
L. plantarum.
(i) Energized cells with HorA activity
and an intact membrane
(PI exclusion) are able to completely exclude
EB. (ii) EB penetrates
viable, deenergized cells by diffusion through
the membrane. Within
1 h, a steady-state level is reached (Fig.
2). (iii) EB penetrates
cells subjected to severe pressure or heat
treatment by a mechanism
comparable to that allowing PI influx into
cells, probably through
gaps in the cytoplasmic membrane. The
steady-state EB fluorescence
of viable, deenergized cells (diffusion
through the cytoplasmic
membrane only) is lower than the EB
fluorescence of cells subjected
to severe
stress.
HP inactivation of L. plantarum: sublethal injury and
cell death.
L. plantarum TMW1.460 was subjected to HP
treatment at 200, 400, 500, and 600 MPa, and cell inactivation was
monitored over time by plating on MRS and MRS-NaCl agar. The results
are shown in Fig. 4. Cell counts of
untreated cells on MRS and MRS-NaCl agars were not different
(P < 0.001); therefore, failure of L. plantarum to grow on MRS-NaCl indicates sublethal injury. Because samples could not be taken during compression and decompression, the
x axis in Fig. 4 indicates the pressure holding time.
View larger version (16K):
[in this window]
[in a new window]
|
FIG. 4.
Kinetics of inactivation of L. plantarum
TMW1.460 incubated at 200 (A) (n = 4), 400 (B)
(n = 3), 500 (C) (n = 3), and 600 (D)
(n = 2) MPa. Shown is the viable cell count on MRS
(nonselective agar) () and MRS-NaCl (selective agar) () compared
to that of untreated cultures. The cell count of control cultures was
(4.27 ± 2.09) × 108 CFU ml1 on
either agar, and the detection limit was 120 CFU ml1.
Symbols represent means ± standard deviations of n
independent experiments.
|
|
As shown in Fig.
4A, the inactivation curves of
L. plantarum
exhibited a sigmoid shape and were characterized by a shoulder,
an
exponential inactivation phase, and a pronounced tailing where
no or
little further inactivation took place. In the initial phase
of
pressurization, the shoulder, no loss of viability was observed.
During
the exponential inactivation phase, cells were sublethally
injured
prior to irreversible cell damage as demonstrated by the
observation
that more than 99.99% of viable cells lost their ability
to grow on
selective media. At longer pressure holding times (more
than 60, 12, or
2 min at 200, 400, or 500 MPa, respectively),
sublethal injury was no
longer observed. Apparently, a fraction
of the population, about 1 in
10
6 cells, was highly resistant to pressure, independent of
whether
the pressure treatment was performed at 200, 400, or 500 MPa.
To evaluate whether this tailing of pressure inactivation curves
stems
from mutants or reflects phenotypic diversity of the population,
survivors of HP treatments were isolated and subcultured once
and their
pressure tolerance was determined. If this selection
cycle was carried
out four times, no increase in barotolerance
was observed (data not
shown).
This characteristic shape of the inactivation curves was not observed
at pressures of 400 and 500 MPa, as the shoulder and
part of the
exponential inactivation occurred already during the
pressure ramps
during which no samples were taken. However, a
barotolerant fraction of
the population exhibiting no sublethal
injury was also observed. At 600 MPa, virtually all cells were
killed during pressure ramps and the
fraction of barotolerant
cells was at or below the detection level (120 CFU ml
1).
HP inactivation of L. plantarum: loss of metabolic
activity.
Cell counts demonstrated sublethal injury during HP
treatment of L. plantarum. To evaluate whether this
phenomenon is related to the loss of metabolic activity, the metabolic
activity of pressurized cells was determined with tetrazolium chloride.
The results are shown in Fig. 5. Whereas
metabolic activity could be detected after a 60-min pressure holding
time at 200 MPa, the pressure ramp to achieve 600 MPa sufficed to
completely inactivate the cultures. Sublethally injured cells exhibited
metabolic activity comparable to that of the untreated controls, but
the loss of metabolic activity was highly correlated with cell death
(r2 = 0.92). However, even upon pressure
treatments killing more than 99.99% of the cells, metabolic activity
above baseline level was observed ([18 ± 7]%, [9 ± 6]%, and [12 ± 4]% at 200 MPa, 60 min; 400 MPa, 1 min; and
500 MPa, 0 min, respectively). This indicates that the loss of
metabolic activity is unlikely to be a primary cause for cell death.
