Clostridium Sporogenes PA Research Articles

Inactivation of Clostridium sporogenes PA 3679 spores by synergistic pressure-assisted thermal processing and antimicrobial compound combinations

Pressure-assisted thermal processing (PATP) technology is an emerging sterilization method for food processing, ensuring microbiological safety with minimal heat damage. This study investigated the potential antimicrobial efficacy of 25 compounds, including enzymes, polysaccharides, cyclodextrins, cationic surfactants, polymers, and plant and fruit extracts, for the inactivation of Clostridium sporogenes PA 3679 spores during PATP. Experiments were conducted using a laboratory-scale, high-pressure processor. Spores suspended in pressure-stable buffer containing a potential antimicrobial agent were subjected to pressure (600 MPa) at 90 °C or 105 °C for a holding time of 3- and 6-min. Spore survivors were enumerated by spread-plating on Trypticase-Peptone-Glucose-Yeast Extract (TPGY) agar and incubated anaerobically at 32 °C for 5 days. PATP treatment at 600 MPa, 90 °C (3-min holding time) and 105 °C (3 or 6-min holding time) inactivated the spore by 1.5-, 3.8-, and 5.8-log spores/mL, respectively. Among all the antimicrobials tested, low- and high-molecular-weight chitosan enhanced PATP (600 MPa, 105 °C, 6-min holding time) inactivation of C. sporogenes spores by 7.9- and 6.9-log reductions, respectively. The treatment also decreased particle size and increased ζ potential of chitosan. Overall, combining PATP with chitosan is a promising synergistic strategy for spore elimination, possibly due to damage to the spore protective layers by PATP followed by chitosan electrostatic interaction.

Role of Dipicolinic Acid in Heat Resistance of Spores of Clostridium botulinum and Clostridium sporogenes PA3679 by Thermal and Pressure-assisted Thermal Processing

Dipicolinic acid (DPA) is a major constituent of spores and reportedly provides protection against inactivation by various thermal processes; however, the relationship between DPA and resistance towards pressure-assisted thermal processing is not well understood. Thermal and pressure-assisted thermal inactivation studies of Clostridium botulinum nonproteolytic strains QC-B and 610-F, proteolytic strain Giorgio-A, and thermal surrogate Clostridium sporogenes PA3679 spores suspended in ACES buffer (0.05 M, pH 7.0) were performed to determine if a relationship exists between DPA release and log reduction of spores. Thermal inactivation at 80, 83, and 87 °C for nonproteolytic strains and 101, 105, and 108 °C for the proteolytic strain and thermal surrogate were conducted. Pressure-assisted thermal inactivation for nonproteolytic strains at 83 °C/600 MPa and for the proteolytic strain and thermal surrogate at 105 °C/600 MPa were performed. Surviving spores were enumerated by 5-tube MPN method for log reductions and analyzed for released DPA by liquid chromatography-tandem mass spectrometry. The correlation between MPN log reductions, released DPA, and D-values were calculated. A positive correlation between released DPA and log reduction of spores was observed for QC-B and 610-F at 80 and 83 °C (r = 0.6073 − 0.7755; P < 0.01). At 87 °C, a positive correlation was detected for 610-F (r = 0.4242, P < 0.05) and no correlation was observed for QC-B (r = 0.1641; P > 0.05). A strong, positive correlation (r = 0.8359 − 0.9284; P < 0.05) between released DPA and log reduction of spores was observed for Giorgio-A at 101, 105, and 108 °C, and a strong, positive correlation (r = 0.8402; P < 0.05) was observed for PA3679 at 101 °C. A positive correlation (r = 0.5646 − 0.6724; P < 0.01) was observed for QC-B, 610-F, and Giorgio-A after pressure-assisted thermal treatment. No correlation (r = 02494; P > 0.05) was found for PA3679 after pressure-assisted thermal treatment. These results suggest a correlation exists between DPA release and heat resistance; however, the level of correlation varied between strains and temperatures. The findings from this research may aid in the development of spore inactivation strategies targeting the thermal resistance profiles of various strains of C. botulinum spores.

