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Appl Environ Microbiol, February 1998, p. 465-471, Vol. 64, No. 2
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Influence of Temperature and Pressure on the
Lethality of Ultrasound
J.
Raso,
R.
Pagán,
S.
Condón, and
F. J.
Sala*
Departamento P.A.C.A.
Tecnología de
los Alimentos, Facultad de Veterinaria, Universidad de Zaragoza,
50013 Zaragoza, Spain
Received 21 October 1996/Accepted 1 November 1997
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ABSTRACT |
A specially designed resistometer was constructed, and the lethal
effect on Yersinia enterocolitica of ultrasonic waves (UW) at different static pressures (manosonication [MS]) and of combined heat-UW under pressure treatments (manothermosonication [MTS]) was
investigated. During MS treatments at 30°C and 200 kPa, the increase
in the amplitude of UW of 20 kHz from 21 to 150 µm exponentially decreased decimal reduction time values (DMS)
from 4 to 0.37 min. When pressure was increased from 0 to 600 kPa at a
constant amplitude (150 µm) and temperature (30°C),
DMS values decreased from 1.52 to 0.20 min. The
magnitude of this decrease in DMS declined
progressively as pressure was increased. The influence of pressure on
DMS values was greater with increased amplitude
of UW. Pressure alone of as much as 600 kPa did not influence the heat
resistance of Y. enterocolitica
(D60 = 0.094; z = 5.65). At
temperatures of as much as 58°C, the lethality of UW under pressure
was greater than that of heat treatment alone at the same temperature.
At higher temperatures, this difference disappeared. Heat and UW under
pressure seemed to act independently. The lethality of MTS treatments
appeared to result from the added effects of UW under pressure and the lethal effect of heat. The individual contributions of heat and of UW
under pressure to the total lethal effect of MTS depended on
temperature. The inactivating effect of UW was not due to titanium particles eroded from the sonication horn. The addition to the MS media
of cysteamine did not increase the resistance of Y. enterocolitica to MS treatment. MS treatment caused cell
disruption.
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INTRODUCTION |
Heat can impair the organoleptic
properties and nutritional values of foods. To avoid the unwanted
effects of heat, efforts are being made to find new methods of food
preservation, either based on new inactivation procedures (22,
35) or by combining others already known (21, 28).
High pressures and high electric field pulses are among the most
intensively studied. The long-known inactivating effect of ultrasound
waves (UW) has drawn very little attention.
UW are generated by mechanical vibrations of frequencies higher than 15 kHz. When these waves propagate into liquid media, alternating
compression and expansion cycles are produced. During the expansion
cycle, high-intensity UW make small bubbles grow in a liquid. When they
attain a volume at which they can no longer absorb enough energy, they
implode violently. This phenomenon is known as cavitation. During
implosion, very high temperatures (approximately 5,500°C) and
pressures (approximately 50 MPa) are reached inside these bubbles
(31, 42, 43). In most authors' opinions (15,
38), this is the ultimate reason for the bactericidal effect of
high-intensity UW.
Some authors have proposed the use of UW in food preservation (2,
12, 16, 26, 44). However, the use of UW for this purpose has not
been adopted, probably because of the adverse effects on food quality
of such high-intensity treatments as those required to inactivate the
most resistant bacterial species and spores (25). The
increase in lethality of UW by increasing static pressure
(manosonication [MS]) was not explored until the 1960s by Neppiras
and Hughes (32). Their investigation was carried out with
very weak acoustic fields, and, as a result, the effect observed was
very slight. More recently, the lethal effect of a combination of heat
with UW (thermoultrasonication) has been reported (17, 33).
The lethality of a combined treatment of heat and UW under pressure
(manothermosonication [MTS]) remains unexplored. The influence of the
amplitude on the lethality of high-intensity UW is also unknown.
The purpose of this investigation was to determine the lethality at
different temperatures of UW under pressure with the food-borne pathogen Yersinia enterocolitica as a model microorganism.
This objective required the design and construction of an instrument that allowed measurement by the multipoint method of the lethality of
these treatments. With this instrument, measurement and control of the
different influencing parameters (temperature, pressure, and amplitude
of UW) were possible.
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MATERIALS AND METHODS |
Bacterial culture and media.
Y. enterocolitica (ATCC
9610) was supplied by the Spanish Type Culture Collection (catalog no.