View larger version (16K):
[in this window]
[in a new window]
|
FIG. 5.
Kinetics of inactivation of L. plantarum
TMW1.460 incubated at 200 (A) (n = 4), 400 (B)
(n = 3), 500 (C) (n = 3), and 600 (D)
(n = 2) MPa. Shown is the INT reduction rate of treated
cells compared to the INT reduction rate of untreated cells. Symbols
represent means ± standard deviations of n independent
experiments.
|
|
HP inactivation of L. plantarum: loss of membrane
integrity.
HP inactivation curves were further characterized by
two different dye exclusion assays based on EB and PI permeation into cells. The results are shown in Fig. 6.
Curves obtained with PI exhibited the same sigmoid shape as those
observed with plate counts (Fig. 4) and staining with INT (Fig. 5);
however, loss of PI exclusion was observed only after the cells were
dead and lost any metabolic activity. This observation was highly
significant at pressure levels of 200, 400, and 500 MPa, where 25 to
50% membrane integrity was observed after HP treatments resulting in
reduction of viable cell counts by 4 to 6 orders of magnitude.
View larger version (20K):
[in this window]
[in a new window]
|
FIG. 6.
Kinetics of inactivation of L. plantarum
TMW1.460 incubated at 200 (A), (n = 4), 400 (B)
(n = 3), 500 (C) (n = 3), and 600 (D)
(n = 2) MPa. Shown is the membrane permeability
determined with the dye PI () or EB () and compared to the
membrane permeability of untreated cells. Symbols represent means ± standard deviations of n independent experiments.
|
|
EB was used as a second probe for determination of membrane integrity.
As opposed to PI, EB also penetrated intact membranes
of viable cells
to a steady-state level in between that of viable,
energized cells and
that of dead cells subjected to severe stress.
Incubation of
deenergized cells with EB excluded the possibility
that ATP- or
membrane potential-dependent transport mechanisms
such as HorA affected
EB uptake. In probing membrane integrity
with EB during HP inactivation
curves, biphasic kinetics were
observed. EB diffusion into 200 MPa-treated cells was facilitated
already after 4 to 20 min of pressure
holding time, resulting
in 50 to 75% membrane integrity, although
cells were not stained
with PI and neither sublethal injury nor loss of
viability was
detectable by plate counts. This facilitated diffusion of
EB through
membranes of pressurized cells indicates that membrane
damage
occurs prior to cell death. An incubation time greater than
3
h at 200 MPa was required for 0% EB membrane integrity (data
not
shown). Accordingly, pressurization at 400 and 500 MPa resulted
in
a rapid drop of EB membrane integrity to about 50% within 1
min
followed by a much slower decrease to 0% within 3 h. Whereas
EB
appeared to be a more sensitive probe than PI for changes in
membrane
structure of viable, PI-excluding cells, longer pressurization
times
were required to obtain 0% membrane integrity as determined
by
EB.
HorA activity of pressurized cells.
HorA activity is a
prerequisite for growth of LAB in the presence of hop bitter compounds
and is therefore crucial for the beer-spoiling capability of LAB.
Therefore, in addition to membrane integrity and metabolic activity,
the HorA activity of pressurized cells was estimated by determination
of EB diffusion into starved and energized cells. The results for HP
treatments at 200 and 600 MPa are shown in Fig.
7. As described above, control cultures (no HP treatment) exhibited a large difference in EB influx depending on the presence of glucose, indicating HorA activity (Fig. 2 and 3).
Pressurization with 600 MPa resulted in complete loss of metabolic activity and membrane integrity (compared to Fig. 5 and 6);
accordingly, EB uptake into cells was facilitated and reached the level
of cells killed by heat (80°C, 10 min) after 6 to 10 min of
pressurization time. A significant effect of glucose on EB uptake was
not observed. Cells pressurized at 200 MPa for 0 to 12 min were able to
maintain an internal low EB concentration although EB diffusion across the membrane was facilitated. During these first 12 min, no loss of
cell viability or sublethal injury was observed by plate counts (Fig.
3). After a 30- and a 60-min pressure holding time, the cultures lost
their ability to exclude EB even in the presence of glucose, indicating
loss of hop resistance. This observation that viable but sublethally
injured cells exhibited reduced HorA activity was more evident if data
were corrected for the systematic difference (±2-log difference)
between HP inactivation curves determined on different days.