Effects of field strength and frequency on inactivation of Clostridium sporogenes PA3679 spores during ohmic heating

The study explored the effects of electric field strength, temperature, and frequency on the inactivation of Clostridium sporogenes PA3679 spores (a surrogate of Clostridium botulinum) within buffer. The experiments were conducted at three different field strengths (30, 40, and 50 V/cm), temperatures (110, 120, and 130 °C), and frequencies (60, 1000, and 5000 Hz). Ohmic heating resulted in significantly greater reduction of C. sporogenes compared to conventional heating. Electric field strength significantly influenced spore inactivation. It was found that lower frequencies led to quicker rates of inactivation. Given that high field strengths have dual benefits of increasing both heating and spore kill rate, the possibility exists to process foods to sterility without a hold. This is expected to result in improved product quality, reduced process times and increased efficiency.

Synergistic effects of pressure, temperature, shear, and their interactions on Clostridium sporogenes PA3679 spore inactivation during ultra-shear processing

Ultra-shear technology (UST) is a semi-continuous high-pressure method for processing liquid foods. By pressurizing liquid foods up to 400 MPa and decompressing them through a shear valve, UST is designed for pasteurizing or sterilizing foods and modifying their structure and rheological characteristics. This study evaluated the lethal effects of pressure, holding time, temperature, shear, and their interactions for inactivating endospores of Clostridium sporogenes PA3679 suspended in pressure-stable 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer solution (0.1 M, pH 7.0, initial concentration = 6.3 log CFU/mL). Results indicated while thermal treatment at 105 °C did not have appreciable spore inactivation, isostatic treatment at 400 MPa and 105 °C for 5 min resulted in a 3.3-log inactivation. UST treatment at 400 MPa and 85 °C for 5 min, followed by 125 °C shear discharge resulted in 3.3-log reduction. Our analysis also revealed that thermal-pressure treatment history as well as their intensities also influence magnitude of spore reduction. This study revealed the sporicidal capability of UST and evaluated the relative importance of different lethal factors on spore inactivation. Industrial relevanceUltra-shear technology (UST) is a novel processing method that has a potential to help produce value-added liquid foods, sauces, gels, and nanoemulsions, while helping to preserve the foods by inactivating undesirable microorganisms using the combination of pressure, shear, and temperature. This research provided an insight on the effects of key UST lethal factors on spores of C. sporogenes PA3679 for commercial sterilization validation. The results of this study could help the industry select the safe-harbor UST processing parameters for sterilizing food products in the future.

Rapid detection and quantitation of dipicolinic acid from Clostridium botulinum spores using mixed-mode liquid chromatography-tandem mass spectrometry.

Analysis of the dipicolinic acid (DPA) released from Clostridium botulinum spores during thermal processing is crucial to obtaining a mechanistic understanding of the factors involved in spore heat resistance and related food safety applications. Here, we developed a novel mixed-mode liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for detection of the DPA released from C. botulinum type A, nonproteolytic types B and F strains, and nonpathogenic surrogate Clostridium sporogenes PA3679 spores. DPA was retained on a mixed-mode C18/anion exchange column and was detected using electrospray ionization (ESI) positive mode within a 4-min analysis time. The intraday and interday precision (%CV) was 1.94-3.46% and 4.04-8.28%, respectively. Matrix effects were minimal across proteolytic type A Giorgio-A, nonproteolytic types QC-B and 202-F, and C. sporogenes PA3679 spore suspensions (90.1-114% of spiked DPA concentrations). DPA recovery in carrot juice and beef broth ranged from 105 to 118%, indicating limited matrix effects of these food products. Experiments that assessed the DPA released from Giorgio-A spores over the course of a 5-min thermal treatment at 108°C found a significant correlation (R = 0.907; P < 0.05) between the log reduction of spores and amount of DPA released. This mixed-mode LC-MS/MS method provides a means for rapid detection of DPA released from C. botulinum spores during thermal processing and has the potential to be used for experiments in the field of food safety that assess the thermal resistance characteristics of various C. botulinum spore types.

