HIGH PRESSUREHIGH TEMPERATURE STERILIZATION:

FROM KINETIC ANALYSIS TO PROCESS VERIFICATION*

TATIANA KOUTCHMA1, BIAO GUO, EDUARDO PATAZCA and
BRIAN PARISI
National Center for Food Safety and Technology
6502 South Archer Road
Summit-Argo, IL 60501-1933
Illinois Institute of Technology
Accepted for Publication September 20, 2005

ABSTRACT
The inactivation of Clostridium sporogenes PA 3679 spores by high
pressure at high temperatures (HPHT) in phosphate buffer was investigated
in a lab-scale temperature-controlled HP system (QFP-6) with an internal
heater to maintain the sample temperature. Some inactivation of spores
occurred during the pressurization come-up time (CUT) and depressurization
time. The inactivation of PA 3679 was found to be exponential during the
adiabatic holding period of the HP cycle at constant pressures and temperatures. The inactivation rate increased with both pressure and temperature. The
kinetic parameters such as D-values at tested temperatures and pressures
that are necessary for the design of process parameters of HP sterilization
process were determined. Within the pressure range of 600800 MPa, the
calculated D-values ranged from 270.3 to 357.4 and 49.0 to 67.6 s at 91 and
108C, respectively. These studies provided basic data on the effects of pressure
and temperature on the inactivation of PA 3679 spores under conditions
applicable to the development of preservation specifications for commercial
HPHT processing of low acid foods. The spore strips of C. sporogenes were
used as indicators for microbiological verification of delivered lethality of
HPHT sterilization process at different processing conditions in a pilot scale
HP vessel.

Corresponding author. TEL: (708) 563-8178; FAX: (708) 563-1873; EMAIL: koutchma@iit.edu
* Mention of trade names and commercial products in this article is solely for the purpose of providing
specific information and does not imply recommendation or endorsement by the National Center for
Food Safety and Technology.

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Journal of Food Process Engineering 28 (2005) 610629. All Rights Reserved.

Copyright 2005, Blackwell Publishing

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INTRODUCTION
High hydrostatic pressure (HP) is an alternative preservation technique
for thermal sterilization of low acid foods (LACF) with a shorter process time.
As defined by LACF regulations (21 CFR 113), in order to establish the
sterilization process, the commercial sterility must be demonstrated in terms of
spoilage-causing organisms and pathogens capable of growing under the conditions of storage and distribution. The LACF regulations mention commercial sterility or essentially no risk without defining the process. For
canning, the problems associated with spoilage, and the solutions to those
problems, have evolved over a long history. The classical heat-resistant, surrogate microorganisms spores of Geobacillus stearothermophilus were sufficiently characterized to design and validate the thermal sterilization process.
Along with G. stearothermophillus, Clostridium sporogenes, a typical heatresistant, mesophilic, spore-forming organism, is used in establishing thermal
process specifications. Spores of the strain PA 3679 were selected because
their heat resistance has been found to be equal to or greater than that of spores
of Clostridium botulinum. These bacterial spores known for their resistances to
heat and irradiation have also been shown to be resistant to pressure (Maggi
et al. 1996). Unless HP in excess of 800 MPa is used, heat combined with HP
is required for HP sterilization of LACF. The regulation that was not intended
for pressure-processed foods can be applicable to HP sterilization of LACF
because of a strong thermal component required for the spores destruction,
and HP processing has not had sufficient usage to demonstrate that pressure
does have an accelerating effect on the spores inactivation kinetics. Thus, for
pressure-sterilized LACF, the requirements in 21 CFR 113 must be fulfilled
before a product can enter commercial production.
The two paths to addressing the issues of spoilage and pathogens of
public health significance (as relating to commercial sterility) are used. First,
the establishment of process temperature, pressure and time for HPhigh
temperature (HT) sterilization processes requires knowledge of inactivation
kinetic parameters of target pathogenic and spoilage-causing spore-forming
bacteria. Second, the process outcome or performance criterion must be
defined as a prerequisite to further validation of the established sterilization
process. Historically the endpoint of sterilization processes has not been
clearly defined. The 12-decimal logs concept established by Stumbo (1973)
also does not establish an endpoint, but merely defines that the process should
accomplish a 12-decimal point reduction of C. botulinum. Pflug (1987) first
defined the thermal process as the probability of survival of either a spoilagecausing organism or pathogen and proposed an alternative approach to calculate the process lethality by describing the outcome. The last essential step in
establishing the process is microbiological validation in product samples to