View larger version (29K):
[in this window]
[in a new window]
|
FIG. 7.
HorA activity of L. plantarum TMW1.460 after
pressure treatment at 200 (n = 4) (squares) and 600 (n = 2) (inverted triangles) MPa. Shown is the EB
fluorescence of cells incubated in phosphate buffer with 20 µmol of
EB liter1 for 90 min in the presence (black symbols) and
absence (open symbols) of glucose. Symbols represent means ± standard deviations of n independent experiments.
|
|
| |
DISCUSSION |
In this study, the pressure-mediated inactivation of L. plantarum was investigated and surviving cells were characterized with respect to viability, metabolic activity, membrane integrity, and
functionality of hop resistance mechanisms. Based on this method,
pressure inactivation of L. plantarum was found to involve a
series of several inactivation steps. Mild-pressure treatment results
in sublethal injury of cells. An increased permeability of the
cytoplasmic membrane and reduced HorA activity were related to
sublethal injury. Subsequently, loss of cell viability and concomitant
loss of metabolic activity were observed. Membrane damage as determined
with PI was observed later than cell death. These data provide a
rationale for the observation that mild pressurization of
microorganisms strongly reduces the ability of microorganisms to
survive in adverse environments. Furthermore, assays developed for
characterization of HP-treated cells have been found to be suitable
methods for rapid determination of cell viability and activity.
First-order kinetics have been used to calculate pressure death-time
data for bacteria and yeasts previously (10, 11, 48).
However, for Bacillus species (16, 28) and for
E. coli (15), it was shown that pressure
inactivation curves may exhibit a pronounced shoulder and tailing. The
shoulder of pressure inactivation curves was explained by phenotypic
heterogeneity in the population, and a mathematical model that used a
Weibull-distributed kinetics for the transition from a stable to a
metastable state during pressure treatments fitted experimental
death-time data well (16). If pressure treatment of bacteria
indeed is a two-stage inactivation process leading to metastable or
sublethally injured cells, it should be possible to identify
physiological changes occurring during the shoulder of pressure
death-time curves. A number of investigators have proposed selective
media for determination of sublethally injured E. coli,
Salmonella species, Staphylococcus aureus, and
Listeria monocytogenes cells after pressure treatments (8, 14, 18, 19, 26). As a selective agent for lactobacilli, the use of 0.6% NaCl was proposed (41), and the failure of
lactobacilli to grow on MRS-NaCl was thought to indicate membrane
damage (40). We used 4% NaCl to inhibit growth of
sublethally injured cells upon HP treatments and observed a shorter
shoulder (200 MPa) or no shoulder at all (400 MPa) in pressure
death-time curves characterized with MRS-NaCl agar. These data thus
provide further physiological proof for a multistage inactivation
process during pressurization of microorganisms. However, as shoulders
were also observed if inactivation curves were characterized with cell
counts on MRS-NaCl agar, this selective medium apparently failed to
indicate cell damage occurring early during inactivation.
The determination of sublethal injury in HP-treated populations may be
of major interest for food preservation, as sublethal injury may
eliminate the ability of a culture to grow during food storage. Indeed,
pressurized cells of E. coli failed to survive in fruit
juices under conditions tolerated by untreated cells (8,
21), although only a minor part of the population was actually
killed by the HP treatment.
Tailing of pressure inactivation curves obtained with spores of
Bacillus subtilis was thought to reflect a heterogeneous
distribution of barotolerance in the population (28) based
on genotypic or phenotypic diversity. Hauben et al. (13)
have isolated barotolerant mutants of E. coli. Eighteen
selection cycles were required to obtain mutant strains that tolerated
pressure treatments resulting in a greater than 8-log inactivation of
the wild-type strain (13). We were unable to isolate
baroresistant mutants after four selection cycles, indicating that
tailing of pressure inactivation reflected phenotypic diversity within
the population rather than the presence of baroresistant mutants.
The staining of cells with tetrazolium salts was proposed as a rapid
method for determination of cell viability (9, 43, 45).