The effect of high pressure combined with moderate temperature and peptidoglycan fragments on spore inactivation

High pressure processing (HPP) is a promising non-thermal processing method for food production. However, extremely high pressure and temperature are often required to achieve spores inactivation and commercial sterilization using HPP. In this study, the combined treatment of HPP, moderate temperature, and peptidoglycan fragments (PGF) for spore inactivation was investigated. The combined treatment of 200 MPa and 1 mg/mL PGF at 80 °C for 20 min resulted in 8.6 log inactivation of Bacillus subtilis 168 and more than 5 log reductions of Clostridium sporogenes PA3679 spores, respectively. A strong synergistic effect on spore inactivation among HPP, PGF, and temperature was observed. By comparing the effect of the treatment on the fluidity of the inner membrane and structural change of spores using fluorescence assay, a probable inactivation mechanism was proposed. It was concluded that the spores were firstly triggered to enter the Stage I of the germination process by HPP and PGF, and then immediately inactivated by the mild heat. This novel processing method could be an alternative to ensure commercial sterilization in the food industry.

Building of Pressure-Assisted Ultra-High Temperature System and Its Inactivation of Bacterial Spores.

The pressure-assisted ultra-high temperature (PAUHT) system was built by using soybean oil as pressure-transmitting medium, and the multiple regression equation of soybean oil temperature change (ΔTP) during pressurization as a function of initial temperature (Ti) and set pressure (P) was developed: ΔTP = -13.45 + 0.46 Ti + 0.0799 P - 0.0037 - 2.83 × 10-5 P2. The fitted model indicated that the temperature of the system would achieve ≥121°C at 600 MPa when the initial temperature of soybean oil was ≥84°C. The PAUHT system could effectively inactivate spores of Bacillus subtilis 168 and Clostridium sporogenes PA3679 (less than 1 min). Treatment of 600 MPa and 121°C with no holding time resulted in a 6.75 log reductions of B. subtilis 168 spores, while treatment of 700 MPa and 121°C with pressure holding time of 20 s achieved more than 5 log reductions of C. sporogenes PA3679 spores. By comparing the PAUHT treatment with high pressure or thermal treatment alone, and also studying the effect of compression on spore inactivation during PAUHT treatment, the inactivation mechanism was further discussed and could be concluded as follows: both B. subtilis 168 and C. sporogenes PA3679 spores were triggered to germinate firstly by high pressure, which was enhanced by increased temperature, then the germinated spores were inactivated by heat.

Effect of electro-activated aqueous solutions, nisin and moderate heat treatment on the inactivation of Clostridium sporogenes PA 3679 spores in green beans puree and whole green beans

In this work, the synergistic effect of electro-activated solutions (EAS) of potassium acetate and potassium citrate, nisin and moderate heat treatment to inactivate C. sporogenes PA 3679 spores was evaluated in green beans puree and whole green beans. Electro-activated solutions (EAS) of potassium acetate and potassium citrate were generated under 400 mA during 60 min. They were characterized by an oxidation-reduction potential (ORP) and pH values ranged from +300 to +1090 mV and 2.8 to 3.67, respectively. Moreover, the EAS were combined with a bacteriocin nisin at concentrations of 250, 500, 750 and 1000 IU/mL and the targeted sporicidal effect was evaluated under moderate heat treatment. The inoculated mixtures were subjected to temperatures of 95, 105 and 115 °C for exposure times of 5, 15 and 30 min. After plate counting, the synergistic effect of the hurdle principle composed of electro-activated solutions, nisin and moderate temperatures was demonstrated. The obtained results showed that the synergistic effect of the used hurdle was able to achieve an inactivation efficacy of 5.9–6.1 log CFU/mL. Furthermore, experiments carried out with whole green beans showed that spore inactivation level was significantly higher and reach 6.5 log CFU/mL. Moreover, spore morphology was examined by transmission electron microscopy and the obtained micrographs showed important damages in all of the treated spores.

Headspace oxygen as a hurdle to improve the safety of in-pack pasteurized chilled food during storage at different temperatures

