high pressure high temperature sterilization

HIGH PRESSUREHIGH TEMPERATURE STERILIZATION:FROM KINETIC ANALYSIS TO PROCESS VERIFICATION*

TATIANA KOUTCHMA1, BIAO GUO, EDUARDO PATAZCA andBRIAN PARISI

National Center for Food Safety and Technology6502 South Archer Road

Summit-Argo, IL 60501-1933Illinois Institute of Technology

Accepted for Publication September 20, 2005

ABSTRACT

The inactivation of Clostridium sporogenes PA 3679 spores by highpressure at high temperatures (HPHT) in phosphate buffer was investigatedin a lab-scale temperature-controlled HP system (QFP-6) with an internalheater to maintain the sample temperature. Some inactivation of sporesoccurred during the pressurization come-up time (CUT) and depressurizationtime. The inactivation of PA 3679 was found to be exponential during theadiabatic holding period of the HP cycle at constant pressures and tempera-tures. The inactivation rate increased with both pressure and temperature. Thekinetic parameters such as D-values at tested temperatures and pressuresthat are necessary for the design of process parameters of HP sterilizationprocess were determined. Within the pressure range of 600800 MPa, thecalculated D-values ranged from 270.3 to 357.4 and 49.0 to 67.6 s at 91 and108C, respectively. These studies provided basic data on the effects of pressureand temperature on the inactivation of PA 3679 spores under conditionsapplicable to the development of preservation specifications for commercialHPHT processing of low acid foods. The spore strips of C. sporogenes wereused as indicators for microbiological verification of delivered lethality ofHPHT sterilization process at different processing conditions in a pilot scaleHP vessel.

1 Corresponding author. TEL: (708) 563-8178; FAX: (708) 563-1873; EMAIL: [email protected]* 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 forFood Safety and Technology.

Journal of Food Process Engineering 28 (2005) 610629. All Rights Reserved. Copyright 2005, Blackwell Publishing610

INTRODUCTION

High hydrostatic pressure (HP) is an alternative preservation techniquefor thermal sterilization of low acid foods (LACF) with a shorter process time.As defined by LACF regulations (21 CFR 113), in order to establish thesterilization process, the commercial sterility must be demonstrated in terms ofspoilage-causing organisms and pathogens capable of growing under the con-ditions of storage and distribution. The LACF regulations mention commer-cial sterility or essentially no risk without defining the process. Forcanning, the problems associated with spoilage, and the solutions to thoseproblems, have evolved over a long history. The classical heat-resistant, sur-rogate microorganisms spores of Geobacillus stearothermophilus were suffi-ciently characterized to design and validate the thermal sterilization process.Along with G. stearothermophillus, Clostridium sporogenes, a typical heat-resistant, mesophilic, spore-forming organism, is used in establishing thermalprocess specifications. Spores of the strain PA 3679 were selected becausetheir heat resistance has been found to be equal to or greater than that of sporesof Clostridium botulinum. These bacterial spores known for their resistances toheat and irradiation have also been shown to be resistant to pressure (Maggiet al. 1996). Unless HP in excess of 800 MPa is used, heat combined with HPis required for HP sterilization of LACF. The regulation that was not intendedfor pressure-processed foods can be applicable to HP sterilization of LACFbecause of a strong thermal component required for the spores destruction,and HP processing has not had sufficient usage to demonstrate that pressuredoes have an accelerating effect on the spores inactivation kinetics. Thus, forpressure-sterilized LACF, the requirements in 21 CFR 113 must be fulfilledbefore a product can enter commercial production.

The two paths to addressing the issues of spoilage and pathogens ofpublic health significance (as relating to commercial sterility) are used. First,the establishment of process temperature, pressure and time for HPhightemperature (HT) sterilization processes requires knowledge of inactivationkinetic parameters of target pathogenic and spoilage-causing spore-formingbacteria. Second, the process outcome or performance criterion must bedefined as a prerequisite to further validation of the established sterilizationprocess. Historically the endpoint of sterilization processes has not beenclearly defined. The 12-decimal logs concept established by Stumbo (1973)also does not establish an endpoint, but merely defines that the process shouldaccomplish a 12-decimal point reduction of C. botulinum. Pflug (1987) firstdefined the thermal process as the probability of survival of either a spoilage-causing organism or pathogen and proposed an alternative approach to calcu-late the process lethality by describing the outcome. The last essential step inestablishing the process is microbiological validation in product samples to

611HIGH PRESSUREHIGH TEMPERATURE STERILIZATION

measure the delivered process by achieved log reductions and converting thisto a process value. Biological indicators (BI) such as spore strips of G.stearothermophilus and C. sporogenes are widely used for sterility testing invarious sterilization environments. Because standard BIs are easy and conve-nient to use for a direct measurement of the lethality, they also can be used toverify adequacy of the delivered process in HPHT sterilization to reaffirm theefficiency of defined process in controlling the microbiological hazard to therequired level.