Tetrazolium reduction to formazan by lactobacilli depends on the
activity of NADH-dependent enzymes, such as NADH oxidase or peroxidase
activities, or specific NADH dehydrogenases that are present in LAB
(4, 20, 24, 30, 37, 39). Reduction of tetrazolium salts by
lactobacilli thus requires NADH generation by an ongoing carbohydrate
metabolism, and staining of cells with INT not only relies on the
activity of a single enzyme system, i.e., NADH-reducing enzymes, but
also may be considered an indicator of the overall metabolic activity
of lactobacilli. Accordingly, we observed a loss of acidification
capacity of pressurized cells of L. plantarum concomitant
with loss of capability for INT reduction (data not shown). The
observation that a loss of metabolic activity of HP-treated LAB is
linked to cell death rather than to sublethal injury does conform with
literature data obtained with different methods. Measuring the
intracellular ATP pool and F0F1-ATPase activity
of HP-treated L. plantarum revealed that ATP-generating glycolytic enzymes retained activity after treatments resulting in 60 to 95% reduction of viable cell counts (46) and that
severe-pressure treatments (0.4% survival) were required to completely
inhibit ATP-generating enzymes (46).
The assessment of membrane integrity by dye exclusion assays has been
proposed by several authors to determine the efficacy of germ-killing
processes, including HP treatments (1, 2, 5). Bunthof et al.
(5) used PI staining for characterization of
Lactococcus lactis stressed with freeze-thaw treatment, bile salts, low pH, and heat treatment. Whereas PI exclusion by L. lactis corresponded to plate counts for most stress treatments, membranes of heat-killed cells were impermeable to PI (5). The observation that HP-treated cells are not recoverable by plate counts but are not stained with PI was interpreted as an indication of
the presence of living, but metabolically inactive, cells
(1). Our data confirm that failure to grow on nonselective
media may precede membrane permeability to PI. Membrane damage was
observed with the PI assay only for treatments resulting in greater
than 5-log reductions of viable cell counts. Thus, membrane
impermeability to PI alone is an inappropriate indicator for cell
viability as proposed by Arroyo et al. (1) but may serve as
fail-safe method to evaluate the effect of pressure treatments.
EB penetrates cells with intact membranes and may therefore serve as an
indicator of transient membrane perturbation. L. lactis and
beer-spoiling LAB are known to possess transport systems mediating proton motive force- and ATP-dependent EB efflux, respectively, against
a concentration gradient (3, 33). Therefore, to take into
consideration both the changes in membrane permeability to EB and the
inactivation of efflux systems, we performed EB transport assays with
energized and starved cells. Membrane damage and inactivation of
membrane-bound transport systems were identified as important events of
pressure inactivation of L. plantarum, Salmonella
enterica serovar Typhimurium, and S. cerevisiae
(27, 29, 42, 46). Remarkably, EB uptake by starved cells
revealed that the permeability of cells to EB was increased by
pressurization. The kinetic resolution of HP inactivation of L. plantarum at 200 MPa indicates that membrane damage is an early
event during pressurization of microorganisms, as it was observed even
upon pressure treatments that did not affect viability or induce
sublethal injury. We expect that membrane response to pressure may be
nearly universal, as it was observed that pressures as low as 100 to
200 MPa result in destruction of lysosomal membranes in bovine muscle
tissue (17).
In addition to increased permeability of HP-treated cells to EB, we
observed a reduction of HorA activity prior to cell death. As the
metabolic activity in these cells was largely unaffected by
pressurization, this finding indicates inactivation of HorA. Modifications of lipid-enzyme interactions are considered to
affect the activity of membrane-bound enzymes in eucaryotic cells.
Membrane-bound Na+-Mg2+-ATPase of
Acholeplasma laidlawii B is active only in association with
liquid-crystalline lipids, and inactivation occurs when its boundary
lipids undergo a phase transition (34). Phase transitions in
the lipids associated with ATPase were considered to determine its
activity at pressures ranging from 30 to 100 MPa (22, 23). Inhibition of Na-K-ATPase by pressure was directly correlated with
increased membrane order (6). Extrapolating these findings with eucaryotic ATPase to corresponding bacterial enzymes suggests that
phase transitions of membrane lipids may contribute to the pressure
inactivation of membrane-bound transport systems such as HorA (this
study) or F1F0-ATPase (46) in
addition to pressure-mediated protein denaturation or subunit
dissociation. Membrane damage and subsequent HorA inactivation thus
result in sublethally injured, hop-sensitive cells that fail to survive
or even grow during beer storage. We could thus demonstrate that
relatively mild-pressure treatments are suitable in applications with
the aim of preventing or delaying the growth of spoilage bacteria
rather than the sterilization of foods.