This study investigated the use of headspace oxygen in a model food system to prevent the growth of anaerobic pathogenic bacteria in in-pack pasteurized food at various storage temperatures. Three model food formulations prepared with tryptic soy broth and three agar concentrations (0.1, 0.4 and 1.0%) were sealed without removing the air from the package in high oxygen barrier pouches (OTR=0.3cm3/m2·day·atm). Important properties influencing bacterial growth, including pH and water activity (aw) were determined. The oxygen sorption kinetics of each model food were obtained at three different storage temperatures (8, 12, and 20°C) using an OxySense Gen III 300 system. An analytical solution of Fick's second law was used to determine the O2 diffusion coefficient. Growth challenge studies at 12 and 20°C were conducted at three selected locations (top, center and bottom layers) in model foods containing 1% agar. Model foods were inoculated with Clostridium sporogenes PA 3679 (300spores/mL), and were classified as low-acid (pH>4.5, aw>0.85). When the storage temperature decreased from 20 to 8°C, the oxygen diffusion decreased from 0.82×10−9m2/s to 0.68×10−9m2/s. As the agar concentration was increased from 0.1 to 1.0%, the effective oxygen permeability decreased significantly (p=0.007) from 0.88×10−9m2/s to 0.65×10−9m2/s. When the inoculated model foods were stored at 12°C for 14days, C. sporogenes PA 3679 was unable to grow. As the storage temperature was increased to 20°C, significant bacterial growth was observed with storage time (p<0.0001), and the C. sporogenes PA 3679 population increased by around 6logCFU/g. However, the location of the food did not influence the growth distribution of C. sporogenes PA 3679. These results demonstrate that oxygen diffusion from the pouch headspace was primarily limited to the food surface. Findings suggest that the air/oxygen present in the package headspace may not be considered as a food safety hurdle in the production of pasteurized packaged food.

Genetic Characterization of the Exceptionally High Heat Resistance of the Non-toxic Surrogate Clostridium sporogenes PA 3679

Clostridium sporogenes PA 3679 is a non-toxic endospore former that is widely used as a surrogate for Clostridium botulinum by the food processing industry to validate thermal processing strategies. PA 3679 produces spores of exceptionally high heat resistance without botulinum neurotoxins, permitting the use of PA 3679 in inoculated pack studies while ensuring the safety of food processing facilities. To identify genes associated with this heat resistance, the genomes of C. sporogenes PA 3679 isolates were compared to several other C. sporogenes strains. The most significant difference was the acquisition of a second spoVA operon, spoVA2, which is responsible for transport of dipicolinic acid into the spore core during sporulation. Interestingly, spoVA2 was also found in some C. botulinum species which phylogenetically cluster with PA 3679. Most other C. sporogenes strains examined both lack the spoVA2 locus and are phylogenetically distant within the group I Clostridium, adding to the understanding that C. sporogenes are dispersed C. botulinum strains which lack toxin genes. C. sporogenes strains are thus a very eclectic group, and few strains possess the characteristic heat resistance of PA 3679.

Effect of Potassium Sorbate Addition on Germination and Outgrowth of Clostridium Sporogenes Pa 3679 Spores in Low Cost Bologna Type Sausage During Ambient Temperature Storage

ObjectivesLow cost Bologna type product reaches high production volumes in Brazil and is marketed at ambient temperature. Previous studies including challenge test with Clostridium sporogenes PA 3679 indicated that water activity (aw) up to 0.96 prevented spores germination. The objective of this study was to evaluate the efficacy of potassium sorbate addition (0.25%) on limiting germination of Clostridium sporogenes PA 3679 in low lost Bologna type product formulated with aw 0.96 and 0.965 during 90 d storage at 27°C.Materials and MethodsThe experiment comprised 4 treatments formulated with mechanically deboned chicken meat (60%), edible offal (2%), pork skin (8%), 80/20 pork trimmings (15%), water (4%), texturized soy protein (3.5%), tapioca starch (5%), 150ppm ingoing sodium nitrite, 500ppm sodium isoascorbate, 0.15% sodium acid pyrophosphate and 0.35% sodium tripolyphosphate. The amount of sodium chloride varied to achieve the desired aw (0.96 and 0.965). Potassium sorbate was added at 0.25% in the final product. Raw material was comminuted in a bowl chopper. The raw batter was vacuum stuffed in 60mm PVDC impermeable casing. The samples were cooked in a cooking chamber with direct steam until 75°C was reached in the center of the product. Cooling was performed in running tap water until 27°C and the samples were kept in a chamber at 27°C (± 2°C) during 90 d. Lactic acid bacteria, Enterobacteriaceae, mesophilic aerobic, sulphite-reducing clostridia (spores and vegetative cells) counts, residual nitrite and pH value were evaluated in 3 samples of each treatment 24h after processing and at 15, 30, 60, and 90 d. Data (log CFU/g, residual sodium nitrite and pH value) were analyzed using GLM model procedure of SAS (SAS Inst. Inc., Cary, NC) as a 2 aw (0.96, 0.965) X 2 potassium sorbate amounts (0, 0.25%) X 5 storage time (0, 15, 30, 60, 90) factorial design with repeated measurements. Interactions and main effects were considered significant at p < 0.05. Least square means for significant effects (p < 0.05) were separated by Tukey’s test.ResultsLactic acid bacteria and Enterobacteriaceae counts were below 1 Log CFU/g during shelf life for all treatments. There was a significant effect (p < 0.05) of the interaction aw X sorbate X storage on mesophilic aerobic and sulphite-reducing counts (spores and total) and residual nitrite concentration. It was observed that sorbate addition prevented mesophilic aerobes growth and sulphite-reducing clostridia spores germination during 90 d storage at aw 0.96 and during 60 d at aw 0.965. Regarding residual nitrite, the addition of potassium sorbate decreased nitrite depletion at both water activities until 30 d and marked difference has been perceived at 0.965.ConclusionAddition of potassium sorbate may enhance microbial stability of this type of emulsified product and prevent spores germination during storage at ambient temperature. The amount of sodium in the product due to its high amount of sodium chloride required to reach 0.95 may be reviewed especially after other experiments including other shelf life enhancers such as sodium or potassium lactate which are effectives at inhibiting different microorganisms.