Most of the published data have dealt with the HP destruction of thethermophilic spores of Geobacillus. Clostridium spores have been assumedto behave in a similar manner to Geobacillus spores under HP. However,recent studies on Clostridium spores had not confirmed this assumption. Golaet al. (1996), evaluating the pressure sensitivity of C. sporogenes PA 3679at 900 MPa for 10 min at 30C, were unable to completely destroy8.4 102 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, showingno significant inactivation. Maggi et al. (1996) demonstrated that 1500 MPa at20C applied for 5 min had no effects on the spores. The pressure treatmentparameters had been examined by Rovere et al. (1996a) for inactivation ofspores of C. sporogenes PA 3679 starting with concentrations of approxi-mately 105 cfu/mL and pressure-hold times of 5 min. Elimination of thesespore levels was possible with processes of 1400 and 800 MPa at 54 and 75C,respectively, in different model food systems. In a study involving sporesuspensions of C. sporogenes PA 3679 in meat broth, the pressure acted as acomplementary synergistic process to allow reduction of the thermal process-ing parameters necessary to eliminate problematic spore-formers in foods.They concluded that it was important to combine HP with HT during pressureprocessing. Knowledge of the HP inactivation kinetics of C. sporogenes isessential to design a safe sterilization process.

The objectives of this study were: (1) to measure the inactivation kineticsof C. sporogenes PA 3679 spores by HP at elevated temperatures in phosphatebuffer for the design of HPHT sterilization process; and (2) to use spore stripsas indicators for microbiological verification of delivered lethality of HPHTsterilization process at different processing conditions in a pilot scale HPvessel.

MATERIALS AND METHODS

MicroorganismFive milliliters of commercially available C. sporogenes PA 3679 spores

(National Food Laboratories Inc., Dublin, CA) with a concentration of

612 T. KOUTCHMA ET AL.

1.2 108 cfu/mL D at 121C of 0.8 min, Z-value of 7.8C was obtained. Thespores were divided in 1-mL aliquots in sterile screw-cap tube and kept frozenat -20C during storage.

Spore Samples PreparationSterile sodium phosphate buffer (pH 7.0) was used to prepare 1:100

dilutions from the stock spore suspension to obtain an initial spore count ofapproximately 106 cfu/mL. Two-milliliter aliquots of this spore preparationwere enclosed in a sterile high barrier Nylon-film pouch (CV Systems,Downers Grove, IL) and sealed using a manual heat sealer. All preparedsamples were kept in ice water prior to HP treatment. One inoculated samplewas used per HPHT treatment.

Spore EnumerationTwo unprocessed samples were diluted and plated as a control for each

experiment in order to obtain the initial spore count. After HP treatment, thepouches were immediately cooled in the water-ice container and kept therebefore analysis. The samples were serially diluted in sodium phosphate buffer.The spores counts with and without treatments were determined by pour-plate enumeration in duplicate, using trypticase soy agar (TSA) plus 0.6%yeast extract (YE) (Difco Laboratories, Detroit, MI). The plates were incu-bated anaerobically at 35C for 5 days before enumeration.

Spore Strip TestingGeobacillus stearothermophilus spore strips ATCC 7953 (Raven Biologi-

cal Laboratories Inc. Omaha, NE) and C. sporogenes ATCC 11437 spore strips(NAMSA Laboratories, Northwood, OH) were used for testing. Spore stripswere 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 thestrips was 1.0 106 cfu per strip.

Bromocresol purple (BCP) (Fisher Scientific, Chicago, IL) was used as agrowth indicator dye. The BCP dye is purple at neutral pH and changes colorto bright yellow at pH 5.2, and starting at pH 6.8 it gradually begins to lightentoward a yellow color. A stock solution of 0.4% BCP (100 mL) was preparedby adding 0.4 g of dehydrated BCP to 100 mL of deionized water. One milli-liter 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 tubesand autoclaved.