| |
ACKNOWLEDGMENT |
This work was supported by the "Wissenschaftsförderung
der Deutschen Brauwirtschaft," grant no. B51.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: TU
München, Lehrstuhl für Technische Mikrobiologie,
Weihenstephaner Steig 16, D-85350 Freising, Germany. Phone: 49 (0)8161
71 3959. Fax: 49 (0)8161 71 3327. E-mail:
michael.gaenzle{at}blm.tu-muenchen.de.
| |
REFERENCES |
| 1.
|
Arroyo, G.,
P. D. Sanz, and G. Prestamo.
1999.
Response to high-pressure, low-temperature treatment in vegetables: determination of survival rates of microbial populations using flow cytometry and detection of peroxidase activity using confocal microscopy.
J. Appl. Microbiol.
86:544-556[CrossRef][Medline].
|
| 2.
|
Benito, A.,
G. Ventoura,
M. Casadei,
T. Robinson, and B. Mackey.
1999.
Variation in resistance on natural isolates of Escherichia coli O157 to high hydrostatic pressure, mild heat, and other stresses.
Appl. Environ. Microbiol.
65:1564-1569[Abstract/Free Full Text].
|
| 3.
|
Bolhuis, H.,
D. Molenaar,
G. Poelarends,
H. W. van Veen,
B. Poolman,
A. J. M. Driessen, and W. N. Konings.
1994.
Proton motive force-driven and ATP-dependent drug extrusion systems in multidrug-resistant Lactococcus lactis.
J. Bacteriol.
176:6957-6964[Abstract/Free Full Text].
|
| 4.
|
Bolotin, A.,
S. Mauger,
K. Malarme,
S. D. Ehrlich, and A. Sorokin.
1999.
Low-redundancy sequencing of the entire Lactococcus lactis IL403 genome.
Antonie Leeuwenhoek
76:27-76[CrossRef][Medline].
|
| 5.
|
Bunthof, C. J.,
S. van den Braak,
P. Breeuwer,
F. M. Rombouts, and T. Abee.
1999.
Rapid fluorescence assessment of the viability of stressed Lactococcus lactis.
Appl. Environ. Microbiol.
65:3681-3689[Abstract/Free Full Text].
|
| 6.
|
Chong, P. L.-G.,
P. A. G. Fortes, and D. M. Jameson.
1988.
Mechanisms of inhibition of (Na,K)-ATPase by hydrostatic pressure studied with fluorescent probes.
J. Biol. Chem.
260:14484-14490[Abstract/Free Full Text].
|
| 7.
|
de Man, J. C.,
M. Rogosa, and M. E. Sharpe.
1960.
A medium for the cultivation of lactobacilli.
J. Appl. Bacteriol.
23:130-135.
|
| 8.
|
Garcia-Graells, C.,
K. J. A. Hauben, and C. W. Michiels.
1998.
High-pressure inactivation and sublethal injury of pressure-resistant Escherichia coli mutants in fruit juices.
Appl. Environ. Microbiol.
64:1566-1568[Abstract/Free Full Text].
|
| 9.
|
Garcia-Lara, J.,
J. Martinez,
M. Vilamu, and J. Vives-Rego.
1993.
Effect of previous growth conditions on the starvation-survival of Escherichia coli in seawater.
J. Gen. Microbiol.
139:1425-1431[Medline].
|
| 10.
|
Gervilla, R.,
E. Sendra,
V. Ferragut, and B. Guamis.
1999.
Sensitivity of Staphylococcus aureus and Lactobacillus helveticus in ovine milk subjected to high hydrostatic pressure.
J. Dairy Sci.
82:1099-1107.
|
| 11.
|
Gervilla, R.,
M. Mor-Mur,
V. Ferragut, and B. Guamis.
1999.
Kinetics of destruction of Escherichia coli and Pseudomonas fluorescens inoculated in ewe's milk by high hydrostatic pressure.
Food Microbiol.
16:173-184.
|
| 12.
|
Hashizume, C.,
K. Kimura, and R. Hayashi.
1995.
Kinetic analysis of yeast inactivation by high pressure treatment at low temperatures.
Biosci. Biotechnol. Biochem.
