40

Journal of Food Protection, Vol. 62, No. 1, 1999, Pages 40–45

Copyright Q, International Association of Milk, Food and Environmental Sanitarians

High-Pressure Destruction Kinetics of

Listeria monocytogenes on Pork
D. M. MUSSA, H. S. RAMASWAMY,* AND J. P. SMITH

Department of Food Science and Agricultural Chemistry, Macdonald Campus, McGill University, 21,111 Lakeshore Road,
Ste. Anne-de-Bellevue, Quebec, H9X-3V9, Canada

MS 98-66: Received 12 March 1998/Accepted 31 July 1998

ABSTRACT
Packaged fresh pork chops (30-g samples) containing an indigenous bacterial population of ;107 CFU/g were inoculated
with 107 CFU of Listeria monocytogenes Scott A per g, heat sealed, and subjected to high-pressure processing at 200 to 400
MPa for up to 90 min. Total counts and the number of surviving L. monocytogenes cells were determined by a spread plate
technique on tryptic soy agar and modified Oxford medium, respectively. The pressure destruction was characterized by a
dual-behavior, consisting of a step change in the number of survivors (Pko) with the application of a pressure pulse and a first-
order rate drop in the number of survivors during the pressure hold period. Higher pressures resulted in higher rates of microbial
inactivation, as indicated by their associated lower D values (and higher k values). The pressure sensitivities of the kinetic
parameters were evaluated on the basis of Arrhenius and pressure death time (PDT)-type models. The results suggested that
L. monocytogenes was more resistant to pressure inactivation than the indigenous microflora (the volume change of activation,
DV± [Arrhenius model]), and zp values (PDT model) were 24.17 3 1025 m3 mole21 and 134 MPa for indigenous microflora
and 23.43 3 1025 m3 mole21 and 163 MPa for L. monocytogenes respectively.