4315) and during this investigation was maintained on tryptic soy agar
slants (Biolife, Milan, Italy).
A 250-ml Erlenmeyer flask containing 50 ml of sterile tryptic soy broth
(Biolife) was inoculated to a final concentration of
106 cells/ml in the flask, with a 12-h broth subculture in
tryptic soy broth at 37°C (obtained from a single colony). This flask was incubated at 4°C under agitation (130 rpm) until the stationary phase of growth was reached (2 weeks) and was then stored for 1 month
in the same medium at 4°C, but without agitation. Cultures grown at
4°C exhibited no loss of viability or of resistance to heat or UW
during storage.
Heat, MS, and MTS treatments: the instrument.
Heat, MS, and
MTS treatments were carried out in a specially designed resistometer
(Fig. 1) built in our laboratory. This instrument was a modified version of the thermoresistometer TR-SC (7, 8).
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FIG. 1.
MTS resistometer. A, Main vessel; B, agitation motor; C,
cooling coil; D, fraction collector; E, pH meter; F, MTS resistometer
main unit; G, ultrasound generator; 1 and 3, top and bottom caps,
respectively; 2, pH electrode; 4, vacuum inlet; 5, pressure inlet; 6, pH electrode pressurized housing; 7, main agitation shaft; 8, temperature sensor; 9, heating element; 10, temperature sensor; 11, manometer; 12, automatic injection syringe; 13, sonication horn and
housing; 14, treatment chamber; 15, filling-emptying tube; 16, one-way
inverted sense valve; 17, solenoid sampling valve; 18, photocell
detector.
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This MTS resistometer, built of stainless steel and Teflon, consisted
of a main vessel (650-ml capacity) with removable top
and bottom caps
with o rings. Caps were held in place by stainless
steel rings screwed
onto the wall of the main vessel. The top
cap, as in the original
instrument, had a security pressure release
valve and connections to
vacuum and pressure. This cap also had
a pressurized housing to hold,
when needed, a pH electrode and
held an agitation shaft, a 1,200-W
heating element, and a temperature
sensor.
The bottom cap had a filling and emptying tube with a valve and a
cooling coil to dissipate heat generated by ultrasound.
A small
treatment chamber (23 ml) was screwed onto this cap. This
chamber also
had a small agitation shaft and blade, coupled to
the agitation shaft
of the main vessel through a Teflon friction
bearing, to prevent any
leakage to or from the main vessel. This
agitation device ensured a
quick and homogeneous distribution
of the inoculum during the
experiment. This small treatment chamber
was connected to a capillary
sampling tube that had a solenoid
sampling valve activated by a timer.
This chamber also had two
one-way inverted sense valves in the top and
three hermetical
ports with o rings in the wall to hold a temperature
sensor, a
tube connected to a manometer, and an automatic injection
syringe
holder. One of the valves allowed the filling of the chamber by
vacuum through the sampling tube before the experiment started,
and the
other allowed refilling it with menstruum from the main
vessel after
every sample extraction.
The bottom of the chamber was reached by the tip of the sonication horn
of an ultrasound generator held in place by a housing
screwed on to the
bottom cap of the main vessel. The remaining
elements of the MTS
resistometer were as described in the original
thermoresistometer TR-SC
(
7,
8).
Pressure during the experiments was supplied by a nitrogen cylinder and
was monitored by the manometer of the treatment chamber.
Temperature control during ultrasonication experiments was achieved by
dissipating excess heat evolved during ultrasonication
by circulating
cold tapwater through the cooling coil.
As in the original instrument, during heat treatments, samples could be
taken manually (as many as 30 samples/min) or automatically
(as many as
32 samples/s) with a fraction collector. To compensate
for the decrease
in pressure in the treatment chamber during automatic
sampling, the
fraction collector was fitted with a photocell detector
(OMRON E32;
Tokyo, Japan) (
18) that opened the solenoid sampling
valve
only during the preset time (0.1 s) every time a tube passing
under the
sampling tip was detected. During MTS treatments, the
maximum sampling
speed used was five samples/s.
Ultrasound generators.
In this investigation, two Branson
Sonifier ultrasound generators (Branson Ultrasonics, Danbury, Conn.)
with maximum outputs of 450 and 2,000 W were used. These
constant-frequency instruments (20 kHz) keep the amplitude constant by
automatically supplying the amount of power needed to maintain the set
amplitude. The higher the pressure applied to the horn, the greater the
power required to maintain a given amplitude. The maximum intensity that a given generator is capable of delivering depends on its maximum
output. The 2,000-W instrument was used to widen the operational range.
