Inactivation of Clostridium botulinum nonproteolytic type B spores by high pressure processing at moderate to elevated high temperatures

The aim of this study was to determine the resistance of multiple strains of the three nonproteolytic types of Clostridium botulinum (seven strains of type E, eight of type B, and two of type F) spores exposed to combined high pressure and thermal processing. The resistance of spores suspended in N-(2-acetamido)-2-aminoethanesulfonic acid (ACES) buffer (0.05 M, pH 7) was determined at a process temperature of 80°C with high pressures of 600, 650, and 700 MPa using a laboratory-scale pressure test system. Spores of C. botulinum serotype E strains demonstrated less resistance than nonproteolytic spores of type B or F strains when processed at 80°C and 600 MPa for up to 15 min. All C. botulinum type E strains were reduced by >6.0 log units within 5 min under these conditions. Among the nonproteolytic type B strains, KAP 9-B was the most resistant, resulting in reductions of 2.7, 5.3, and 5.5 log, coinciding with D-values of 7.7, 3.4, and 1.8 min at 80°C and 600, 650, and 700 MPa, respectively. Of the two nonproteolytic type F strains, 610F was the most resistant, showing 2.6-, 4.5-, and 5.3-log reductions with D-values of 8.9, 4.3, and 1.8 min at 80°C and 600, 650, and 700 MPa, respectively. Pulsed-field gel electrophoresis was performed to examine the genetic relatedness of strains tested and to determine if strains with similar banding patterns also exhibited similar D-values. No correlation between the genetic fingerprint of a particular strain and its resistance to high pressure processing was observed.

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Combined High Pressure and Thermal Processing on Inactivation of Type A and Proteolytic Type B Spores of Clostridium botulinum

Journal of Food Protection ◽

10.4315/0362-028x.jfp-12-538 ◽

2013 ◽

Vol 76 (8) ◽

pp. 1384-1392 ◽

Cited By ~ 16

Author(s):

N. RUKMA REDDY ◽

KRISTIN M. MARSHALL ◽

TRAVIS R. MORRISSEY ◽

VIVIANA LOEZA ◽

EDUARDO PATAZCA ◽

...

Keyword(s):

High Pressure ◽

Thermal Processing ◽

Clostridium Botulinum ◽

High Pressures ◽

Type A ◽

Test System ◽

High Pressure Processing ◽

Type B ◽

Botulinum Type ◽

D Values

The aim of this study was to determine the resistance of multiple strains of Clostridium botulinum type A and proteolytic type B spores exposed to combined high pressure and thermal processing and compare their resistance with Clostridium sporogenes PA3679 and Bacillus amyloliquefaciens TMW-2.479-Fad-82 spores. The resistance of spores suspended in N-(2-acetamido)-2-aminoethanesulfonic acid (ACES) buffer (0.05 M, pH 7.0) was determined at a process temperature of 105°C, with high pressures of 600, 700, and 750 MPa by using a laboratory-scale pressure test system. No surviving spores of the proteolytic B strains were detected after processing at 105°C and 700 MPa for 6 min. A >7-log reduction of B. amyloliquefaciens spores was observed when processed for 4 min at 105°C and 700 MPa. D-values at 105°C and 700 MPa for type A strains ranged from 0.57 to 2.28 min. C. sporogenes PA3679 had a D-value of 1.48 min at 105°C and 700 MPa. Spores of the six type A strains with high D-values along with C. sporogenes PA3679 and B. amyloliquefaciens were further evaluated for their pressure resistance at pressures 600 and 750 MPa at 105°C. As the process pressure increased from 600 to 750 MPa at 105°C, D-values of some C. botulinum strains and C. sporogenes PA3679 spores decreased (i.e., 69-A, 1.91 to 1.33 min and PA3679, 2.35 to 1.29 min). Some C. botulinum type A strains were more resistant than C. sporogenes PA3679 and B. amyloliquefaciens to combined high pressure and heat, based on D-values determined at 105°C. Pulsed-field gel electrophoresis (PFGE) was also performed to establish whether strains with a similar restriction banding pattern also exhibited similar D-values. However, no correlation between the genomic background of a strain and its resistance to high pressure processing was observed, based on PFGE analysis. Spores of proteolytic type B strains of C. botulinum were less resistant to combined high pressure and heat (700 MPa and 105°C) treatment when compared with spores of type A strains.