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measure the delivered process by achieved log reductions and converting this
to a process value. Biological indicators (BI) such as spore strips of G.
stearothermophilus and C. sporogenes are widely used for sterility testing in
various sterilization environments. Because standard BIs are easy and convenient to use for a direct measurement of the lethality, they also can be used to
verify adequacy of the delivered process in HPHT sterilization to reaffirm the
efficiency of defined process in controlling the microbiological hazard to the
required level.
Most of the published data have dealt with the HP destruction of the
thermophilic spores of Geobacillus. Clostridium spores have been assumed
to behave in a similar manner to Geobacillus spores under HP. However,
recent studies on Clostridium spores had not confirmed this assumption. Gola
et al. (1996), evaluating the pressure sensitivity of C. sporogenes PA 3679
at 900 MPa for 10 min at 30C, were unable to completely destroy
8.4 102 cfu/mL in truffle cream. Mills et al. (1998) reported that spores of C.
sporogenes were resistant to pressure at 600 MPa for 30 min at 20C, showing
no significant inactivation. Maggi et al. (1996) demonstrated that 1500 MPa at
20C applied for 5 min had no effects on the spores. The pressure treatment
parameters had been examined by Rovere et al. (1996a) for inactivation of
spores of C. sporogenes PA 3679 starting with concentrations of approximately 105 cfu/mL and pressure-hold times of 5 min. Elimination of these
spore levels was possible with processes of 1400 and 800 MPa at 54 and 75C,
respectively, in different model food systems. In a study involving spore
suspensions of C. sporogenes PA 3679 in meat broth, the pressure acted as a
complementary synergistic process to allow reduction of the thermal processing parameters necessary to eliminate problematic spore-formers in foods.
They concluded that it was important to combine HP with HT during pressure
processing. Knowledge of the HP inactivation kinetics of C. sporogenes is
essential to design a safe sterilization process.
The objectives of this study were: (1) to measure the inactivation kinetics
of C. sporogenes PA 3679 spores by HP at elevated temperatures in phosphate
buffer for the design of HPHT sterilization process; and (2) to use spore strips
as indicators for microbiological verification of delivered lethality of HPHT
sterilization process at different processing conditions in a pilot scale HP
vessel.
MATERIALS AND METHODS
Microorganism
Five milliliters of commercially available C. sporogenes PA 3679 spores
(National Food Laboratories Inc., Dublin, CA) with a concentration of

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613

1.2 108 cfu/mL D at 121C of 0.8 min, Z-value of 7.8C was obtained. The
spores were divided in 1-mL aliquots in sterile screw-cap tube and kept frozen
at -20C during storage.
Spore Samples Preparation
Sterile sodium phosphate buffer (pH 7.0) was used to prepare 1:100
dilutions from the stock spore suspension to obtain an initial spore count of
approximately 106 cfu/mL. Two-milliliter aliquots of this spore preparation
were enclosed in a sterile high barrier Nylon-film pouch (CV Systems,
Downers Grove, IL) and sealed using a manual heat sealer. All prepared
samples were kept in ice water prior to HP treatment. One inoculated sample
was used per HPHT treatment.
Spore Enumeration
Two unprocessed samples were diluted and plated as a control for each
experiment in order to obtain the initial spore count. After HP treatment, the
pouches were immediately cooled in the water-ice container and kept there
before analysis. The samples were serially diluted in sodium phosphate buffer.
The spores counts with and without treatments were determined by pourplate enumeration in duplicate, using trypticase soy agar (TSA) plus 0.6%
yeast extract (YE) (Difco Laboratories, Detroit, MI). The plates were incubated anaerobically at 35C for 5 days before enumeration.
Spore Strip Testing
Geobacillus stearothermophilus spore strips ATCC 7953 (Raven Biological Laboratories Inc. Omaha, NE) and C. sporogenes ATCC 11437 spore strips
(NAMSA Laboratories, Northwood, OH) were used for testing. Spore strips
were in Schleicher and Schuell filter paper (3470), size 6.4 mm 38.1 mm
packaged in a peel-open glassine paper pouch. The initial population of the
strips was 1.0 106 cfu per strip.
Bromocresol purple (BCP) (Fisher Scientific, Chicago, IL) was used as a
growth indicator dye. The BCP dye is purple at neutral pH and changes color
to bright yellow at pH 5.2, and starting at pH 6.8 it gradually begins to lighten
toward a yellow color. A stock solution of 0.4% BCP (100 mL) was prepared
by adding 0.4 g of dehydrated BCP to 100 mL of deionized water. One milliliter of the BCP stock solution was added to 250-mL tryptic soy broth (TSB)
(Difco Laboratories). The resultant media was TSB with 0.002% BCP dye
(TSB/BCP). Nine milliliters of the TSB/BCP was transferred into test tubes
and autoclaved.