The main advantages of the application of high pres-
sure (HP) to food systems include uniform transmission of
pressure regardless of the size and shape of the samples
during processing and the possibilities for low-temperature
treatments that result in high-quality products. Normally,
HP technology utilizes water compressed by an air-driven
pump that transmits the pressure into foods packaged in
flexible containers; there is no waste generated and the pro-
cess is therefore environmentally friendly (12). However, it
has taken almost a century to overcome the technical chal-
lenges related to commercial application of HP technology
and for the high-pressure-processed food products to appear
on the market since the concept was first tested by Hite in
1899 (8). The first HP-processed commercial product was
fruit jam which was introduced by the Meidi-ya company
in Japan in 1990 (3). Since then, several other products
have been introduced in the Japanese market. The majority
of these products are high-acid foods such as fruit juices,
fruit jams and jellies, fruit yogurt, salad dressing, and wine.
Meat was one of the first food products that was tested
in the early studies by Hite (8). In those studies, HP pro-
cessing was found to be beneficial for extending the shelf
life of meat through inactivation of microorganisms. Sub-
sequent studies have focused on the application of high
pressure as an alternative method for accelerated meat con-
ditioning (2, 13). These latter studies suggested that high-
pressure technology should be limited to prerigor meat,
which perhaps limited the scope of HP research in this area.
Recent studies, however, have shown that high-pressure
* Author for correspondence. Tel: 514-398-7919; Fax: 514-398-7977;
E-mail: ramaswamy@agradm.lan.mcgill.ca.
treatment can improve the textural properties of both pre-
and postrigor muscle (10, 16). Although there is still no
practical commercial application of HP technology in the
meat industry, the Japanese companies Fuji Chiku and Mut-
terham have introduced HP-processed ham, and Yaizu Fish-
eries Company, Japan, has introduced HP-processed fish
sausages to the market. These products are reported to have
been introduced mainly for market testing while commer-
cial approval is being sought (3). The acceptance of HP
processing technology in the food industry requires the as-
surance of safety of these processed products with respect
to pathogenic microorganisms (11). Listeria monocytogenes
is one of the pathogens of serious concern to meat proces-
sors, consumers, and regulatory authorities, because it has
the ability to grow well at refrigeration temperatures and is
highly pathogenic to certain groups of individuals (6). As-
surance of the safety of HP technology would therefore be
impossible without properly designed and controlled pro-
cessing conditions to ensure complete destruction of this
pathogen. Reports on HP kinetic data on microorganisms
and quality attributes of foods are very limited in the lit-
erature. The unavailability of kinetic data and the lack of
understanding of mechanisms involved in high-pressure ef-
fects on food systems are among the scientific challenges
that need to be overcome before HP processing conditions
can be successfully established (12). The objective of this
study was, therefore, to obtain kinetic data on the patho-
genic microorganism Listeria monocytogenes Scott A on
pork in relation to the kinetics of the indigenous microflora.
MATERIALS AND METHODS
High-pressure equipment. The equipment used in this study
was an ABB Cold Isostatic Press, model CIP42260 (Autoclave
J. Food Prot., Vol. 62, No. 1 HIGH-PRESSURE DESTRUCTION OF L. MONOCYTOGENES ON PORK
TABLE 1. Pressure and time data points for HP processing of
pork
Processing parameters
Pressure (MPa) Time (min)
200 0 30 60 90
250 0 20 40 60
300 0 15 30 45
350 0 10 15 20
400 0 2 3 5
Engineers, Subsidiary of ABB Autoclave System, Columbus,
Ohio) with a cylindrical camber of 10 cm in diameter and 55 cm
in height for pressure treatment. The equipment was rated for
operation up to a maximum pressure of 414 MPa. Water contain-
ing 2% water-soluble oil (Autoclave Engineers, part no. 5019) was
used as the hydrostatic fluid in which packaged test samples were
submerged for the pressure treatment.
Preparation of the inoculum. Listeria monocytogenes Scott
A (human isolate) was obtained from the Microbial Hazards Bu-
reau (Health Protection Branch, Health and Welfare Canada, Ot-
tawa). The cultures were maintained on tryptic soy agar (TSA)
slants (Difco Laboratories, Detroit, Mich., USA) at 48C and were
transferred monthly to ensure viability. The inoculum was pre-
pared by transferring an isolated colony of L. monocytogenes Scott
A from TSA into 5 ml of tryptic soy broth with 0.6% yeast extract
(TSBYE) (Difco) and incubated at 308C for 24 h to give a working
suspension of 109 CFU/ml. Appropriate dilutions were made in
0.1% peptone broth (Difco) and inoculated to pork samples.
Pork sample preparation, inoculation, and processing.
Pork chops from the loin center cut (2 days postrigor) containing
an indigenous bacterial population of about 107 CFU/g were ob-
tained from Provigo market, Montreal, Quebec. The center cores
of the chops were cut into pieces weighing 30 g and were inoc-
ulated with 1.0 ml of the prepared culture of L. monocytogenes
Scott A to give a final listerial concentration of 107 CFU/g. The
inoculum was placed at four different locations on the meat sur-
face and the samples were heat sealed in Dual Peel sterilization
pouches (Baxter Corp., Missisauga, Ont.). The bags were then
given a gentle massage to distribute the test cells evenly on the
meat surface (although the pressure effect would be the same no
matter where the cells were located on the product.) The inocu-
lated samples as well as the uninoculated controls were subjected
to HP processing under the selected conditions detailed in Table
1. The pressure come up period during processing varied between
1 and 2 min, and the come down period was about 0.5 min. Un-
treated control samples were maintained at low temperature (48C)
while the experimental samples were being processed.
Survivor growth. Immediately following the pressure treat-
ment, the pouches were asceptically opened and the samples ho-
mogenized with 270 ml of 0.1% peptone water in a stomacher
(Model 400, A. J. Seeward, London, UK) for 2 min (4). All sub-
sequent dilutions were made from this 1021 dilution. L. monocy-
togenes cells were enumerated on a selective medium, modified
Oxford agar (MOX, Difco), by using a spread plate technique.
Total microbial count (which included listeria) was determined by
spread plating appropriate dilutions on a nonselective medium,
tryptic soy agar (TSA). All prepared plates were incubated at 308C
for 48 h before being counted.
41
Data analyses. To obtain the concentration of indigenous
microflora in test samples of the inoculated pork, L. monocyto-