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INACTIVATION OF CLOSTRIDIUM BOTULINUM TYPE E SPORES BY HIGH PRESSURE PROCESSING

Journal of Food Safety ◽

10.1111/j.1745-4565.1999.tb00252.x ◽

1999 ◽

Vol 19 (4) ◽

pp. 277-288 ◽

Cited By ~ 75

Author(s):

N.R. REDDY ◽

H. M. SOLOMON ◽

G.A. FINGERHUT ◽

E.J. RHODEHAMEL ◽

V.M. BALASUBRAMANIAM ◽

...

Keyword(s):

High Pressure ◽

Clostridium Botulinum ◽

High Pressure Processing ◽

Botulinum Type ◽

Clostridium Botulinum Type E

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Non-linear pressure/temperature-dependence of high pressure thermal inactivation of proteolytic Clostridium botulinum type B in foods

PLoS ONE ◽

10.1371/journal.pone.0187023 ◽

2017 ◽

Vol 12 (10) ◽

pp. e0187023 ◽

Cited By ~ 3

Author(s):

Maximilian B. Maier ◽

Christian A. Lenz ◽

Rudi F. Vogel

Keyword(s):

High Pressure ◽

Temperature Dependence ◽

Clostridium Botulinum ◽

Thermal Inactivation ◽

Type B ◽

Botulinum Type ◽

Non Linear

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Thermal and Pressure-Assisted Thermal Destruction Kinetics for Spores of Type A Clostridium botulinum and Clostridium sporogenes PA3679

Journal of Food Protection ◽

10.4315/0362-028x.jfp-15-310 ◽

2016 ◽

Vol 79 (2) ◽

pp. 253-262 ◽

Cited By ~ 5

Author(s):

N. RUKMA REDDY ◽

EDUARDO PATAZCA ◽

TRAVIS R. MORRISSEY ◽

GUY E. SKINNER ◽

VIVIANA LOEZA ◽

...

Keyword(s):

High Pressure ◽

High Temperature ◽

High Temperatures ◽

Clostridium Botulinum ◽

Pilot Scale ◽

Inactivation Kinetics ◽

Clostridium Sporogenes ◽

Log Reduction ◽

Pressure Test ◽

D Values

ABSTRACT The purpose of this study was to determine the inactivation kinetics of the spores of the most resistant proteolytic Clostridium botulinum strains (Giorgio-A and 69-A, as determined from an earlier screening study) and of Clostridium sporogenes PA3679 and to compare the thermal and pressure-assisted thermal resistance of these spores. Spores of these strains were prepared using a biphasic medium method. C. sporogenes PA3679 spores were heat treated before spore preparation. Using laboratory-scale and pilot-scale pressure test systems, spores of Giorgio-A, 69-A, and PA3679 suspended in ACES [N-(2-acetamido)-2-aminoethanesulfonic acid] buffer (pH 7.0) were exposed to various combinations of temperature (93 to 121°C) and pressure (0.1 to 750 MPa) to determine their resistance. More than a 5-log reduction occurred after 3 min at 113°C for spores of Giorgio-A and 69-A and after 5 min at 117°C for spores of PA3679. A combination of high temperatures (93 to 121°C) and pressures yielded greater log reductions of spores of Giorgio-A, 69-A, and PA3679 compared with reduction obtained with high temperatures alone. No survivors from initial levels (>5.0 log CFU) of Giorgio-A and 69-A were detected when processed at a combination of high temperature (117 and 121°C) and high pressure (600 and 750 MPa) for <1 min in a pilot-scale pressure test system. Increasing pressure from 600 to 750 MPa at 117°C decreased the time from 2.7 to 1 min for a >4.5-log reduction of PA3679 spores. Thermal D-values of Giorgio-A, 69-A, and PA3679 spores decreased (i.e., 29.1 to 0.33 min for Giorgio-A, 40.5 to 0.27 min for 69-A, and 335.2 to 2.16 min for PA3679) as the temperature increased from 97 to 117°C. Pressure-assisted thermal D-values of Giorgio-A, 69-A, and PA3679 also decreased as temperature increased from 97 to 121°C at both pressures (600 and 750 MPa) (i.e., 17.19 to 0.15 min for Giorgio-A, 9.58 to 0.15 min for 69-A, and 12.93 to 0.33 min for PA3679 at 600 MPa). At higher temperatures (117 or 121°C), increasing pressure from 600 to 750 MPa had an effect on pressure-assisted thermal D-values of PA3679 (i.e., at 117°C, pressure-assisted thermal D-value decreased from 0.55 to 0.28 min as pressure increased from 600 to 750 MPa), but pressure had no effect on pressure-assisted thermal D-values of Giorgio-A and 69-A. When compared with Giorgio-A and 69-A, PA3679 had higher thermal and pressure-assisted thermal D-values. C. sporogenes PA3679 spores were generally more resistant to combinations of high pressure and high temperature than were the spores of the C. botulinum strains tested in this study.