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Three spore strips were prepared by using sterile forceps to withdraw

them from their protective pouch. Each strip was placed within a tube with
TSB/BCP media. Three tubes from a batch of G. stearothermophilus were
incubated for 24 h at 55C. The tubes with C. sporogenes spore strips were
incubated anaerobically for 24 h at 35C. Anaerobic conditions were obtained
by using anaerobic jars and system envelopes (Gas Pak, Becton Dickinson,
Franklin Lakes, NJ) equipped with a palladium catalyst. The control unit
exhibits a color change toward yellow. If the control unit does not show signs
of growth, the test is considered invalid. A test unit that retains its original
purple color indicates that test parameters have been met. A failed sterilization
cycle is indicated by a color change to or toward yellow.
Food Samples
Commercial round scrambled egg patties (Michael Foods Egg Products
Co., Gaylord, MN) were used for the study. The average mass of the egg patty
was 42.5 g 7.1 g. Handling and shipping procedure of the scrambled egg
patties was performed as in an industrial setting, where patties would be stored
in a frozen state before HPHT treatment. Frozen samples from a single lot
were received and stored frozen at -30C. Each patty was then repackaged in
special 89 mm 89 mm pouches (ALCAN, Chicago, IL) under vacuum and
defrosted overnight at 5C.
For tests in food samples the spore strips were left inside their envelopes
and placed or sandwiched between two egg patties. The patties were placed in
a high barrier pouch (ALCAN) and vacuum-sealed to 15 mbar. Immediately
after sealing the egg patties with the strips inside, the package were subjected
to HPHT using the maximum pressure of 688 MPa and processing temperature from 90 to 121C. Three sets of double patties were placed in three
different locations throughout the vessel (top, middle and bottom). Following
processing, packages were immersed into an iced water bath. After cooling,
the strips were removed and transferred to the tubes and incubated at 55C for
7 days. The results were checked daily after the second day of incubation.
HP Inactivation Treatments of Spore Samples
The spore samples were subjected to HP processing using a Quintus Food
Processing Isostatic Press (model QFP-6, ABB Autoclave Systems Inc.,
Columbus, OH) supplemented with an internal heater arrangement (Flow
Pressure Systems AB, Vsters, Sweden). The equipment was rated for operation of up to a maximum pressure of 120 kpsi (828 MPa). The pressure
come-up time (CUT) depended on the final pressure (600800 MPa) and
varied from 110 to 160 s. The depressurization time was less than 4 s at

HIGH PRESSUREHIGH TEMPERATURE STERILIZATION

615

all pressures levels. Deionized water was used as the pressure-transmitting

medium. The system was equipped with two K-type thermocouples (Omega
Engineering Inc., Stamford, CT) attached to a data logger to monitor and
record the temperature in the pressure-transmitting medium and in the sample
throughout the process. The internal heater arrangement made it possible to
attain higher temperatures during processing time, with a recommended
maximum temperature of 110C. With the adiabatic heating effect, the
temperature increase was approximately equivalent to 4C per 100 MPa. To
simultaneously record the pressuretemperature profiles of the water-jacket
temperature, the control panel of the equipment was connected to a data
acquisition card (Agilent 34970A, Agilient, Palo Alto, CA) that converted the
signals that will be read and recorded by a computer equipped with the
appropriate software (Laboratory VIEW version 6, National Instruments Corporation, Austin, TX). Pressure, temperature and time were recorded every
second during the entire treatment.
Treatment Protocol
The pressure vessel (water jacket) and pressure-transmitting medium
(deionized water) were preheated to the desired starting temperature prior to
HP tests using the heater element set point. The initial preheat temperatures of
the samples prior to HP treatment were determined first through preliminary
testing that aims to achieve particular process temperatures because of the
work of compression at pressures of 600, 700 and 800 MPa. By varying the
initial temperature before the start of pressurization, it was possible to study
the inactivation kinetics of the spores at the nominal process temperatures of
91, 100 and 108C. When the set temperature was reached, the inoculated
sample and control pouches were preheated to the initial temperature in an
external water bath. A thermocouple was placed in the control pouch and the
temperature was recorded throughout the process. The samples were subjected
to the HP treatments as given in Table 1. The zero time samples were taken as
soon as possible (0.6 s) after the process pressure reached the preprogrammed
maximum.
HP Treatments of Food Samples
The HP treatments of packed egg patties were carried out in a pilot scale
35-L HP Quintus Press (QFP 35 L S, Avure Technologies, Kent, WA). The
apparatus is designed for operation of up to 690 MPa and temperatures of up
to 130C. The pressure medium used was water. The system is comprised of
multiple subsystems including a preheating tank, cooling tank, wire-wound
vessel, low-pressure fill system, HP pump and control system. Another four