Acidification of potassium acetate and potassium citrate with/without KCl by electro-activation and impact of the solution on spores of Clostridium sporogenes PA 3679 at ambient temperature

Acidity and ORP of electro-activated potassium acetate and potassium citrate with and without KCl was studied and the effect of these solutions on spores of C. sporogenes was evaluated at ambient temperature. The ORP and amount of formed acid were proportional to the intensity of the applied electric current and duration of the electro-activation process. Electro-activation at 400 mA during 60 min yielded solution with ORP values of +417.50 to +1043.33 mV and pH of 3.18–3.47. The four solutions were used to study their effect on spores of C. sporogenes PA 3679 at ambient temperature. The application of electro-activated solutions resulted in a reduction level of >7 log CFU/mL of the spores. The sporicidal effect of the electro-activated solutions was demonstrated by transmission electron microscopy analysis. The micrographs showed several damages in the spores components such as core, coat and spore cortex.

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

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.

Application of electro-activated potassium acetate and potassium citrate solutions combined with moderate heat treatment on the inactivation of Clostridium sporogenes PA 3679 spores

Combined effects of moderate temperatures and the electro-activated aqueous solutions of potassium acetate and potassium citrate on the inactivation of C. sporogenes PA 3679 spores (D121°C = 1.18 min) were studied. Four types of solutions (potassium acetate with/without KCl and potassium citrate with/without KCl) were activated at 400 mA for 60 min. The oxidation reduction potential (ORP) and pH values ranged from + 417.50 to + 1043.33 mV and 3.18 to 3.47, respectively. The combination of these solutions with a moderate heat treatment (95 °C, 105 °C, and 115 °C) for different time (5, 10, 20, and 30 min) was sufficient to reach a 100% of spore destruction (inactivation) in a medium with an initial contamination level comprised between 7.0 and 7.8 log CFU/mL. The sporicidal effect of solutions was also present even if activated solutions were applied alone against spores without being combined with heat treatment. Spore morphology was determined under transmission electron microscopy and showed that there were important damages, such as rupture of spores and release of spore components in all of the treated spores. Thus, the sporicidal effect detected was the result of inactivation mechanisms of electro-activated solutions on spores. In almost all of observed micrographs, there were coreless spores, deformed spores, or debris of spores. The current investigation can be used for achieving further studies in order to better understand the mechanisms of inactivation of C. sporogenes spores by electro-activated solutions. This research article aims to study the combined effect of electro-activated potassium acetate and citrate solutions and moderate heat treatment on the viability of Clostridium sporogenes in model solutions as a non-pathogenic surrogate of Clostridium botulinum. The objective was to use hurdle technology to produce nutritious, minimally processed foods while ensuring food safety. Moreover, this approach allowed for a reduced level of sodium in canned foods since the solutions were sodium-free.