genes Scott A counts obtained on MOX were subtracted from the
total count found on TSA. Data for L. monocytogenes Scott A
and indigenous microbial counts obtained for unpressurized sam-
ples were used as controls (containing approximately 107 CFU/
g). Small variations in the initial concentrations were unavoidable
since tests were carried out over several days, and therefore the
residual counts were normalized. A common procedure for nor-
malization is to express the surviving microbial cell numbers after
each treatment as a fraction of their initial counts or to multiply
the survivor fraction by their nominal initial counts (107 CFU/g,
found and used in this study).
The pressure inactivation of the microorganisms was ana-
lyzed on the basis of the dual-effect pressure inactivation behavior
reported by Basak and Ramaswamy (1) for the enzyme pectin
methyl esterase (PME) in orange juice. Those authors observed
that the pressure inactivation of PME followed an instantaneous
change in the concentration, which was the result of application
of an initial pressure pulse, followed by the conventional first-
order inactivation during the pressure hold time. This instanta-
neous change in activity was defined (1) as the instantaneous pres-
sure kill (IPK) value. These values were originally obtained from
the regression intercepts of the first-order rate hold time model
extended to time zero. In this study, a zero-hold time condition
was included in the experimental model (i.e., the samples were
pressurized until the desired pressure level was attained (;2 min)
and then immediately depressurized without any holding time).
The logarithmic change in the microbial concentration from a
pressure-pulse kill with zero holding time was designated Pko. Pko
values were also calculated as in the Basak and Ramaswamy (1)
approach by using the regression data.
The pressure inactivation kinetics of microorganisms during
the pressure hold time was analyzed assuming a first-order kinetic
process, expressed as
ln(CFU/CFU0) 5 2kt, [1]
where CFU refers to the number of viable cells in pressure-treated
samples, CFU0 is the number of survivors following Pko, t is time
in minutes and k is the reaction rate constant (min21). The k values
obtained were negative slopes of the plots of the linear regressions
of the natural logarithm of the normalized number of microbial
survivors versus time:
k 5 2[slope]. [2]
The D value or decimal reduction time (i.e., the time required
to reduce the number of survivors by 90%) was obtained as the
negative reciprocal slope of the semilogarithmic survivor curve
(or time taken to traverse one decimal log cycle). D is reciprocally
related to k:
D 5 2[1/slope] or 2.303/k. [3]
The pressure dependence of the kinetic parameters was analyzed
by two approaches, an Arrhenius-type model and the pressure
death time (PDT) models. In the Arrhenius-type model, the pres-
sure sensitivity of the rate constant was analyzed by plotting the
ln k values against the pressure. The volume change of activation
or DV± (a measure of the net effect of pressure on reactions caus-
ing the physiological change at constant temperature) was ob-
tained from the regression slope:
DV± 5 2RT(slope) or 2RT[D(ln k)/DP], [4]
where P is the pressure (MPa), reactants k is the rate constant
(min21), T is the absolute temperature (K), R is the gas constant
42 MUSSA ET AL.
FIGURE 1. Survivor curves of L. monocytogenes Scott A on pork as influenced by HP treatment.
(8.314 3 1026 m3 mole21 MPaK21), and DV± is the volume
change of activation (m3 mole21) (5).
In the pressure destruction time or PDT model, the pressure
sensitivity of D values was obtained from the plot of log D versus
pressure. The pressure z value zp (the pressure range between
which the decimal reduction time changes by a factor of 10) was
obtained as the negative reciprocal slope of the regression line:
zp 5 2[1/slope] or (P2 2 P1)/log(D1/D2) [5]
where P1 and P2 (MPa) are pressures corresponding to D1 and D2
(min).
RESULTS AND DISCUSSION
Residual survivor curves for the pressure destruction
of L. monocytogenes and the indigenous microflora of the
pork are shown in Figures 1 and 2, respectively. In both
cases, pressure destruction of the microorganisms was char-
acterized by dual behavior, with a first-order rate of destruc-
tion prevailing during the pressure hold time after a step
change in the number of survivors that was apparently the
result of a pressure pulse (i.e., pressurization and immediate
depressurization with no holding time). The experimental
Pko values as well as those predicted from the intercept
coefficients of hold time regression data as suggested by
Basak and Ramaswamy (1) and the kinetic parameters (rate
constant k and decimal reduction time D) obtained are sum-
marized in Table 2.
An increase in pressure resulted in an increase in Pko
for both types of microorganisms. However, L. monocyto-
genes appeared to be slightly more sensitive to pressure
pulse application than the indigenous microflora, as shown
by the marginally higher Pko values (Table 2). It is possible
J. Food Prot., Vol. 62, No. 1
to suggest why the instantaneous pressure kill is due to the
rapid pressurization-depressurization cycle. It is understood
that pressurization causes the compression of bacterial cell
material, the magnitude of compression being proportional
to the amount of applied pressure. A quick depressurization
of compressed microbial cells will cause an adiabatic ex-
pansion of the cytoplasmic material that may cause the de-
struction of the cell wall and therefore the death of the cell
(7). For bacterial spores, effective Pko values have been
observed at elevated temperatures of about 708C, which
were expected to soften the cell coat (7). This study was
carried out at room temperature, and a large number of
bacterial spores were not expected in the pork samples (6).
The Pko values observed may appear to be relatively small,
about 1 log-cycle reduction at 350 to 400 MPa for both
listeriae and the indigenous microflora. It is interesting to
note that the experimental values of Pko nearly matched the
predicted or calculated value of Pko from the regression of
hold time data. This also suggests that the influence of the
short come-up time (;2 min) on Pko is rather small.
Figures 1 and 2 show that higher pressure levels were
more effective in bringing about the destruction of the mi-
crobial cells, as is indicated by the increase in the slopes
of the curves as the pressure level was increased. However,
from the curves it is also apparent that holding time had a
significant effect in bringing about the microbial destruc-
tion. Different time and pressure combinations can there-
fore bring about same destructive pressure effect, i.e., lon-
ger holding periods at lower pressure or shorter holding
times at higher pressures (Figs. 1 and 2).
The kinetic parameters (rate constant k and decimal
J. Food Prot., Vol. 62, No. 1 HIGH-PRESSURE DESTRUCTION OF L. MONOCYTOGENES ON PORK 43