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TABLE 1.
EXPERIMENTAL CONDITIONS USED FOR THE KINETIC STUDIES ON
PRESSURETEMPERATURE INACTIVATION OF CLOSTRIDIUM SPOROGENES PA 3679
SPORES IN PHOSPHATE BUFFER
Pressure
(MPa 1.5)

Initial temperature
(C 1)

Process temperature*
(C 1)

Holding time (s)

600

67
74
82
62
70
78
58
66
74

91
100
108
91
100
108
91
100
108

0, 60, 120, 180, 240, 300

0, 60, 120, 180, 240
0, 30, 60, 90, 120
0, 60, 120, 180, 240
0, 60, 120, 180, 240
0, 30, 60, 90, 120
0, 60, 120, 180, 240
0, 60, 120, 180, 240
0, 30, 60, 90, 120

700

800

* Values are means of three replicates.

K-type thermocouples (Omega Engineering Inc.) were used to measure the

temperature of the pressure medium as well as inside of the patty. HP treatments were carried out in the temperature range from 105 to 121C at a pressure
of 688 MPa. The initial temperature in the pressure vessel was adjusted to
achieve the process temperature and determined by subtracting the temperature increase attributed to the compression heating from the desired final
process temperature.
Data Analysis
The Microsoft Excel 7.0 and MINITAB 14 statistical software (Minitab
Inc., State College, PA) were used to perform mathematical and statistical
analysis of data.

RESULTS AND DISCUSSION

Kinetic Analysis of PressureTemperature Effects
The kinetic analysis of C. sporogenes PA 3679 spore inactivation was
carried out at isobaric and isothermal conditions after the CUT portion of the
pressure cycle was completed and attained the maximum experimentalprocess pressure. The effect of preheat on the inactivation of spores was not
considered because no significant change in spore counts was found in the
samples after equilibration to initial process conditions. However, analysis of
the effect of CUT or pressurization period in the HP treatment cycle followed

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617

TABLE 2.
EFFECT OF PRESSURIZATION COME-UP TIME (CUT) AND DEPRESSURIZATION
COME-DOWN TIME (CDT) ON CLOSTRIDIUM SPOROGENES PA 3679 VIABLE SPORES
SURVIVAL OF LOG10(N/N0)
CUT (s)
600 MPA-91C
96
700 MPa-91C
112
800 MPa-91C
125

Log (N/N0)*
0.35 0.17
0.49 0.13
0.49 0.06

CUT (s)
600 MPa-100C
95
700 MPa-100C
111
800 MPa-100C
128

Log (N/N0)*
0.39 0.220
0.37 0.150
0.65 0.004

CUT (s)
600 MPa-108C
95
700 MPa-108C
111
800 MPa-108C
127

Log (N/N0)*
0.53 0.09
0.66 0.16
0.81 0.02

* Values are means of three replicates.

by instant depressurization or come-down time (CDT) on the survival of the

tested spores showed that significant inactivation could occur. Results demonstrating the effect of CUT and CDT on spore survival are summarized in
Table 2 for all tests conducted. Examining the mean of log reductions values
at zero holding time (tp of 0.01) at various pressuretemperature-CUT parameters suggests that some inactivation of PA 3679 spores occurred during the
pressurization and depressurization and varied from 0.35 to 0.53 log reductions at 600 MPa and achieved up to 0.81 logs at 800 MPa at process temperature of 108C. Inactivation is therefore already noticeably advanced at
zero holding times.
The results of the HPHT treatments of C. sporogenes PA 3679 spores are
shown in Figs. 13 as plots of the decimal logarithm of survival fraction (log10
[N/N0]) versus holding time at constant pressures of 600, 700 and 800 MPa for
constant temperatures of 91, 100 and 108C. It can be seen that during holding
time of up to 5 min the log survival plots of isothermal and isobaric inactivation of PA 3679 spores can be adequately described by a first-order kinetic
model (R2 0.95) in the tested temperature and pressure range. The first-order
kinetics inactivation of spores of G. stearothermophillus was also reported by
Clery-Barraud et al. (2004), Heij et al. (2005) and Patazca et al. (2005).
As expected, the rate of PA 3679 spore inactivation was parallel with
increases in both temperature and pressure. The temperature was important
and increased the inactivation at the applied identical pressure. For 600 MPa,
as an example, the inactivation was equal to 1.18 and 3.25 logs at 91 and 108C
both for 300 s, respectively. Accordingly, the D-values decreased from 357.4 s
at 600 MPa and 91 C to 67.6 s at 600 MPa and 108C. The effect of pressure
upon these spores was less pronounced especially at 91C and pressures of 700
and 800 MPa. The inactivation rates at 700 and 800 MPa did not vary widely

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T. KOUTCHMA ET AL.