Three spore strips were prepared by using sterile forceps to withdrawthem from their protective pouch. Each strip was placed within a tube withTSB/BCP media. Three tubes from a batch of G. stearothermophilus wereincubated for 24 h at 55C. The tubes with C. sporogenes spore strips wereincubated anaerobically for 24 h at 35C. Anaerobic conditions were obtainedby using anaerobic jars and system envelopes (Gas Pak, Becton Dickinson,Franklin Lakes, NJ) equipped with a palladium catalyst. The control unitexhibits a color change toward yellow. If the control unit does not show signsof growth, the test is considered invalid. A test unit that retains its originalpurple color indicates that test parameters have been met. A failed sterilizationcycle is indicated by a color change to or toward yellow.

Food SamplesCommercial round scrambled egg patties (Michael Foods Egg Products

Co., Gaylord, MN) were used for the study. The average mass of the egg pattywas 42.5 g 7.1 g. Handling and shipping procedure of the scrambled eggpatties was performed as in an industrial setting, where patties would be storedin a frozen state before HPHT treatment. Frozen samples from a single lotwere received and stored frozen at -30C. Each patty was then repackaged inspecial 89 mm 89 mm pouches (ALCAN, Chicago, IL) under vacuum anddefrosted overnight at 5C.

For tests in food samples the spore strips were left inside their envelopesand placed or sandwiched between two egg patties. The patties were placed ina high barrier pouch (ALCAN) and vacuum-sealed to 15 mbar. Immediatelyafter sealing the egg patties with the strips inside, the package were subjectedto HPHT using the maximum pressure of 688 MPa and processing tempera-ture from 90 to 121C. Three sets of double patties were placed in threedifferent locations throughout the vessel (top, middle and bottom). Followingprocessing, packages were immersed into an iced water bath. After cooling,the strips were removed and transferred to the tubes and incubated at 55C for7 days. The results were checked daily after the second day of incubation.

HP Inactivation Treatments of Spore SamplesThe 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 (FlowPressure Systems AB, Vsters, Sweden). The equipment was rated for opera-tion of up to a maximum pressure of 120 kpsi (828 MPa). The pressurecome-up time (CUT) depended on the final pressure (600800 MPa) andvaried from 110 to 160 s. The depressurization time was less than 4 s at


all pressures levels. Deionized water was used as the pressure-transmittingmedium. The system was equipped with two K-type thermocouples (OmegaEngineering Inc., Stamford, CT) attached to a data logger to monitor andrecord the temperature in the pressure-transmitting medium and in the samplethroughout the process. The internal heater arrangement made it possible toattain higher temperatures during processing time, with a recommendedmaximum temperature of 110C. With the adiabatic heating effect, thetemperature increase was approximately equivalent to 4C per 100 MPa. Tosimultaneously record the pressuretemperature profiles of the water-jackettemperature, the control panel of the equipment was connected to a dataacquisition card (Agilent 34970A, Agilient, Palo Alto, CA) that converted thesignals that will be read and recorded by a computer equipped with theappropriate software (Laboratory VIEW version 6, National Instruments Cor-poration, Austin, TX). Pressure, temperature and time were recorded everysecond 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 toHP tests using the heater element set point. The initial preheat temperatures ofthe samples prior to HP treatment were determined first through preliminarytesting that aims to achieve particular process temperatures because of thework of compression at pressures of 600, 700 and 800 MPa. By varying theinitial temperature before the start of pressurization, it was possible to studythe inactivation kinetics of the spores at the nominal process temperatures of91, 100 and 108C. When the set temperature was reached, the inoculatedsample and control pouches were preheated to the initial temperature in anexternal water bath. A thermocouple was placed in the control pouch and thetemperature was recorded throughout the process. The samples were subjectedto the HP treatments as given in Table 1. The zero time samples were taken assoon as possible (0.6 s) after the process pressure reached the preprogrammedmaximum.

HP Treatments of Food SamplesThe 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). Theapparatus is designed for operation of up to 690 MPa and temperatures of upto 130C. The pressure medium used was water. The system is comprised ofmultiple subsystems including a preheating tank, cooling tank, wire-woundvessel, low-pressure fill system, HP pump and control system. Another four


K-type thermocouples (Omega Engineering Inc.) were used to measure thetemperature of the pressure medium as well as inside of the patty. HP treat-ments were carried out in the temperature range from 105 to 121C at a pressureof 688 MPa. The initial temperature in the pressure vessel was adjusted toachieve the process temperature and determined by subtracting the tempera-ture increase attributed to the compression heating from the desired finalprocess temperature.