FIGURE 2. Survivor curves for indigenous microflora of pork inoculated with L. monocytogenes Scott A as influenced by HP treatment.

reduction time D) obtained from these curves were well the indigenous organisms of meats are primarily pressure-
described by the loglinear equation model with R2 . 0.96 sensitive gram-negative bacteria.
(Table 2). Pressure inactivation of microorganisms at a first- Pressure sensitivity of the reaction rate constants k and
order rate has been reported in previous studies involving D values modeled by the Arrhenius and PDT models are
various media, foods, and various types of microorganisms shown in Figures 3 and 4, respectively. Regression results
(14, 15, 17). For both L. monocytogenes and indigenous of these reaction rate constants, k and D, as a function of
microflora higher lethal effects were observed at higher pressure with the associated volume change of activation
pressures, as shown by higher k values and lower D values. (DV±) and pressure z values (zp) (equations 4 and 5) are
L. monocytogenes appeared to be more resistant to inacti- detailed in Table 3. High R2 values were observed with both
vation during the pressure hold period than the indigenous models, indicating their suitability in explaining the pres-
microorganisms of pork, as shown by lower k values and sure sensitivity of the kinetic parameters. For both micro-
higher D values at each pressure level, probably because organisms, a negative value of volume change of activation
(DV±) was obtained indicating that as the pressure level was
TABLE 2. Pko and kinetic parameters of L. monocytogenes Scott increased, the pressure destruction rate of these microor-
A and the indigenous microflora of the pressure-treated pork ganisms also increased. This trend conforms to the Braun-
Le Chatelier’s principle (9). Again, L. monocytogenes ap-
Pko (log)
k value peared to be more resistant to pressure change than the
Pressure Experi- (3 1022 D value indigenous microflora, as indicated by the less negative
(MPa) mental Predicted min21) (min) R2 DV± and higher zp values (Table 3).
Values for quantitative pressure destruction kinetics
Listeria monocytogenes data for different microorganisms are of limited availability
200 0.319 0.305 4.34 63.1 0.973 in the literature. Comparison of the results of this study
250 0.658 0.712 7.49 30.8 0.984
300 0.975 1.03 14.3 16.2 0.994
350 1.31 1.26 27.0 8.52 0.985 TABLE 3. Pressure z values (zp) and volume change of activation
400 1.52 1.61 65.2 3.52 0.968 (DV±) for L. monocytogenes Scott A and indigenous microflora
Indigenous microflora of the pressure-treated pork
200 0.271 0.000 5.39 42.7 0.986 DV± (3 1025
250 0.488 0.449 11.4 20.2 0.994 Type of microorganism zp (MPa) m3 mole21 R2
300 0.624 0.658 23.1 9.97 0.997
350 1.05 0.975 54.5 4.23 0.995 L. monocytogenes Scott A 163 23.43 0.988
400 1.21 1.20 178 1.29 0.983 Indigenous microflora 134 24.17 0.996
44 MUSSA ET AL. J. Food Prot., Vol. 62, No. 1