No statistically significant differences (P 
0.05)
were observed between the DMS values (where
D is decimal + reduction time) obtained with either
instrument under the same operational parameters.
Heat, MS, and MTS resistance measurements: the method.
The
general handling procedure was as already described for the
thermoresistometer TR-SC (7, 8). Once temperature and pressure (and amplitude of UW in MS and MTS treatments) had attained stability, 0.2 ml of the Y. enterocolitica suspension was
injected into the treatment chamber containing citrate-phosphate buffer (pH 7) (13). At preset intervals, one 0.1-ml sample for each treatment time was directly collected into tubes of melted sterile nutrient agar (Biolife) with 500 mg of Bacto-Dextrose (Difco, Detroit,
Mich.) per liter. These tubes were immediately plated. Survival curves
(obtained by plotting the log of the number of survivors versus time of
treatment) were plotted with 8 to 15 separate samples collected over
time.
Incubation of heated samples and survival counting.
Colonies
were counted after incubation at 37°C for 48 h. Previous
experiments showed that longer incubation times did not influence the
slope of survival curves. After incubation, the plates were counted
with an Image Analyzer Automatic Counter (Protos; Analytical Measuring
Systems, Cambridge, United Kingdom) fitted with a 70-mm objective to
facilitate the count of plates with high numbers of CFU (as much as
3 × 104 CFU per plate) (9).
Microscopic observations.
Microscopic counts and
observations of the effects of heat, MS, and MTS treatments were
carried out with a Nikon (Nippon Kogaku KK, Tokyo, Japan) microscope
and Thoma counting chamber.
Heat, MS, and MTS resistance parameters.
The lethality of
treatments, single or combined, was measured by D values,
which were defined as minutes of a given treatment for the number of
survivors to decrease one log cycle (DT,
DMS, and DMTS, values for
heat, MS, and MTS, respectively). D values were calculated
from the slope of the regression line plotted with the counts of the
straight portion of the survival curve. Only survival curves with a
correlation coefficient (r0) of 0.98 and with
more than four values in the straight portion were used. Survival
curves with a straight portion including less than one log cycle were
rejected.
Decimal reduction time curves (DRTC) were obtained by plotting
D values versus their corresponding heating temperatures.
z values (measured as the increase in temperature [in
degrees Celsius]
for the
DT value to decrease
by one log cycle) were calculated
from the slope of DRTC.
r0s and 95% confidence limits (CL) were
calculated by the appropriate statistical package (StatView SE plus
Graphics; Abacus
Concepts Inc., Berkeley, Calif.). The coefficients of
variation
(CV%) and the statistical significance of differences
(
P 0.05)
among
DT,
DMS, and
DMTS values were
calculated and checked, respectively,
as described by Steel and Torrie
(
40).
The individual contributions of heat and of UW to the lethal effect of
MTS treatments at different temperatures were evaluated
by the degree
to which experimental
DMTS values matched the
theoretical
DRTC.
DMTS values used to plot
theoretical DRTC were calculated
by assuming that heat and UW acted
independently and that heat,
MS, and MTS destruction of
Y. enterocolitica were single reactions
ruled by first-order
kinetics. In this way the logarithmic order
of death of microorganisms
would be expressed by the following
equations: log
Nt = log
N0 
(1/
DT) ×
t for heat treatments, log
Nt = log
N0 (1/
DMS) ×
t for MS treatments, and
log
Nt = log
N0 (1/
DMTS) ×
t for MTS treatments,
where
Nt is the number of
surviving cells after
t minutes of treatment,
N0 is the
number
of living cells at
t = 0, and
DT,
DMS, and
DMTS are the corresponding
decimal reduction
times.
If heat and UW acted independently, MTS inactivation rates could be
calculated by adding the inactivation rate of UW under
pressure to the
heat inactivation rate by the following equations:
1/
DMTS = 1/
DT + 1/
DMS and
DMTS = (
DT ×
DMS)/(
DT +
DMS).
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RESULTS |
Performance of the MTS resistometer.