Data AnalysisThe Microsoft Excel 7.0 and MINITAB 14 statistical software (Minitab

Inc., State College, PA) were used to perform mathematical and statisticalanalysis of data.

RESULTS AND DISCUSSION

Kinetic Analysis of PressureTemperature EffectsThe kinetic analysis of C. sporogenes PA 3679 spore inactivation was

carried out at isobaric and isothermal conditions after the CUT portion of thepressure cycle was completed and attained the maximum experimental-process pressure. The effect of preheat on the inactivation of spores was notconsidered because no significant change in spore counts was found in thesamples after equilibration to initial process conditions. However, analysis ofthe effect of CUT or pressurization period in the HP treatment cycle followed

TABLE 1.EXPERIMENTAL CONDITIONS USED FOR THE KINETIC STUDIES ON

PRESSURETEMPERATURE INACTIVATION OF CLOSTRIDIUM SPOROGENES PA 3679SPORES IN PHOSPHATE BUFFER

Pressure(MPa 1.5)

Initial temperature(C 1)

Process temperature*(C 1)

Holding time (s)

600 67 91 0, 60, 120, 180, 240, 30074 100 0, 60, 120, 180, 24082 108 0, 30, 60, 90, 120

700 62 91 0, 60, 120, 180, 24070 100 0, 60, 120, 180, 24078 108 0, 30, 60, 90, 120

800 58 91 0, 60, 120, 180, 24066 100 0, 60, 120, 180, 24074 108 0, 30, 60, 90, 120

* Values are means of three replicates.


by instant depressurization or come-down time (CDT) on the survival of thetested spores showed that significant inactivation could occur. Results demon-strating the effect of CUT and CDT on spore survival are summarized inTable 2 for all tests conducted. Examining the mean of log reductions valuesat zero holding time (tp of 0.01) at various pressuretemperature-CUT param-eters suggests that some inactivation of PA 3679 spores occurred during thepressurization and depressurization and varied from 0.35 to 0.53 log reduc-tions at 600 MPa and achieved up to 0.81 logs at 800 MPa at process tem-perature of 108C. Inactivation is therefore already noticeably advanced atzero holding times.

The results of the HPHT treatments of C. sporogenes PA 3679 spores areshown 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 forconstant temperatures of 91, 100 and 108C. It can be seen that during holdingtime of up to 5 min the log survival plots of isothermal and isobaric inactiva-tion of PA 3679 spores can be adequately described by a first-order kineticmodel (R2 0.95) in the tested temperature and pressure range. The first-orderkinetics inactivation of spores of G. stearothermophillus was also reported byClery-Barraud et al. (2004), Heij et al. (2005) and Patazca et al. (2005).

As expected, the rate of PA 3679 spore inactivation was parallel withincreases in both temperature and pressure. The temperature was importantand 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 108Cboth for 300 s, respectively. Accordingly, the D-values decreased from 357.4 sat 600 MPa and 91 C to 67.6 s at 600 MPa and 108C. The effect of pressureupon these spores was less pronounced especially at 91C and pressures of 700and 800 MPa. The inactivation rates at 700 and 800 MPa did not vary widely

TABLE 2.EFFECT OF PRESSURIZATION COME-UP TIME (CUT) AND DEPRESSURIZATION

COME-DOWN TIME (CDT) ON CLOSTRIDIUM SPOROGENES PA 3679 VIABLE SPORESSURVIVAL OF LOG10(N/N0)

CUT (s) Log (N/N0)* CUT (s) Log (N/N0)* CUT (s) Log (N/N0)*

600 MPA-91C 600 MPa-100C 600 MPa-108C96 0.35 0.17 95 0.39 0.220 95 0.53 0.09

700 MPa-91C 700 MPa-100C 700 MPa-108C112 0.49 0.13 111 0.37 0.150 111 0.66 0.16800 MPa-91C 800 MPa-100C 800 MPa-108C125 0.49 0.06 128 0.65 0.004 127 0.81 0.02

* Values are means of three replicates.


at 91C and were equal to 0.0034 and 0.0380/s, respectively. The pressureaccelerated the inactivation of the C. sporogenes PA 3679 spores at tempera-tures 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 versustime/temperature/pressure showed that temperature was the most significantfactor in the inactivation of PA 3679 spores.