59:1455-1458[Medline].
|
| 13.
|
Hauben, K. J. A.,
D. H. Bartlett,
C. C. F. Soontjens,
K. Cornelis,
E. Y. Wuytack, and C. W. Michiels.
1997.
Escherichia coli mutants resistant to inactivation by high hydrostatic pressure.
Appl. Environ. Microbiol.
63:945-950[Abstract].
|
| 14.
|
Hauben, K. J. A.,
E. Y. Wuytack,
C. C. F. Soontjens, and C. W. Michiels.
1996.
High-pressure transient sensitiation of Escherichia coli to lysozyme and nisin by disruption of outer-membrane permeability.
J. Food Prot.
59:350-355.
|
| 15.
|
Hauben, K. J. A.,
K. Bernaerts, and C. W. Michiels.
1998.
Protective effect of calcium on inactivation on Escherichia coli by high hydrostatic pressure.
J. Appl. Microbiol.
85:678-684[CrossRef][Medline].
|
| 16.
|
Heinz, V., and D. Knorr.
1996.
High pressure inactivation kinetics of Bacillus subtilis cells by a three-state-model considering distributed resistance mechanisms.
Food Biotechnol.
10:149-161.
|
| 17.
|
Jung, S.,
M. de Lamballerie-Anton,
P. Courcoux, and M. Ghoul.
1998.
High pressure improvement of the meat aging enzymes activity, p. 295-303.
In
N. S. Isaacs (ed.), High pressure food science, bioscience and chemistry Royal Society of Chemistry, Cambridge, United Kingdom.
|
| 18.
|
Kalchayanand, N.,
A. Sikes,
C. P. Dunne, and B. Ray.
1998.
Factors influencing death and injury of foodborne pathogens by hydrostatic pressure-pasteurization.
Food Microbiol.
15:207-214[CrossRef].
|
| 19.
|
Kalchayanand, N.,
T. Sikes,
C. P. Dunne, and B. Ray.
1994.
Hydrostatic pressure and electroporation have increased bactericidal efficiency in combination with bacteriocins.
Appl. Environ. Microbiol.
60:4174-4177[Abstract/Free Full Text].
|
| 20.
|
Kanbe, C., and K. Uchida.
1987.
NADH dehydrogenase activity of Pediococcus halophilus as a factor determining its reducing force.
Agric. Biol. Chem.
51:507-514.
|
| 21.
|
Linton, M.,
J. M. J. McClements, and M. F. Patterson.
1999.
Survival of Escherichia coli O157:H7 during storage in pressure-treated orange juice.
J. Food Prot.
62:1038-1040[Medline].
|
| 22.
|
Macnaughtan, W., and A. G. Macdonald.
1982.
Effects of pressure and pressure antagonists on the growth and membrane-bound ATP-ase of Acholeplasma laidlawii B.
Comp. Biochem. Physiol. A
72:405-414[Medline].
|
| 23.
|
Marquis, R. E.
1984.
Reversible actions of hydrostatic pressure and compressed gases on microorganisms, p. 273-302.
In
A. Hurst, and A. Nasim (ed.), Repairable lesions in microorganisms. Academic Press, Inc., New York, N.Y.
|
| 24.
|
Marty-Teysset, F.,
F. de la Torre, and J. R. Garel.
2000.
Increased production of hydrogen peroxide by Lactobacillus delbrueckii subsp. bulgaricus upon aeration: involvement of an NADH oxidase in oxidative stress.
Appl. Environ. Microbiol.
66:262-267[Abstract/Free Full Text].
|
| 25.
|
Niven, G. W.,
C. A. Miles, and B. M. Mackey.
1999.
The effects of hydrostatic pressure on ribosome conformation in Escherichia coli: an in vivo study using differential scanning calorimetry.
Microbiology
145:419-425[Abstract/Free Full Text].
|
| 26.
|
Patterson, M. F.,
M. Quinn,
R. Simpson, and A. Gilmour.
1995.
Sensitivity of vegetative pathogens to high hydrostatic pressure treatment in phosphate-buffered saline and foods.
J. Food Prot.
58:524-529.
|
| 27.
|
Perrier-Cornet, J.-M.,
M. Hayert, and P. Gervais.
1999.
Yeast cell mortality related to a high-pressure shift: occurrence of cell membrane permeabilization.
J. Appl. Microbiol.
87:1-7[CrossRef][Medline].
|
| 28.
|
Raso, J.,
G. Barbosa-Canovas, and B. G. Swanson.
1998.