Time (s)
0

100

200

300

400

0.0

Log10 (N/N0)

1.0
2.0
3.0
4.0

600 MPa 91C

700 MPa 91C

5.0
800 MPa 91C
6.0
FIG. 1. SURVIVOR CURVES OF CLOSTRIDIUM SPOROGENES PA 3679 SPORES AT 91C
PROCESS TEMPERATURE AND PRESSURES OF 600, 700 AND 800 MPA

at 91C and were equal to 0.0034 and 0.0380/s, respectively. The pressure
accelerated the inactivation of the C. sporogenes PA 3679 spores at temperatures of 100 and 108C.
The analysis of variance for spore survival showed that all three factors
time, temperature and pressure were significant and with a very low P-value
(P 0.001) for pressure. However, the regression analysis of survival versus
time/temperature/pressure showed that temperature was the most significant
factor in the inactivation of PA 3679 spores.
Next, the D-values at tested processing pressures and temperatures were
determined and summarized in Table 3. From Table 3, it can be concluded that
the highest D-value of 357.4 s was found at 600 MPA and 91C. The D-values
at 108C were 67.6, 58.3 and 49.0 s for 600, 700 and 800 MPa, respectively.
The lowest D-values were observed at 108C and 800 MPa. In other words,

HIGH PRESSUREHIGH TEMPERATURE STERILIZATION

619

Time (s)
0

100

200

300

400

0.0

Log10 (N/N0)

1.0
2.0
3.0
4.0

600 MPa 100C

700 MPa 100C

5.0
800 MPa 100C
6.0
FIG. 2. SURVIVORS CURVES OF CLOSTRIDIUM SPOROGENES PA 3679 SPORES AT 100C
PROCESS TEMPERATURE AND PRESSURES OF 600, 700 AND 800 MPA

HPHT condition at 800 MPa and 108C proved to be the most effective for
inactivating PA 3679 spores. The D-values of PA 3679 spores obtained in this
study were in good agreement with those reported by Rovere et al. (1998).
They reported that processing at 108C and 800 MPa was the most effective
treatment with a calculated D-value of 0.695 min (41.7 s) whereas heat treatment (110C) alone yielded a D-value of 13.300 min. The obtained D-values
were further used for HPHT sterilization process calculation.
Figure 4 illustrates the log of D-values of PA 3679 spores as a function of
temperature at constant pressures of 600, 700 and 800 MPa. The pressure
dependence at constant temperatures of 91, 100 and 108C of D-values is
shown in Fig. 5. Thermal resistance (ZP) at constant pressures and pressure
resistance (ZT) at constant temperatures were calculated as the negative inverse
of the slope of the linear plots of the temperature and pressure dependency of

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T. KOUTCHMA ET AL.

Time (s)
0

100

200

300

400

0.0
600 MPa 108C

1.0

Log10 (N/N0)

700 MPa 108C

2.0

800 MPa 108C

3.0
4.0
5.0
6.0

FIG. 3. SURVIVOR CURVES OF CLOSTRIDIUM SPOROGENES PA 3679 SPORES AT 108C

PROCESS TEMPERATURE AND PRESSURES OF 600, 700 AND 800 MPA

TABLE 3.
D-VALUES OF CLOSTRIDIUM SPOROGENES PA 3679 AFTER
HIGH PRESSUREHIGH TEMPERATURE TREATMENTS
Pressure (MPa)

Temperature (C)

D-value (s)

600
600
600
700
700
700
800
800
800

91
100
108
91
100
108
91
100
108

357.4
192.3
67.6
294.0
169.5
58.3
270.3
136.9
49.0

HIGH PRESSUREHIGH TEMPERATURE STERILIZATION

621

2.8
2.6
2.4

Log D

2.2
2.0
1.8
600 MPa
1.6
700 MPa

1.4

800 MPa
1.2
1.0
90

95

100

105

110

Temperature (C)
FIG. 4. TEMPERATURE DEPENDENCE OF D-VALUES FOR HIGH PRESSURE
INACTIVATION OF CLOSTRIDIUM SPOROGENES PA 3679