FIGURE 3. Arrhenius plots for L. monocytogenes Scott A and indigenous microflora on HP-treated pork.

with values in the literature is difficult, since the few avail-
able reports involved different processing conditions. For
example, Styles et al. (15) reported a D value of 9.3 min
for L. monocytogenes Scott A in raw milk treated at 340
MPa. On the other hand, Szczawinski et al. (17) reported
D values for L. monocytogenes in three types of ripened
cheeses, Edamski, Podlaski, and Gouda, processed at 350
MPa to be 9.32, 9.16, and 8.33 min, respectively. In this
study, a D value of 8.52 min at 350 MPa was found for L.
monocytogenes Scott A on pork chops (Table 2) which is
an excellent match with published results (15, 17). Other
pressure levels, however, resulted in larger deviations,
yielding different DV± and zp values. For example, Szcza-
winski et al. (17) found greater zp values for the indigenous
microflora in the three types of cheeses than for L. mono-
cytogenes. This reversal of sensitivity may be due to dif-

FIGURE 4. Decimal reduction time curves for L. monocytogenes Scott A and indigenous microflora on HP-treated pork.
J. Food Prot., Vol. 62, No. 1 HIGH-PRESSURE DESTRUCTION OF L. MONOCYTOGENES ON PORK
ferences in the makeup of indigenous microflora on cheese
and pork.
In general, the results of our study suggest a higher
pressure resistance for L. monocytogenes than for the in-
digenous microflora and therefore indicates that it may be
prudent to target listerial destruction for establishing pres-
sure processing procedures. Thus the gathered pressure de-
struction kinetic data for listeria would be more useful than
those for the natural microflora. However, it is too risky to
deal with L. monocytogenes in routine microbiological pro-
cedures because of its associated high pathogenicity. It is,
therefore, recommended that alternative indicators, such as
L. innocua or enzymes, be explored.
ACKNOWLEDGMENTS
This research was supported by the Natural Sciences and Engineer-
ing Research Council of Canada (NSERC) Strategic Grants Program and
CORPAQ.
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