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Effect of Sporulation Temperature on the Resistance of Clostridium botulinum Type A Spores to Thermal and High Pressure Processing

Journal of Food Protection ◽

10.4315/0362-028x.jfp-14-186 ◽

2015 ◽

Vol 78 (1) ◽

pp. 146-150 ◽

Cited By ~ 5

Author(s):

KRISTIN M. MARSHALL ◽

LOUIS NOWACZYK ◽

TRAVIS R. MORRISSEY ◽

VIVIANA LOEZA ◽

LINDSAY A. HALIK ◽

...

Keyword(s):

High Pressure ◽

Heat Resistance ◽

Clostridium Botulinum ◽

Type A ◽

Test System ◽

High Pressure Processing ◽

Decimal Reduction Time ◽

Optimum Growth Temperature ◽

Botulinum Type ◽

Log Linear

The purpose of this study was to determine the effect of sporulation temperature on the resistance of Clostridium botulinum type A spores of strains 62A and GiorgioA to thermal and high pressure processing (HPP). Spore crops produced in Trypticase–peptone–glucose–yeast extract broth at four incubation temperatures (20, 27, 37, and 41°C) were harvested, and heat resistance studies were conducted at 105°C (strain 62A) and 100°C (strain GiorgioA). Resistance to HPP was evaluated by subjecting the spores to a high pressure (700 MPa) and temperature combination (105°C, strain 62A; 100°C strain GiorgioA) in a laboratory-scale pressure test system. The decimal reduction time (D-value) was calculated using the log-linear model. Although the time to sporulation for GiorgioA was shorter and resulted in higher spore concentrations than for 62A at 20, 27, and 37°C, GiorgioA did not produce a sufficient spore crop at 41°C to be evaluated. The heat resistance of 62A spores was greatest when produced at 27°C and decreased for spore crops produced above or below 27°C (D105°C-values: 20°C, 1.9 min; 27°C, 4.03 min; 37°C, 3.66 min; and 41°C, 3.5 min; P < 0.05). Unlike 62A, the heat resistance behavior of GiorgioA spores increased with rising sporulation temperature, and spores formed at the organism's optimum growth temperature of 37°C were the most resistant (D100°C-values: 20°C, 3.4 min; 27°C, 5.08 min; and 37°C, 5.65 min; P < 0.05). Overall, all spore crops were less resistant to pressure-assisted thermal processing than thermal treatment alone. Sporulation temperature has an effect on the resistance of C. botulinum spores to heat and HPP, and is characteristic to a particular strain. Knowledge of the effect of sporulation temperature on the resistance of C. botulinum spores is vital for the production of spores utilized in thermal and high pressure inactivation studies.

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Inactivation of Clostridium botulinum Type A Spores by High-Pressure Processing at Elevated Temperatures

Journal of Food Protection ◽

10.4315/0362-028x-66.8.1402 ◽

2003 ◽

Vol 66 (8) ◽

pp. 1402-1407 ◽

Cited By ~ 73

Author(s):

N. R. REDDY ◽

H. M. SOLOMON ◽

R. C. TETZLOFF ◽

E. J. RHODEHAMEL

Keyword(s):

High Pressure ◽

Phosphate Buffer ◽

Clostridium Botulinum ◽

Elevated Temperatures ◽

Type A ◽

High Pressure Processing ◽

Maximum Temperature ◽

Maximum Pressure ◽

Botulinum Type ◽

Pressure Maximum

The effects of high-pressure treatments at various temperature-time combinations on the inactivation of spores of Clostridium botulinum type A strains 62-A and BS-A in phosphate buffer (0.067 M, pH 7.0) and in a crabmeat blend were investigated. The log unit reduction of strain 62-A spores increased significantly as the processing pressure increased from 417 to 827 MPa (from 60,000 to 120,000 lb/in2) at 75°C. The reduction of BS-A and 62-A spores in either medium increased as processing temperatures increased from 60 to 75°C and processing times increased from 5 to 15 or 20 min at a maximum pressure of 827 MPa. Approximately 2- and 3-log reductions of BS-A and 62-A spores, respectively, in phosphate buffer were obtained at the maximum pressure–maximum temperature combination of 827 MPa and 75°C for a processing time of 20 min. Processing for 15 min at the maximum pressure–maximum temperature combination resulted in maximum reductions of 3.2 and 2.7 log units for BS-A and 62-A spores, respectively, in the crabmeat blend. Results obtained in this study indicate that the crabmeat blend did not protect BS-A and 62-A spores against inactivation by high-pressure processing.