D-values, respectively. Regression coefficient (R2) values ranged from 0.98 to

1.00, indicating the suitability of these parameters for describing temperature
and pressure resistance of D-values in HP process calculations. It was found
that the thermal resistance of PA 3679 spores (ZP-values) did not vary with
pressure and the mean ZP-value obtained was 23.7C at 600, 700 and 800 MPa.
C. sporogenes PA 3679 are very pressure-stable spores. The ZT-values did not
vary with pressure at 91, 100 and 108C, signifying that the increase in pressure
does not accelerate the destruction of PA 3679 spores. The mean value
obtained was a ZT of 1500.7 MPa.
There have been no published reports containing ZP- or ZT-values under
HPHT treatments for the inactivation of C. sporogenes PA 3679. For G.
stearothermophilus spores, Patazca et al. (2005) found a decrease in thermal
resistance when pressure increased from 500 to 600 MPa. The results indicated a decrease in ZT-values with an increase in temperature, clearly indicating a lower resistance of spores to pressure at higher temperatures. The

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T. KOUTCHMA ET AL.

2.8
2.6
2.4

Log D

2.2
2.0
1.8
1.6

91C

1.4

100C

1.2

108C

1.0
500

600

700

800

900

Pressure (MPa)
FIG. 5. PRESSURE DEPENDENCE OF D-VALUES FOR HIGH PRESSURE INACTIVATION
OF CLOSTRIDIUM SPOROGENES PA 3679

pressure resistance of G. stearothermophilus was significantly lower than that

of C. sporogenes PA 3679, slightly decreasing with increase of temperature.
Fo-Value of HPHT Sterilization
The current evidence and earlier reported studies (Rovere et al. 1998;
Heij et al. 2005; Patazca et al. 2005) demonstrated that a linear model is
suitable to predict the microbial inactivation of both classical surrogates such
as G. stearothermophilus and C. sporogenes PA 3679 in thermally assisted HP
process when processed at isobaric and isothermal conditions during holding
time. Consequently, the approaches used in thermal processing can be applied
for HPHT process specification. Pflugs concept (Pflug and Zeghman 1985)
was adapted for determining the Fo-value for the HPHT sterilization of foods
in the current study. The spoilage failure rate of 10-6 was selected for mesophilic spores represented by C. sporogenes PA 3679 and 10-3 for thermophilic

HIGH PRESSUREHIGH TEMPERATURE STERILIZATION

623

3
PA 3679 700 MPa
G. sterothermophollis 700 MPa

Log 10 D-value

2.5

PA 3679 600 MPa

G. sterothermophillis 600 MPa

2
1.5
1
0.5
90

100

110

120

Temperature (C)
FIG. 6. TEMPERATURE DEPENDENCE OF HIGH PRESSURE INACTIVATION OF
CLOSTRIDIUM SPOROGENES PA 3679 AND GEOBACILLUS STEAROTHERMOPHILLUS
SPORES AT 600 AND 700 MPA

spores. As indicated above, G. stearothermophillus is the critical species of

nonpathogenic thermophilic bacteria that is traditionally used in establishing
thermal process specification. In addition, the calculation of Fo-value requires
the knowledge of initial microbial load (pathogenic and nonpathogenic mesophilic and thermophilic spores) and their D-value at 121C. The initial number
of resistant spores is a variable. However, according to Pflug, the initial
number (N0) of 10 and 100 per unit is adequately high for mesophilic and
thermophilic spores in canned foods relatively.
Because of technical limitations of a lab-scale QFP-6 HP unit to reach
temperatures higher than 110C, D-values at 121C of C. sporogenes PA 3679 at
600 and 700 MPa were calculated using the D-value at 108C as a reference
D-value and obtained a ZP-value of 23.7. The linear relationships or thermal
resistance curves are presented graphically for both spore-forming species in
Fig. 6. Table 4 summarizes the D- and Fo-values of G. stearothermophilus and
C. sporogenes PA 3679 at 121C and pressures of 0.1, 600.0 and 700.0 MPa.
The pressure used in a standard thermal process is 0.1 MPa. The obtained

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T. KOUTCHMA ET AL.

TABLE 4.
DECIMAL REDUCTION TIMES AND Fo-VALUES OF SPORE-FORMERS AT 121C OF
VARIOUS PRESSURES
Clostridium sporogenes PA 3679

Geobacillus stearothermophilus

Pressure
(MPa)

D-value
at 121C (s)

Fo (s)

Pressure
(MPa)

D-value
at 121C (s)

Fo (s)

0.1*
600.0
700.0
800.0

48.0
20.8
19.2
14.5

336.0 (5.60 min)

145.6 (2.43 min)
134.4 (2.24 min)
101.5 (1.70 min)

0.1
600.0
700.0
800.0

330.0
5.7
5.7
NA

1650.0 (27.5 min)

28.5
28.5
NA

* Conditions represent heat inactivation used in conventional thermal sterilization at 121C.