The temperature stability
of the MTS resistometer instrument during heat treatments was
±0.05°C and during experiments including UW was ±0.2°C. The
heating-up rate was 10°C/min. In the range of 40 to 140°C, the
instrument stabilized to a temperature 10°C higher in approximately 3 min.
The alternative compression and decompression cycles generated by UW
might force the contents of the treatment chamber to
leak out to the
main vessel. An experiment was carried out to
evaluate the significance
for the results of possible leaks occurring
during an MS treatment at
the highest amplitude (250 kPa, 30°C,
and 150 µm of amplitude).
Since microorganisms were unsuitable
for this purpose, the detection of
leaks was performed with radioactive
[
14C]ascorbic acid.
After 0.2 ml of a concentrated solution of radioactive
ascorbate had
been injected into the treatment chamber, one 0.1-ml
sample per each
treatment time was extracted. These samples were
counted in a liquid
scintillation counter (LKB-Wallac; 1211ß)
as described elsewhere
(
34). The results of this experiment
are shown in Fig.
2. As seen in this figure, radioactivity
(in
counts per minute) decreased exponentially with time (
t)
according
to the equation log CPM/ml = 5.3
0.036
t.
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FIG. 2.
Concentration of [14C]ascorbic acid (in
counts per minute per milliliter) in the treatment chamber during MS
treatment (250 kPa, 30°C, 150 µm of amplitude, 20 kHz).
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The regression line in Fig.
2 had an
r0 of 0.98, and the 95% CL of the slope ranged from
0.030 to
0.042. According
to these
results, the expected leak to the main vessel was evaluated to
be approximately 10% of the contents of the treatment chamber/minute
of treatment.
The lowest
D value measurable by a mixing method
(
3) is limited by the time needed for the inoculum to attain
a homogeneous
distribution and also by its sampling speed. Time
required for
a homogeneous distribution was determined in three
consecutive
experiments by the procedure already described
(
8). The homogeneous
distribution of inoculum was attained
in less than 0.2 s (data
not shown).
During MTS experiments, to keep the treatment parameters stable during
automatic sampling and to minimize the dilution effect
caused by
menstruum from the main vessel replenishing the chamber
after every
sample extraction, a photocell tube detector was installed.
This device
opened the sampling valve during a preset time (0.1
s) when a tube was
detected. To check the performance of this
sampling device, 0.2 ml of a
suspension of
Y. enterocolitica was
automatically injected
into the menstruum at room temperature.
After injection, one 0.1-ml
sample per treatment time was automatically
collected. Counts obtained
in two successive experiments of 2-s
duration with a sampling speed of
five tubes/s had a CV of 1%
(data not shown). This variation is
considered normal in microbiological
counts (
24). Therefore,
this sampling method should not influence
precision of results.
Figure
3 shows reproducibility obtained
in five consecutive MS treatments at room temperature, at 200 kPa, and
at 117 µm of
amplitude. The CV% of
D values obtained in
these experiments was
7%.
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FIG. 3.
Survival curves of Y. enterocolitica
corresponding to five different experiments under the same experimental
conditions (200 kPa, 30°C, 150 µm of amplitude, 20 kHz).
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Lethal effect of heat, MS, and MTS treatments.
Our strain of
Y. enterocolitica had a D59 of 0.138 min (95% CL ranged from 0.122 to 0.154) and a z value of
5.65°C (95% CL ranged from 5.32 to 6.02) as calculated from the
corresponding DRTC (see below). Pressure (to as much as 600 kPa) did
not influence heat resistance (data not shown).
At ambient temperature and pressure, the
D value of
Y. enterocolitica corresponding to an ultrasonication treatment at
the
highest amplitude setting (150 µm) was 1.5 min.
Figure
4A shows the survival curves of
Y. enterocolitica corresponding to MS treatments of
different amplitudes, at 200 kPa
and 30°C. In panel B, the
relationship between amplitude and log
D value is shown. The
lethality of these treatments increased
exponentially with amplitude
(
A) according to a regression line
(
r0 = 0.98; 95% CL of the slope ranged from
0.01 to
0.006) that
followed the equation log
DMS = 0.806
0.008
A.
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FIG. 4.
Influence of amplitude of ultrasonication on the
lethality of MS treatment. (A) Survival curves of Y. enterocolitica corresponding to MS treatments (200 kPa, 30°C, 20 kHz) at the following amplitudes of ultrasonication: 21 ( ), 63 ( ), 76 ( ), 117 ( ), and 150 ( ) µm. (B) Relationship
between amplitude of ultrasonication and lethality of MS treatments
(log D).