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Effect of Packaging Systems and Pressure Fluids on Inactivation of Clostridium botulinum Spores by Combined High Pressure and Thermal Processing

Journal of Food Protection ◽

10.4315/0362-028x.jfp-12-181 ◽

2013 ◽

Vol 76 (3) ◽

pp. 448-455 ◽

Cited By ~ 8

Author(s):

EDUARDO PATAZCA ◽

TRAVIS R. MORRISSEY ◽

VIVIANA LOEZA ◽

N. RUKMA REDDY ◽

GUY E. SKINNER ◽

...

Keyword(s):

High Pressure ◽

Thermal Processing ◽

Clostridium Botulinum ◽

Pilot Scale ◽

Test System ◽

High Pressure Processing ◽

Laboratory Scale ◽

Packaging System ◽

Test Systems ◽

Pressure Test

Several studies have been published on the inactivation of bacterial spores by using high pressure processing in combination with heat. None of the studies investigated the effect of the packaging system or the pressurizing fluid on spore inactivation. The objective of this study was to select and validate an appropriate packaging system and pressure transfer fluid for inactivation of Clostridium botulinum spores by using high pressure processing in combination with thermal processing. Inactivation of spores packaged in three packaging systems (plastic pouches, cryovials, and transfer pipettes) was measured in two pressure test systems (laboratory-scale and pilot-scale) at 700 MPa and >105°C. Total destruction (>6.6-log reduction) of the spores packaged in the graduated tube part of transfer pipettes was obtained after processing for up to 10 min at 118°C and 700 MPa in both pressure test systems, compared with the spores packaged either in plastic pouches or cryovials. Reduction of spores packaged in plastic pouches was lowest (<4.8 log) for both pressure test systems when processed at the same conditions (i.e., 700 MPa and 118°C). Within the pilot-scale pressure system, increasing the process temperature from 118 to 121°C at 700 MPa for 10 min resulted in only a small increase in spore reduction (<5.1 log) for spores packaged in plastic pouches, whereas there were no recoverable spores for either of the other two packaging systems. Use of plastic pouches for packaging spores in inactivation kinetic studies could lead to erroneous conclusions about the effect of high pressure in combination with heat. BioGlycol is the pressure–heat transfer fluid of choice, as compared with Duratherm oil, to maximize the temperature response rate during pressurization within the laboratory-scale pressure test system.

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High-Pressure-Mediated Survival of Clostridium botulinum and Bacillus amyloliquefaciens Endospores at High Temperature

Applied and Environmental Microbiology ◽

10.1128/aem.72.5.3476-3481.2006 ◽

2006 ◽

Vol 72 (5) ◽

pp. 3476-3481 ◽

Cited By ~ 156

Author(s):

Dirk Margosch ◽

Matthias A. Ehrmann ◽

Roman Buckow ◽

Volker Heinz ◽

Rudi F. Vogel ◽

...

Keyword(s):

High Pressure ◽

Temperature Effects ◽

Bacillus Amyloliquefaciens ◽

Clostridium Botulinum ◽

Ambient Pressure ◽

Isothermal Holding ◽

Bacterial Spores ◽

Type B ◽

Bacterial Endospores ◽

Heat Tolerant

ABSTRACT Endospores of proteolytic type B Clostridium botulinum TMW 2.357 and Bacillus amyloliquefaciens TMW 2.479 are currently described as the most high-pressure-resistant bacterial spores relevant to food intoxication and spoilage in combined pressure-temperature applications. The effects of combined pressure (0.1 to 1,400 MPa) and temperature (70 to 120ï¿½C) treatments were determined for these spores. A process employing isothermal holding times was established to distinguish pressure from temperature effects. An increase in pressure (600 to 1,400 MPa) and an increase in temperature (90 to 110ï¿½C) accelerated the inactivation of C. botulinum spores. However, incubation at 100ï¿½C, 110ï¿½C, or 120ï¿½C with ambient pressure resulted in faster spore reduction than treatment with 600 or 800 MPa at the same temperature. This pressure-mediated spore protection was also observed at 120ï¿½C and 800, 1,000, or 1,200 MPa with the more heat-tolerant B. amyloliquefaciens TMW 2.479 spores. Inactivation curves for both strains showed a pronounced pressure-dependent tailing, which indicates that a small fraction of the spore populations survives conditions of up to 120ï¿½C and 1.4 GPa in isothermal treatments. Because of this tailing and the fact that pressure-temperature combinations stabilizing bacterial endospores vary from strain to strain, food safety must be ensured in case-by-case studies demonstrating inactivation or nongrowth of C. botulinum with realistic contamination rates in the respective pressurized food and equipment.

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