NA, no available data.

D-values at 121C of PA 3679 were more than twice lower at 600 and 700 MPa
levels of HP treatments as compared to heat inactivation. The Fo-values shown
in Table 4 were calculated for a reduction of 7 logs for PA 3679 spores and a
5 log reduction for G. stearothermophilus spores. The calculations showed that
a process of 2.24 min at 121C and at pressures of 600 or 700 MPa or that of
1.7 min at 800 MPa and 121C will be adequate to destroy spoilage-causing
mesophilic and thermophilic spore-forming organisms using HPHT sterilization process. It is evident that the duration of the HPHT process is drastically
reduced to 1.72.24 min as opposed to conventional thermal sterilization that
takes 27 min. According to the D-value differences between HPHT and
thermal process, combining temperature with pressure is very important for
reducing the treatment times. Nevertheless, the design of HPHT processes to
make foods safe from a public-health point of view will require additional
knowledge of D-value at 121 of C. botulinum spores at similar HP conditions.
Microbiological Verification of HPHT Process Lethality Using
Spore Bioindicators
Microbiological spore methods are used as direct process-validation
methods. They rely on measuring the delivered process by achieved log reductions for a process using a nonpathogenic microorganism such as spores of G.
stearothermophilus or C. sporogenes and converting this to a process value. As
evident from comparison of the HPHT kinetic parameters of both traditional
surrogates, C. sporogenes is the more pressure-resistant of the two. Thus, the
spore strips of C. sporogenes were used to verify adequacy of the delivered
lethality of HPHT process in a pilot scale sterilization unit QFP-35 L at
690 MPa and process temperatures of 100 to 121C, and holding times of 3 to
5 min.

HIGH PRESSUREHIGH TEMPERATURE STERILIZATION

130

625

800
700

120
110

500

100

400
300

90

Tc 1

80

Tc 2

200

Pressure

100

70
0

50

100

150

200

250

300

350

Pressure (MPa)

Temperature (C)

600

0
400

Time (s)
FIG. 7. TIME-TEMPERATURE PROFILE IN THE EGG PATTY DURING HIGH
PRESSUREHIGH TEMPERATURE CYCLE IN A 35-L HP UNIT AT 688 MPA AND 121C
Water was used as compression medium.

The thermal treatment of spore strips was investigated first. Three tubes
with the spores were heated in a water bath at 100C for 15 min and steamsterilized at 121C for 60 min using the autoclaves preset kill cycle. The
results obtained were as follows: untreated control tube and heat-shocked tube
were yellow-colored indicating a positive result. Whereas, the autoclaved
tubes were purple-colored indicating a negative result. Next, a similar experiment was run except the spore strips were left inside their envelopes and
placed or sandwiched between two egg patties. Two patties were placed in a
high barrier pouch and vacuum-sealed to 15 mbar. Three sets of double patties
were sealed and treated immediately in a 100C water bath for 18 min and
subsequently sterilized using a steam autoclave set at 121C for 60 min. Following 48 h of incubation, the heat-shocked package indicated a positive
result; whereas the autoclaved package showed a negative result.
After the preliminary tests, the spore strips were subjected to HPHT
treatments in the package with the egg patties as described above in a pilot
scale unit. The HPHT processing cycle in the 35-L HP machine at 121C and
688 MPa is illustrated in Fig. 7. The packages were treated immediately after
packaging to minimize the oxygen exposure of the strips. The results are
reported in Table 5 as a number of positive samples to the total number of

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T. KOUTCHMA ET AL.

TABLE 5.
HPHT INACTIVATION OF CLOSTRIDIUM SPOROGENES BIOINDICATOR SPORES IN EGG
PATTIES PROCESSED IN 35-L HP VESSEL AT 688 MPA
Initial temperature (C)

73
80
85
90

Process temperature (C)

105
110
115
121

Number of positive samples after holding

3 min

5 min

4 min

3(+)/3
3(+)/3
3(+)/3
0/3

3(+)/3
3(+)/3; 2(+)/6
1(+)/6
0/3

3(+)/3
0/3; 0/6
0/3; 0/6
0/3

HPHT, high pressurehigh temperature.