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The influence of pressure on the lethality of UW of 117 and 150 µm of
amplitude is shown in Fig.
5. At 150 µm
of amplitude,
an increase in pressure from 0 to 300 kPa reduced the
DMS from
1.5 to 0.28 min. A further increase
from 300 to 600 kPa reduced
DMS only from 0.28 to 0.20 min. The relationship between amplitude
and pressure is also
illustrated in this figure. While at 100
kPa the increase of amplitude
from 117 to 150 µm reduced
DMS from
0.95 to
0.77 min, at 300 kPa this increase reduced
DMS
from 0.60
to 0.28 min.
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FIG. 5.
Relationship between pressure and lethality of MS
treatments (log D) at the following amplitudes: 117 ( )
and 150 () µm.
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The lethality of UW of 117 µm of amplitude at 200 kPa but at
different temperatures was investigated. The results of these
experiments are shown in Fig.
6, which
shows the DRTC corresponding
to heat treatments as well as
DMTS values at different temperatures.
The
theoretical DRTC corresponding to MTS treatment has been included
(dotted line) to illustrate the discussion. This theoretical DRTC
was
calculated as described in Materials and Methods.
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FIG. 6.
Influence of temperature on the lethality of MS
treatment. Experimental values were for heat () and MS () (117 µm, 200 kPa, and 20 kHz) treatments. Curved line, theorical
inactivation curve obtained by the formula DMTS = (DT × DMS)/(DT + DMS).
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Inactivation mechanisms of MS and MTS treatments.
The ultimate
reason for the lethal effect on microorganisms of high-intensity UW is
thought to be the very high temperatures and pressures developed
(15, 38) and/or free radicals released into the medium
(20, 36) by cavitation. Although titanium is supposed to be
a very stable material, titanium particles eroded from the tip of the
horn during ultrasonication treatments might also somehow contribute to
the lethal effect of MS and MTS treatments.
Several experiments were performed to explore the inactivation
mechanisms of MS and MTS treatments. In the first one, an aliquot
of a
Y. enterocolitica cell suspension was kept for 15 min in
a
medium that previously had been intensely MS treated for 10
min (200 kPa, 117 µm). At preset intervals, samples of menstruum
were
extracted to assess the number of survivors. No lethal effect
from the
presence of titanium particles in this medium was observed.
This
experiment did not rule out the possible contribution to
lethality of
short-lived free radicals. In another experiment,
the lethality of an
MS treatment with a medium containing different
concentrations (2, 50, and 100 mM) of the free radical scavenger
cysteamine was measured.
Cysteamine did not increase
D values
(
P 0.05) and did not influence the profile of survival curves
(data not
shown).
The cell suspension was given the following three different treatments:
one heat treatment at 63°C for 7 s; one MS treatment
at 30°C,
200 kPa, and 117 µm of amplitude during 3 min; and one
MTS treatment
at 63°C, 200 kPa, and 117 µm of amplitude for 7
s. A
microscopic observation of suspensions was carried out after
each
treatment to try to establish a relationship between percentages
of
disrupted and live cells. Percentages of disrupted cells were
calculated by measuring the difference between the number of
undisrupted
cells before and after treatment. The percentage of
inactivated
cells in all three treatments was very high (>99%). This
percentage
was calculated by the difference between plate counts before
and
after each treatment. The percentage of disrupted cells was
different
in each treatment. No disruption was observed after heat
treatment.
After MS treatment, no undisrupted cell could be seen. After
MTS
treatment, approximately 80% of cells were undisrupted.
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DISCUSSION |
Performance of the instrument.
The temperature stability of
this instrument in heat resistance experiments was similar to the best
reported result for other heat resistance determination methods
(5, 41). In MS and MTS experiments, stability was slightly
less since it required manual adjustment of the flow of cold water
through the cooling coil. However, it was still better than that of
other heat resistance determination methods (18, 23).
DT values at successively higher temperatures
can be obtained by reinoculating the same menstruum. Since
stabilization to
a higher temperature is fast, the DRTC could be
obtained in the
same working session. By this procedure, the possible
influence
of uncontrolled factors was avoided. The temperature probe
and
the manometer of the treatment chamber allowed the monitoring
of
temperature and pressure during the experiments. The agitation
system
ensured a quick and homogeneous distribution of the inoculum
and a fast
heating-up rate and temperature stabilization.