Sporulation temperature affects initiation of germination and inactivation by high hydrostatic pressure of Bacillus cereus.
J. Appl. Microbiol.
85:17-24[CrossRef][Medline].
|
| 29.
|
Ritz, M.,
M. R. P. Pilet,
J. L. Tholozan, and M. Federighi.
1999.
High hydrostatic pressure effects on Salmonella typhimurium. Physiological and morphological damages, p. 295-298.
In
A. C. J. Tuijtelaars, R. A. Samson, F. M. Rombouts, and S. Notermans (ed.), Food microbiology and food safety into the next millennium. Foundation for Food Microbiology, Zeist, The Netherlands.
|
| 30.
|
Sakamoto, M., and K. Komagata.
1996.
Aerobic growth of and activities of NADH oxidase and NADH peroxidase in lactic acid bacteria.
J. Ferment. Bioeng.
82:210-216[CrossRef].
|
| 31.
|
Sami, M.,
H. Yamashita,
H. Kadokura,
K. Kitamoto,
K. Yoda, and M. Yamasaki.
1997.
A new and rapid method for determination of beer-spoilage ability of lactobacilli.
J. Am. Soc. Brew. Chem.
55:137-140.
|
| 32.
|
Sami, M.,
H. Yamashita,
T. Hirono,
H. Kadokura,
K. Kitamoto,
K. Yoda, and M. Yamasaki.
1997.
Hop-resistant Lactobacillus brevis contains a novel plasmid harboring a multidrug resistance-like gene.
J. Ferment. Bioeng.
84:1-6.
|
| 33.
|
Sami, M.,
K. Suzuki,
K. Sakamoto,
H. Kadokura,
K. Kitamoto, and K. Yoda.
1998.
A plasmid pRH45 of Lactobacillus brevis confers hop resistance.
J. Gen. Appl. Microbiol.
44:361-363.
|
| 34.
|
Silvius, J. R., and R. N. McElhaney.
1980.
Membrane lipid physical state and modulation of the Na+, Mg2+-ATP-ase activity in Acholeplasma laidlawii B.
Proc. Natl. Acad. Sci. USA
77:1255-1259[Abstract/Free Full Text].
|
| 35.
|
Simpson, W. J., and A. R. W. Smith.
1992.
Factors affecting antibacterial activity of hop compounds and their derivatives.
J. Appl. Bacteriol.
72:327-334[Medline].
|
| 36.
|
Simpson, W. J., and J. L. Fernandez.
1994.
Mechanism of resistance of lactic acid bacteria to trans-isohumulone.
J. Am. Soc. Brew. Chem.
52:9-11.
|
| 37.
|
Smart, J. B., and T. D. Thomas.
1987.
Effect of oxygen on lactose metabolism in lactic streptococci.
Appl. Environ. Microbiol.
53:533-541[Abstract/Free Full Text].
|
| 38.
|
Sonoike, K.,
T. Setoyama,
Y. Kuma, and S. Kobayashi.
1992.
Effect of pressure and temperature on the death rates of Lactobacillus casei and Escherichia coli, p. 297-301.
In
C. Balny, R. Hayashi, K. Heremans, and P. Masson (ed.), High pressure and biotechnology. 1992 Colloque INSERM/John Libbey EurotextMontrouge, France.
|
| 39.
|
Stolz, P.,
R. F. Vogel, and W. P. Hammes.
1995.
Utilization of electron acceptors by lactobacilli isolated from sourdough.
Z. Lebensm.-Unters.-Forsch.
201:402-410.
|
| 40.
|
Teixeira, P.,
H. Castro, and R. Kirby.
1995.
Spray drying as a method for preparing concentrated cultures of Lactobacillus bulgaricus.
J. Appl. Bacteriol.
78:456-462.
|
| 41.
|
Teixeira, P.,
H. Castro,
C. Mohacsi-Farkas, and R. Kirby.
1997.
Identification of sites of injury in Lactobacillus bulgaricus during heat stress.
J. Appl. Microbiol.
83:219-226[CrossRef][Medline].
|
| 42.
|
ter Steeg, P. F.,
J. C. Hellemons, and A. E. Kok.
1999.
Synergistic actions of nisin, sublethal ultrahigh pressure, and reduced temperature on bacteria and yeast.
Appl. Environ. Microbiol.