Next, the D-values at tested processing pressures and temperatures weredetermined and summarized in Table 3. From Table 3, it can be concluded thatthe highest D-value of 357.4 s was found at 600 MPA and 91C. The D-valuesat 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,

6.0

5.0

4.0

3.0

2.0

1.0

0.00 100 200 300 400

Time (s)

Log 1

0 (N

/N0)

600 MPa 91C

700 MPa 91C

800 MPa 91C

FIG. 1. SURVIVOR CURVES OF CLOSTRIDIUM SPOROGENES PA 3679 SPORES AT 91CPROCESS TEMPERATURE AND PRESSURES OF 600, 700 AND 800 MPA


HPHT condition at 800 MPa and 108C proved to be the most effective forinactivating PA 3679 spores. The D-values of PA 3679 spores obtained in thisstudy were in good agreement with those reported by Rovere et al. (1998).They reported that processing at 108C and 800 MPa was the most effectivetreatment with a calculated D-value of 0.695 min (41.7 s) whereas heat treat-ment (110C) alone yielded a D-value of 13.300 min. The obtained D-valueswere further used for HPHT sterilization process calculation.

Figure 4 illustrates the log of D-values of PA 3679 spores as a function oftemperature at constant pressures of 600, 700 and 800 MPa. The pressuredependence at constant temperatures of 91, 100 and 108C of D-values isshown in Fig. 5. Thermal resistance (ZP) at constant pressures and pressureresistance (ZT) at constant temperatures were calculated as the negative inverseof the slope of the linear plots of the temperature and pressure dependency of

6.0

5.0

4.0

3.0

2.0

1.0

0.00 100 200 300 400

Time (s)Lo

g 10

(N/N

0)

600 MPa 100C

700 MPa 100C

800 MPa 100C

FIG. 2. SURVIVORS CURVES OF CLOSTRIDIUM SPOROGENES PA 3679 SPORES AT 100CPROCESS TEMPERATURE AND PRESSURES OF 600, 700 AND 800 MPA


0.00 100 200 300 400

Time (s)

600 MPa 108C

700 MPa 108C

800 MPa 108C

6.0

5.0

4.0

3.0

2.0

1.0

Log 1

0 (N

/N0)

FIG. 3. SURVIVOR CURVES OF CLOSTRIDIUM SPOROGENES PA 3679 SPORES AT 108CPROCESS 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 91 357.4600 100 192.3600 108 67.6700 91 294.0700 100 169.5700 108 58.3800 91 270.3800 100 136.9800 108 49.0


D-values, respectively. Regression coefficient (R2) values ranged from 0.98 to1.00, indicating the suitability of these parameters for describing temperatureand pressure resistance of D-values in HP process calculations. It was foundthat the thermal resistance of PA 3679 spores (ZP-values) did not vary withpressure 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 notvary with pressure at 91, 100 and 108C, signifying that the increase in pressuredoes not accelerate the destruction of PA 3679 spores. The mean valueobtained was a ZT of 1500.7 MPa.

There have been no published reports containing ZP- or ZT-values underHPHT treatments for the inactivation of C. sporogenes PA 3679. For G.stearothermophilus spores, Patazca et al. (2005) found a decrease in thermalresistance when pressure increased from 500 to 600 MPa. The results indi-cated a decrease in ZT-values with an increase in temperature, clearly indicat-ing a lower resistance of spores to pressure at higher temperatures. The

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

2.6

2.8

90 95 100 105 110

Temperature (C)

Log

D

600 MPa

700 MPa

800 MPa

FIG. 4. TEMPERATURE DEPENDENCE OF D-VALUES FOR HIGH PRESSUREINACTIVATION OF CLOSTRIDIUM SPOROGENES PA 3679


pressure resistance of G. stearothermophilus was significantly lower than thatof C. sporogenes PA 3679, slightly decreasing with increase of temperature.