During heat treatments under pressure, no influence of dilution and
possible leaks on the
DT values obtained was
observed.
No statistically significant differences (
P 0.05) were detected
between
DT values
measured with this instrument and the TR-SC
(
7,
8). Under
the most drastic ultrasonication conditions
used during this research,
the concentration of the contents of
the chamber decreased by 10% per
min (Fig.
2). Of course, the
significance of dilution plus leaks on the
accuracy of
D values
obtained would be greater for longer
experiments. The duration
of the experiment will be greater to the
extent that the resistance
of the microorganism involved and the number
of decimal reductions
of the survival curve to be obtained are higher.
Under the most
frequent ultrasonication conditions in this study (150 µm, 250
kPa), the maximum
D value measured was 0.77 min.
Therefore, the
largest expected error was a 10% underestimation of the
D value.
This error is within the precision range of most
heat resistance
determination methods (
11,
18,
29). Only
when much larger
D values are measured would a correction be
required.
In mixing methods, the menstruum at treatment temperatures was
inoculated with a microbial suspension. In these methods, the
lowest
measurable
D value was limited by the distribution time
of
the inoculum and by the sampling speed. With this instrument,
the
homogeneous distribution of the inoculum (0.2 ml) was obtained
in less
than 0.2 s. This distribution speed was even greater than
that of
the original TR-SC thermoresistometer (
8). This was
most
probably due to the much smaller size of the treatment chamber
(23 ml
instead of 350 ml).
The precision of
D values obtained with this instrument is
among the greatest reported by authors using different methods
(
11,
18,
29) as judged by the CV% of
D values of
the five
survival curves of Fig.
3 (7%) and the 95% CL of
D and
z values
shown in the figures.
This instrument proved to be a very versatile instrument to measure
with accuracy, by the multipoint method, the sensitivities
of
microorganisms to heat, UW, MS, and MTS treatments in a wide
range of
temperatures, pressures, and amplitudes. The small volume
of the
treatment chamber makes it especially suitable to deal
with small
volume samples or expensive compounds (some enzymes).
Lethal effect of heat, MS, and MTS treatments.
Survival curves
in this investigation sometimes had shoulders and tails. The rather
frequent appearance of shoulders and tails has prompted some authors to
question the logarithmic order of death and to propose alternative
mathematical models (6, 37). However, none of these models
has been widely accepted. Most authors still use the traditional
logarithmic death rate model. In this investigation, resistance to
heat, MS, and MTS treatments is shown as D and z
values.
The heat resistance of our strain was much less
(
DT/5
DT/8) than
that reported for
Y. enterocolitica by other authors, but
the
z value was similar (
27,
39). Perhaps our
DT values were
less because our strain had been
grown at a lower temperature.
It is known that, in most species, lower
growth temperatures lead
to lower
DT values
(
14,
19).
At ambient temperature and pressure, the lethal effect of UW on
microorganisms is small. This is probably why UW have not
been used as
a method for microbial inactivation. The
D value
of 1.5 min
obtained with our strain with UW at 150 µm is similar
to those
obtained for other gram-negative species such as
Pseudomonas aeruginosa (
38) and
Escherichia coli
(
1). By increasing static
pressure, the lethality of UW
increased drastically. At 30°C,
600 kPa, and the same amplitude (150 µm), the
D value was 0.22
min.
It has been reported that the energy of the UW decreases with the
distance from the emission source and increases with amplitude
(
10). However, there are no data in the literature on the
influence
of amplitude on the lethal effect of UW on microorganisms. As
shown by our results (Fig.
4), the resistance of
Y. enterocolitica decreased exponentially when the amplitude was
increased linearly.
Ten-micrometer increments in amplitude led to
steady decreases
of approximately 20% in
DMS.
The effect of pressure on the lethality of UW (MS) has been reported
(with yeasts) only with UW of low intensity (
32). The
mechanism by which pressure increases lethality is not known (
4,
30). As shown in Fig.
5, the lethality of UW of 117 and 150
µm
of amplitude increased with pressure. However, these increments
became
progressively smaller as the pressure increased, tending
to disappear.