65:4148-4154[Abstract/Free Full Text].
|
| 43.
|
Thom, S. M.,
R. W. Horobin,
E. Seidler, and M. R. Barer.
1993.
Factors affecting the selection and use of tetrazolium salts as cytochemical indicators of microbial viability and activity.
J. Appl. Bacteriol.
74:433-443[Medline].
|
| 44.
|
van Veen, H. W., and W. N. Konings.
1998.
The ABC family of multidrug transporters in microorganisms.
Biochim. Biophys. Acta
1365:31-36[Medline].
|
| 45.
|
Walsh, S.,
H. M. Lappin-Scott,
H. Stockdale, and B. N. Herbert.
1995.
An assessment of the metabolic activity of starved and vegetative bacteria using two redox dyes.
J. Microbiol. Methods
24:1-9.
|
| 46.
|
Wouters, P. C.,
E. Glaasker, and J. P. P. M. Smelt.
1998.
Effects of high pressure on inactivation kinetics and events related to proton efflux in Lactobacillus plantarum.
Appl. Environ. Microbiol.
64:509-514[Abstract/Free Full Text].
|
| 47.
|
Yano, Y.,
A. Nakayama,
K. Ishihara, and H. Saito.
1998.
Adaptive changes in membrane lipids of barophilic bacteria in response to changes in growth pressure.
Appl. Environ. Microbiol.
64:479-485[Abstract/Free Full Text].
|
| 48.
|
Zook, C. D.,
M. E. Parish,
R. J. Braddock, and M. O. Balaban.
1999.
High pressure inactivation kinetics of Saccharomyces cerevisiae ascospores in orange and apple juices.
J. Food Sci.
64:533-535[CrossRef].
|
Applied and Environmental Microbiology, September 2000, p. 3966-3973, Vol. 66, No. 9
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Somolinos, M., Garcia, D., Pagan, R., Mackey, B.
(2008). Relationship between Sublethal Injury and Microbial Inactivation by the Combination of High Hydrostatic Pressure and Citral or tert-Butyl Hydroquinone. Appl. Environ. Microbiol.
74: 7570-7577
[Abstract]
[Full Text]
-
Bowman, J. P., Bittencourt, C. R., Ross, T.
(2008). Differential gene expression of Listeria monocytogenes during high hydrostatic pressure processing. Microbiology
154: 462-475
[Abstract]
[Full Text]
-
Moussa, M., Perrier-Cornet, J.-M., Gervais, P.
(2007). Damage in Escherichia coli Cells Treated with a Combination of High Hydrostatic Pressure and Subzero Temperature. Appl. Environ. Microbiol.
73: 6508-6518
[Abstract]
[Full Text]
-
Behr, J., Ganzle, M. G., Vogel, R. F.
(2006). Characterization of a Highly Hop-Resistant Lactobacillus brevis Strain Lacking Hop Transport.. Appl. Environ. Microbiol.
72: 6483-6492
[Abstract]
[Full Text]
-
Margosch, D., Ganzle, M. G., Ehrmann, M. A., Vogel, R. F.
(2004). Pressure Inactivation of Bacillus Endospores. Appl. Environ. Microbiol.
70: 7321-7328
[Abstract]
[Full Text]
-
Molina-Hoppner, A., Doster, W., Vogel, R. F., Ganzle, M. G.
(2004). Protective Effect of Sucrose and Sodium Chloride for Lactococcus lactis during Sublethal and Lethal High-Pressure Treatments. Appl. Environ. Microbiol.
70: 2013-2020
[Abstract]
[Full Text]
-
Molina-Gutierrez, A., Stippl, V., Delgado, A., Ganzle, M. G., Vogel, R. F.
(2002). In Situ Determination of the Intracellular pH of Lactococcus lactis and Lactobacillus plantarum during Pressure Treatment. Appl. Environ. Microbiol.
68: 4399-4406
[Abstract]
[Full Text]
-
Ulmer, H. M., Herberhold, H., Fahsel, S., Ganzle, M. G., Winter, R., Vogel, R. F.
(2002). Effects of Pressure-Induced Membrane Phase Transitions on Inactivation of HorA, an ATP-Dependent Multidrug Resistance Transporter, in Lactobacillus plantarum. Appl. Environ. Microbiol.
68: 1088-1095
[Abstract]
[Full Text]
Top
Abstract
Introduction