Genetic Diversity of Clostridium sporogenes PA 3679 Isolates Obtained from Different Sources as Resolved by Pulsed-Field Gel Electrophoresis and High-Throughput Sequencing.

Clostridium sporogenes PA 3679 is a nonpathogenic, nontoxic model organism for proteolytic Clostridium botulinum used in the validation of conventional thermal food processes due to its ability to produce highly heat-resistant endospores. Because of its public safety importance, the uncertain taxonomic classification and genetic diversity of PA 3679 are concerns. Therefore, isolates of C. sporogenes PA 3679 were obtained from various sources and characterized using pulsed-field gel electrophoresis (PFGE) and whole-genome sequencing. The phylogenetic relatedness and genetic variability were assessed based on 16S rRNA gene sequencing and whole-genome single nucleotide polymorphism (SNP) analysis. All C. sporogenes PA 3679 isolates were categorized into two clades (clade I containing ATCC 7955 NCA3679 isolates 1961-2, 1990, and 2007 and clade II containing PA 3679 isolates NFL, UW, FDA, and Campbell and ATCC 7955 NCA3679 isolate 1961-4). The 16S maximum likelihood (ML) tree clustered both clades within proteolytic C. botulinum strains, with clade I forming a distinct cluster with other C. sporogenes non-PA 3679 strains. SNP analysis revealed that clade I isolates were more similar to the genomic reference PA 3679 (NCTC8594) genome (GenBank accession number AGAH00000000.1) than clade II isolates were. The genomic reference C. sporogenes PA 3679 (NCTC8594) genome and clade I C. sporogenes isolates were genetically distinct from those obtained from other sources (University of Wisconsin, National Food Laboratory, U.S. Food and Drug Administration, and Campbell's Soup Company). Thermal destruction studies revealed that clade I isolates were more sensitive to high temperature than clade II isolates were. Considering the widespread use of C. sporogenes PA 3679 and its genetic information in numerous studies, the accurate identification and genetic characterization of C. sporogenes PA 3679 are of critical importance.

Simulation and evaluation of different statistical functions for describing lag time distributions of a bacterial growth curve

The objective of this study was to simulate the lag time distribution of individual bacterial cells in a population incubated under an isothermal condition. Spores of Clostridium sporogenes PA 3679 inoculated to ground beef were incubated at 37 °C to develop a growth curve and determine its apparent lag time. The lag times of individual cells were simulated using normal, lognormal, Gumbel, gamma, Weibull, and exponential distributions. The growth process was modeled using the McKellar model. A 4th order Runge–Kutta method was used to solve the McKellar model in conjunction with the Nelder–Mead method. The objective of the numerical analysis was to search for the parameters in each lag time distribution model and the kinetic parameters (specific growth rate and mean lag time) of the growth model such that a minimized residual sum of squared errors (RSS) was found.Although different in shapes, the simulation results showed that the performance of normal, lognormal, Gumbel, and gamma distributions was practically the same when used to fit the growth curve, yielding the same estimates of initial and stationary phase bacterial concentrations, specific growth rate, and mean lag time as well as RSS. The lognormal, Gumbel, and gamma distributions of the lag times were slightly skewed to the right of the mean. The standard deviations of the lag times with these distributions were relatively small, suggesting that cells may leave the lag phase almost simultaneously. The Weibull and exponential distributions were also suitable for describing the distribution of lag times, but with larger standard deviations. In general, all these statistical distributions were found suitable for simulating the distribution of lag times of individual cells in a population.

Mathematical modeling and growth kinetics of Clostridium sporogenes in cooked beef