Fo-Value of HPHT SterilizationThe current evidence and earlier reported studies (Rovere et al. 1998;

Heij et al. 2005; Patazca et al. 2005) demonstrated that a linear model issuitable to predict the microbial inactivation of both classical surrogates suchas G. stearothermophilus and C. sporogenes PA 3679 in thermally assisted HPprocess when processed at isobaric and isothermal conditions during holdingtime. Consequently, the approaches used in thermal processing can be appliedfor HPHT process specification. Pflugs concept (Pflug and Zeghman 1985)was adapted for determining the Fo-value for the HPHT sterilization of foodsin the current study. The spoilage failure rate of 10-6 was selected for meso-philic spores represented by C. sporogenes PA 3679 and 10-3 for thermophilic

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

2.6

2.8

500 600 700 800 900

Pressure (MPa)

Log

D

91C100C108C

FIG. 5. PRESSURE DEPENDENCE OF D-VALUES FOR HIGH PRESSURE INACTIVATIONOF CLOSTRIDIUM SPOROGENES PA 3679


spores. As indicated above, G. stearothermophillus is the critical species ofnonpathogenic thermophilic bacteria that is traditionally used in establishingthermal process specification. In addition, the calculation of Fo-value requiresthe knowledge of initial microbial load (pathogenic and nonpathogenic meso-philic and thermophilic spores) and their D-value at 121C. The initial numberof resistant spores is a variable. However, according to Pflug, the initialnumber (N0) of 10 and 100 per unit is adequately high for mesophilic andthermophilic spores in canned foods relatively.

Because of technical limitations of a lab-scale QFP-6 HP unit to reachtemperatures higher than 110C, D-values at 121C of C. sporogenes PA 3679 at600 and 700 MPa were calculated using the D-value at 108C as a referenceD-value and obtained a ZP-value of 23.7. The linear relationships or thermalresistance curves are presented graphically for both spore-forming species inFig. 6. Table 4 summarizes the D- and Fo-values of G. stearothermophilus andC. 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

0.5

1

1.5

2

2.5

3

90 100 110 120Temperature (C)

Log

10 D

-v

alue

PA 3679 700 MPaG. sterothermophollis 700 MPaPA 3679 600 MPaG. sterothermophillis 600 MPa

FIG. 6. TEMPERATURE DEPENDENCE OF HIGH PRESSURE INACTIVATION OFCLOSTRIDIUM SPOROGENES PA 3679 AND GEOBACILLUS STEAROTHERMOPHILLUS

SPORES AT 600 AND 700 MPA


D-values at 121C of PA 3679 were more than twice lower at 600 and 700 MPalevels of HP treatments as compared to heat inactivation. The Fo-values shownin Table 4 were calculated for a reduction of 7 logs for PA 3679 spores and a5 log reduction for G. stearothermophilus spores. The calculations showed thata process of 2.24 min at 121C and at pressures of 600 or 700 MPa or that of1.7 min at 800 MPa and 121C will be adequate to destroy spoilage-causingmesophilic and thermophilic spore-forming organisms using HPHT steriliza-tion process. It is evident that the duration of the HPHT process is drasticallyreduced to 1.72.24 min as opposed to conventional thermal sterilization thattakes 27 min. According to the D-value differences between HPHT andthermal process, combining temperature with pressure is very important forreducing the treatment times. Nevertheless, the design of HPHT processes tomake foods safe from a public-health point of view will require additionalknowledge of D-value at 121 of C. botulinum spores at similar HP conditions.

Microbiological Verification of HPHT Process Lethality UsingSpore Bioindicators

Microbiological spore methods are used as direct process-validationmethods. They rely on measuring the delivered process by achieved log reduc-tions for a process using a nonpathogenic microorganism such as spores of G.stearothermophilus or C. sporogenes and converting this to a process value. Asevident from comparison of the HPHT kinetic parameters of both traditionalsurrogates, C. sporogenes is the more pressure-resistant of the two. Thus, thespore strips of C. sporogenes were used to verify adequacy of the deliveredlethality of HPHT process in a pilot scale sterilization unit QFP-35 L at690 MPa and process temperatures of 100 to 121C, and holding times of 3 to5 min.

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-valueat 121C (s)

Fo (s) Pressure(MPa)

D-valueat 121C (s)

Fo (s)

0.1* 48.0 336.0 (5.60 min) 0.1 330.0 1650.0 (27.5 min)600.0 20.8 145.6 (2.43 min) 600.0 5.7 28.5700.0 19.2 134.4 (2.24 min) 700.0 5.7 28.5800.0 14.5 101.5 (1.70 min) 800.0 NA NA

* Conditions represent heat inactivation used in conventional thermal sterilization at 121C.NA, no available data.