TABLE 6.
CALCULATED PROCESS VALUE AT 700 MPA
Clostridium sporogenes

Geobacillus stearothermophilus

T (C)

D-value (s)

F = 6D (s)

T (C)

D-value (s)

F = 6D (s)

105
110
115
121

86.8
54.1
33.8
19.2

520.8 (8.70 min)

324.6 (5.41 min)
202.8 (3.38 min)
115.2 (1.92 min)

100
105
111
121

17.7
14.9
11.5
6.6

106.2 (1.80 min)

89.4 (1.50 min)
69.0 (1.15 min)
39.6 (0.66 min)

T, temperature.

samples treated at the defined process conditions. In addition, the process

lethality was calculated as time to achieve 6 logs reduction of the PA 3679
spores or 6D process (Table 6). It was found that a process temperature of
105C was not sufficient to destroy the PA 3679 spores during the holding times
tested. However, the increase of process temperature up to 110 and 115C
achieved sterility after 5 min of treatment at 688 MPa. The HPHT treatment
at 121C was sufficient to destroy 6 logs of PA 3679 spores after a minimum
holding time of 3 min.
The experimental observations were in agreement with calculated processing times given in Table 6. For example, the minimum calculated process
time at 110 and 115C was 5.41 and 3.38 min, respectively. An increase of the
process temperature of up to 121C reduced the required process time to
1.92 min.
Furthermore, the spore strips of G. stearothermophilus used for verification of process temperature were located at the top, center and bottom of the
HP vessel. The selected process temperature was 105C and the holding time
varied from 2 to 5 min. Holding for 4 min was needed to destroy 6 logs of G.
stearothermophilus spores on strips placed in the center of the HP vessel.

HIGH PRESSUREHIGH TEMPERATURE STERILIZATION

627

TABLE 7.
HPHT INACTIVATION OF GEOBACILLUS STEAROTHERMOPHILUS ATCC 7953
BIOINDICATOR SPORES IN EGG PATTIES PROCESSED IN 35-L HP VESSEL AT 105C AND
688 MPA
Placement in HP vessel

Top
Middle
Bottom

Number of positive samples after holding at 688 MPa

2 min

3 min

4 min

5 min

3(+)/3
3(+)/3
3(+)/3

3(+)/3
3(+)/3
3(+)/3

1(+)/3
0/3
1(+)/3

0/3
0/3
0/3

Some survivors were found in the samples placed at the top and the bottom of
the carrier, indicating some temperature variations because of possible convection heat transfer in the HP vessel (Table 7). Increasing the holding time up
to 5 min resulted in the complete destruction of spores in all locations. It
should be pointed out that experimentally observed holding time was longer
than the calculated process time of 1.5 min at 105C for the 6D process.
CONCLUSIONS
The results of the HPHT tests carried out showed that HP inactivation
kinetics of C. sporogenes PA 3679 spores in phosphate buffer was a function
of pressure and temperature. In order to get the desired processing temperature
and by taking compression heating into account, preheating conditions need to
be established. A treatment that combines pressure with temperature was
necessary to inactivate PA 3679 spores. The inactivation kinetics during
holding period of HP cycle at constant pressures and temperatures was well
described by a log-linear regression model similar to thermal processing. The
rate of clostridial-spore inactivation increased with both pressure and temperature. The D-values ranged from 49.0 to 357.4 s in the pressure range of
600800 MPa and the testing temperatures of 91, 100, 108C. As expected with
increasing temperature and pressure, the D-values decreased. It was demonstrated that a combination of pressure and temperature is very important in
reducing the processing time. The kinetic parameters were more pressuresensitive at higher temperatures. However, the pressure enhancement effects
observed were significantly less than those seen using G. stearothermophilus
spores. It is also noteworthy to see that PA 3679 spores are less resistant
thermally than G. stearothermophilus spores used in previous studies. The
degree of acceleration in inactivation observed under HP conditions with C.
sporogenes spores was significantly lower than that seen using G. stearothermophilus spores.

628

T. KOUTCHMA ET AL.

Using Pflugs concept of using the endpoint of a sterilization to specify a

process, it becomes apparent that the duration of the HPHT process required
to achieve commercial sterility in terms of spoilage-causing spore-formers is
drastically reduced compared to thermal sterilization. The microbiological
verification runs in packed egg patties confirmed the adequacy of the HPHT
process at 110121C to destroy clostridial spores. Additional lethality
achieved during the pressurization and depressurization cycle contributed to
the overall lethality of the HPHT sterilization process. The obtained results
confirmed that spore bioindicators can be successfully used to measure the
actual delivered process in HPHT sterilization.

ACKNOWLEDGMENTS
Research support was provided in part through a grant from a consortium
of companies participating in a U.S. Army Dual Use Science and Technology
(DUST) High Pressure Processing of Low Acid Foods Program and funds
received from the Combat Rations Network for Technology Implementation
(CORANET).

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