Clostridium sporogenes PA 3679 is a common surrogate for proteolytic Clostridium botulinum for thermal process development and validation. However, little information is available concerning the growth kinetics of C. sporogenes in food. Therefore, the objective of this study was to investigate the growth kinetics of C. sporogenes in cooked beef under different temperature conditions.Ground beef samples, inoculated with C. sporogenes spores, were incubated at temperatures between 8 and 47 °C to examine the growth of this microorganism. Two primary models (Huang and Baranyi models) were used to analyze the growth data. The Ratkowsky square-root model was used as the secondary model to evaluate the effect of temperature on bacterial growth rate and lag time. The USDA IPMP 2013, a free data analysis tool for predictive microbiology, was used in data analysis.No growth of C. sporogenes was observed at temperatures below 15 °C for up to 25 days. At temperatures between 20 and 47 °C, C. sporogenes grew in cooked beef. The growth curves could be analyzed by both Huang and Baranyi models. The root mean squared error (RMSE) was 0.375 for the Huang model, and 0.441 for the Baranyi model with a global h0 of 10.46. The nominal minimum growth temperature (T0) estimated from the Huang model was 15.5 °C, which was 12.7 °C for the Baranyi model. The maximum growth temperature was 48.0 and 48.3 °C for the Baranyi and Huang models, respectively. The lag times and specific growth rates of C. sporogenes observed in this study were reasonably close to the data reported in the literature for C. botulinum under comparable conditions, suggesting that C. sporogenes may be used as a potential surrogate for evaluating the growth of C. botulinum in cooked meats during cooling. However, a direct comparison of growth kinetics between the two microorganisms is needed to confirm the suitability of C. sporogenes as a surrogate of C. botulinum.

Meta-analysis of D-values of proteolytic Clostridium botulinum and its surrogate strain Clostridium sporogenes PA 3679

Foodborne botulism is a serious disease resulting from ingestion of preformed Clostridium botulinum neurotoxin in foodstuff. Since the 19th century, the heat resistance of this spore forming bacteria has been extensively studied in order to guarantee the public health associated with low acidic, ambient stable products. The most largely used heat resistance parameters in thermal settings of such products are the D121.1°C values (time required to have a 10-fold decrease of the spore count, at 121.1°C) and the z-values (temperature increase to have a 10-fold decrease of D-values).To determine D121.1°C and z-values of proteolytic C. botulinum and its nontoxigenic surrogate strain C. sporogenes PA3679, a dataset of 911 D-values was collected from 38 scientific studies. Within a meta-analysis framework, a mixed-effect linear model was developed with the log D-value (min) as response and the heat treatment temperature as explicative variable. The studies (38), the C. botulinum strains (11), and the heat treatment media (liquid media and various food matrices, split into nine categories in total) were considered as co-variables having a random effect. The species (C. botulinum and C. sporogenes) and the pH (five categories) were considered as co-variables having a fixed effect.Overall, the model gave satisfactory results with a residual standard deviation of 0.22. The heat resistance of proteolytic C. botulinum was found significantly lower than the C. sporogenes PA 3679 one: the mean D-values at the reference temperature of 121.1°C, in liquid media and pH neutral, were estimated to 0.19 and 1.28min for C. botulinum and C. sporogenes, respectively. On the other hand, the mean z-values of the two species were similar: 11.3 and 11.1°C for C. botulinum and C. sporogenes, respectively. These results will be applied to thermal settings of low-acid ambient stable products.

Effect of Bacterial Cell-Free Supernatants on Infectivity of Norovirus Surrogates

Bacterial metabolic products were evaluated for inhibitory effects on viral propagation in cell culture. Cell-free supernatants (CFS) were prepared from growth of Enterococcus faecalis ATCC 19433, Pseudomonas fluorescens ATCC 13525, Escherichia coli 08, Staphylococcus epidermidis ATCC 12228, Bacillus subtilis 168, Bacillus coagulans 185A, B. coagulans 7050, Clostridium sporogenes PA3679, and a commercial probiotic mixture of Lactobacillus acidophilus, Lactobacillus rhamnosus, Bifidobacterium bifidum, Lactobacillus salivarius, and Streptococcus thermophilus in microbiological medium or milk. The inhibitory effects of CFS on the propagation of murine norovirus 1 and Tulane virus in RAW 264.7 and LLCMK2 cells, respectively, were evaluated in the continuous presence of CFS or after exposure of host cells to CFS. Slight inhibition of viral propagation was observed for murine norovirus and Tulane virus in the continuous presence of CFS of B. subtilis 168 and E. faecalis 19433, respectively. CFS cytotoxicity was also determined by microscopic examination. Virus persisted in the CFS that demonstrated cytotoxic effects, suggesting a lack of direct effect of CFS on virions. The viral propagation indicates a general lack of competitive inhibition by bacterial extracellular products and bears significance in understanding the persistence of virus in food and human systems shared by bacteria that are recognized for their colonization and competitive capabilities.

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

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-(2acetamido)-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.