The thermal treatment of spore strips was investigated first. Three tubeswith the spores were heated in a water bath at 100C for 15 min and steam-sterilized at 121C for 60 min using the autoclaves preset kill cycle. Theresults obtained were as follows: untreated control tube and heat-shocked tubewere yellow-colored indicating a positive result. Whereas, the autoclavedtubes were purple-colored indicating a negative result. Next, a similar experi-ment was run except the spore strips were left inside their envelopes andplaced or sandwiched between two egg patties. Two patties were placed in ahigh barrier pouch and vacuum-sealed to 15 mbar. Three sets of double pattieswere sealed and treated immediately in a 100C water bath for 18 min andsubsequently sterilized using a steam autoclave set at 121C for 60 min. Fol-lowing 48 h of incubation, the heat-shocked package indicated a positiveresult; whereas the autoclaved package showed a negative result.

After the preliminary tests, the spore strips were subjected to HPHTtreatments in the package with the egg patties as described above in a pilotscale unit. The HPHT processing cycle in the 35-L HP machine at 121C and688 MPa is illustrated in Fig. 7. The packages were treated immediately afterpackaging to minimize the oxygen exposure of the strips. The results arereported in Table 5 as a number of positive samples to the total number of

70

80

90

100

110

120

130

0 50 100 150 200 250 300 350 400Time (s)

Tem

pera

ture

(C)

0

100

200

300

400

500

600

700

800

Pres

sure

(MPa

)

Tc 1

Tc 2

Pressure

FIG. 7. TIME-TEMPERATURE PROFILE IN THE EGG PATTY DURING HIGHPRESSUREHIGH TEMPERATURE CYCLE IN A 35-L HP UNIT AT 688 MPA AND 121C

Water was used as compression medium.


samples treated at the defined process conditions. In addition, the processlethality was calculated as time to achieve 6 logs reduction of the PA 3679spores or 6D process (Table 6). It was found that a process temperature of105C was not sufficient to destroy the PA 3679 spores during the holding timestested. However, the increase of process temperature up to 110 and 115Cachieved sterility after 5 min of treatment at 688 MPa. The HPHT treatmentat 121C was sufficient to destroy 6 logs of PA 3679 spores after a minimumholding time of 3 min.

The experimental observations were in agreement with calculated pro-cessing times given in Table 6. For example, the minimum calculated processtime at 110 and 115C was 5.41 and 3.38 min, respectively. An increase of theprocess temperature of up to 121C reduced the required process time to1.92 min.

Furthermore, the spore strips of G. stearothermophilus used for verifica-tion of process temperature were located at the top, center and bottom of theHP vessel. The selected process temperature was 105C and the holding timevaried 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.

TABLE 5.HPHT INACTIVATION OF CLOSTRIDIUM SPOROGENES BIOINDICATOR SPORES IN EGG

PATTIES PROCESSED IN 35-L HP VESSEL AT 688 MPA

Initial temperature (C) Process temperature (C) Number of positive samples after holding

3 min 5 min 4 min

73 105 3(+)/3 3(+)/3 3(+)/380 110 3(+)/3 3(+)/3; 2(+)/6 0/3; 0/685 115 3(+)/3 1(+)/6 0/3; 0/690 121 0/3 0/3 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 86.8 520.8 (8.70 min) 100 17.7 106.2 (1.80 min)110 54.1 324.6 (5.41 min) 105 14.9 89.4 (1.50 min)115 33.8 202.8 (3.38 min) 111 11.5 69.0 (1.15 min)121 19.2 115.2 (1.92 min) 121 6.6 39.6 (0.66 min)

T, temperature.


Some survivors were found in the samples placed at the top and the bottom ofthe carrier, indicating some temperature variations because of possible con-vection heat transfer in the HP vessel (Table 7). Increasing the holding time upto 5 min resulted in the complete destruction of spores in all locations. Itshould be pointed out that experimentally observed holding time was longerthan 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 inactivationkinetics of C. sporogenes PA 3679 spores in phosphate buffer was a functionof pressure and temperature. In order to get the desired processing temperatureand by taking compression heating into account, preheating conditions need tobe established. A treatment that combines pressure with temperature wasnecessary to inactivate PA 3679 spores. The inactivation kinetics duringholding period of HP cycle at constant pressures and temperatures was welldescribed by a log-linear regression model similar to thermal processing. Therate of clostridial-spore inactivation increased with both pressure and tempera-ture. The D-values ranged from 49.0 to 357.4 s in the pressure range of600800 MPa and the testing temperatures of 91, 100, 108C. As expected withincreasing temperature and pressure, the D-values decreased. It was demon-strated that a combination of pressure and temperature is very important inreducing the processing time. The kinetic parameters were more pressure-sensitive at higher temperatures. However, the pressure enhancement effectsobserved were significantly less than those seen using G. stearothermophilusspores. It is also noteworthy to see that PA 3679 spores are less resistantthermally than G. stearothermophilus spores used in previous studies. Thedegree of acceleration in inactivation observed under HP conditions with C.sporogenes spores was significantly lower than that seen using G. stearother-mophilus spores.