No statistically significant differences (
P 0.05)
were found between
DMS values obtained at 400 and 600 kPa. Our
results are partly in agreement with those of the very
few investigations
reported in the literature (
4,
30). The
authors in these
investigations also reported an increase in the
physicochemical
effects of UW when pressure was increased. However,
after an optimum
pressure at which maximum effect was obtained, any
further pressure
increase led to a decrease in efficacy. We did not
observe the
occurrence of any decrease in our working range (of as much
as
600 kPa).
No reports are found in the literature on the interaction between
amplitude and pressure on the effects of UW. As shown in
Fig.
5, the
increase in lethality by increasing pressure (to as
much as 600 kPa)
was greater when the amplitude of UW was higher.
The individual contributions of heat and of UW under pressure (MS) to
the lethal effect of MTS can be deduced from Fig.
6.
As seen in this
figure, the
D value of
Y. enterocolitica
corresponding
to UW under pressure was the same (up to 50°C)
regardless of the
temperature of treatment. From this temperature to
58°C,
D values
decreased rapidly and were always smaller
than
D values for heat
treatment alone at the same
temperature. At temperatures higher
than 58°C,
D values
corresponding to heat and to MTS were equal.
The results of the experiments on the contribution to the lethal effect
of UW of free radicals and of titanium particles and
the microscopical
observation of suspensions after heat, MS, or
MTS treatments appeared
to indicate that the inactivation of MS
was due to one single
mechanism, the mechanical disruptions of
cells. This mechanism of
action has also been suggested by other
authors (
15,
31).
Unlike MS, the lethality of MTS treatments appeared to be due to two
different mechanisms acting independently, one of heat
and the other of
MS. The lethal effect of MTS treatments would
be the result of adding
the lethal effect of MS to the lethal
effect of heat. The inactivation
rate of each mechanism would
be determined by temperature. Therefore,
which one would prevail
would depend on the temperature of treatment.
Up to 50°C, the
D value of MS treatments was constant
(Fig.
6), because in this range the inactivation rate of UW under
pressure
was not influenced by temperature and because, at these
temperatures,
the lethality of heat would be negligible. The rapid
decrease
in
D value at temperatures higher than 50°C (MTS)
would be due
only to the exponential increase in the lethality of heat
by linear
increases in temperature. This exponential increase in the
lethality
of heat (while that of MS treatment remained constant) would
finally
make (at 58°C) the lethality of UW under pressure negligible.
At temperatures higher than 58°C,
D values of MTS and of
heat
would be equal. At these temperatures, the inactivating effect
would be solely due to heat.
The good match of
D values corresponding to MS-MTS
treatments with the theoretical DRTC (dotted line in Fig.
6) supports
this
hypothesis. The values of this curve were calculated by adding
the
lethal effect of MS treatment to that of heat. Furthermore,
microscopic
observations of cell disruption are also in agreement.
After an MS
treatment at 30°C that caused a three-log reduction
in the number of
survivors, all cells were disrupted. However,
an MTS treatment at
63°C that caused the same reduction in survivors
disrupted only 20%
of cells.
As shown by our results, static pressure is a very efficient means of
increasing lethality of UW (MS). This increase becomes
greater when the
amplitude of UW is higher. Between 50 and 58°C,
the lethality of heat
can be increased by combining heat treatments
with UW under pressure
(MS). The lethality of this treatment (MTS)
is equivalent to the
additive lethal effect of heat and UW. MS
and MTS treatments could
become an alternative for the inactivation,
in heat-sensitive media
(i.e., liquid egg), of
Y. enterocolitica and possibly other
microorganisms. It might also find applications
in foods in which the
high intensity of heat treatments required
(i.e., low-water-activity
foods) would impair food quality.
| |
ACKNOWLEDGMENTS |
This study was supported by the CICYT (Project ALI90-900) and by
the Ministerio Español de Educación y Ciencia which
provided J. Raso and R. Pagán with a grant to carry out this
investigation.
Our thanks to S. Kennelly and R. Levene for their collaboration in the
English correction of this work.
| |
FOOTNOTES |
*
Corresponding author. Mailing address:
Tecnología de los Alimentos, Facultad de Veterinaria,
Universidad de Zaragoza, C/ Miguel Servet 177, 50013 Zaragoza, Spain.
Phone: 76-761581. Fax: 76-761612. E-mail:
pacosala{at}mvet.unizar.es.
| |
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Abstract
Introduction