TABLE 7.HPHT INACTIVATION OF GEOBACILLUS STEAROTHERMOPHILUS ATCC 7953

BIOINDICATOR SPORES IN EGG PATTIES PROCESSED IN 35-L HP VESSEL AT 105C AND688 MPA

Placement in HP vessel Number of positive samples after holding at 688 MPa

2 min 3 min 4 min 5 min

Top 3(+)/3 3(+)/3 1(+)/3 0/3Middle 3(+)/3 3(+)/3 0/3 0/3Bottom 3(+)/3 3(+)/3 1(+)/3 0/3


Using Pflugs concept of using the endpoint of a sterilization to specify aprocess, it becomes apparent that the duration of the HPHT process requiredto achieve commercial sterility in terms of spoilage-causing spore-formers isdrastically reduced compared to thermal sterilization. The microbiologicalverification runs in packed egg patties confirmed the adequacy of the HPHTprocess at 110121C to destroy clostridial spores. Additional lethalityachieved during the pressurization and depressurization cycle contributed tothe overall lethality of the HPHT sterilization process. The obtained resultsconfirmed that spore bioindicators can be successfully used to measure theactual delivered process in HPHT sterilization.

ACKNOWLEDGMENTS

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

REFERENCES

CLERY-BARRAUD, C., GAUBERT, A., MASSON, P. and VIDAL, D. 2004.Combined effects of high pressure and temperature for inactivation ofBacillus anthracis spores. Appl. Environ. Microb. 70(1), 635637.

GOLA, S., FOMAN, C., CARPI, G., MAGGI, A. and ROVERE, P. 1996.Inactivation of bacterial spores in phosphate buffer and in vegetablecream treated at high pressures. Problems Biotechnol. 13, 253259.

HEIJ, W., VAN DEN BERG, R., VAN SCHEPDAEL, L. and HOOGLAND,H. 2005. Sterilization only better. New Food 2, 5661.

MAGGI, A., GOLA, S., ROVERE, P., MIGLIOLI, L., DALLAGLIO, G. andLONNEBORG, N.G. 1996. Effects of combined high pressure-temperature treatments on Clostridium sporogenes spores in liquidmedia. Ind. Conserve 71, 814.

MILLS, G., EARNSHAW, R. and PATTERSON, M.F. 1998. Effects of highhydrostatic pressure on Clostridium sporogenes spores. Lett. Appl.Microbiol. 26, 227230.

PATAZCA, E., DUNN, J., KOUTCHMA, T. and RAMASWAMY, H. 2005.Inactivation Kinetics of Bacillus stearothermophilus Spores in WaterUsing High Pressure Processing at Elevated Temperatures, submitted forpublication.


PFLUG, I.J. 1987. Using the straight-line semi logarithmic microbial destruc-tion model as an engineering design model for determining the F-valuefor heat processes. J. Food Protect. 50(4), 342246.

PFLUG, I.J. and ZEGHMAN, L.G. 1985. Microbial Death Kinetics in theHeat Processing of Food: Determining an F-value. Proceedings ofAseptic Processing and Packaging of Foods, Sept 912, 1985, Tylosand,Sweden, pp. 211220.

ROVERE, P., MAGGI, A., SCARAMUZZA, N., GOLA, N., MIGLIOLI, L.,CARPI, G. and DALLAGLIO, G. 1996a. High-pressure heat treatments:Evaluation of the sterilizing effect and of thermal damage. Ind. Conserve71, 473483.

ROVERE, P., MIGLIOLI, L., LONNEBOLG, N.G. and GOLA, N. 1998.Modeling and calculation of the sterilizing effect in high pressure heat-treatments. Ind. Conserve 73, 303314.

STUMBO, C.R. 1973. Thermobacteriology in Food Processing. AcademicPress, New York, NY.


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