Clostridium Sporogenes PA Research Articles

Determination of thermal process schedule for emulsion type buffalo meat block in retort pouch.

The process temperature for buffalo met blocks processed in retort pouches calculated based on the heat resistance of Clostridium sporogenes PA 3679 in Phosphate buffer saline (PBS- Ph 7.0) as reference medium and in buffalo meat block (pH 6.28) was in the range of 110-121°C. The D values and Z values calculated for C.sporogenes PA 3679 confirmed that the suspension was best suited for conducting thermal resistance studies. The experiment for indirect confirmation of microbial safety of the products involving inoculating the buffalo meat emulsion filled in pouches with C.sporogenes PA 3679 and processed at Fo 12.13 min showed no growth of microorganisms.

Clostridium sporogenes PA 3679 and its uses in the derivation of thermal processing schedules for low-acid shelf-stable foods and as a research model for proteolytic Clostridium botulinum.

The putrefactive anaerobe Clostridium sporogenes PA 3679 has been widely used as a nontoxigenic surrogate for proteolytic Clostridium botulinum in the validation of thermal processes for low-acid shelf-stable foods, as a target organism in the derivation of thermal processes that reduce the risk of spoilage of such foods to an acceptable level, and as a research model for proteolytic strains of C. botulinum. Despite the importance of this organism, our knowledge of it has remained fragmented. In this article we draw together the literature associated with PA 3679 and discuss the identity of this organism, the phylogenetic relationships that exist between PA 3679 and various strains of C. sporogenes and proteolytic C. botulinum, the heat resistance characteristics of PA 3679, the advantages and limitations associated with its use in the derivation of thermal processing schedules, and the knowledge gaps and opportunities that exist with regard to its use as a research model for proteolytic C. botulinum. Phylogenetic analysis reviewed here suggests that PA 3679 is more closely related to various strains of proteolytic C. botulinum than to selected strains, including the type strain, of C. sporogenes. Even though PA 3679 is demonstrably nontoxigenic, the genetic basis of this nontoxigenic status remains to be elucidated, and the genetic sequence of this microorganism appears to be the key knowledge gap remaining to be filled. Our comprehensive review of comparative heat resistance data gathered for PA 3679 and proteolytic strains of C. botulinum over the past 100 years supports the practice of using PA 3679 as a (typically fail-safe) thermal processing surrogate for proteolytic C. botulinum.

Evaluation of peracetic acid sanitizers efficiency against spores isolated from spoiled cans in suspension and on stainless steel surfaces.

The aim of this study was to determine the inactivation effect of industrial formulations of peracetic acid biocides on bacterial spores adhering to stainless steel surfaces. A standardized protocol was used to validate biocide activity against spores in suspension. To validate sporicidal activity under practical conditions, we developed an additional protocol to simulate industrial sanitization of stainless steel surfaces with a foam sanitizer. Spores of three spore-forming bacteria, Clostridium sporogenes PA3679, Geobacillus stearothermophilus, and Moorella thermoacetica/thermoautotrophica, were sprayed onto stainless steel as bioaerosols. Sporicidal activity was high against the C. sporogenes spore suspension, with more than 5 log CFU ml(-1) destroyed at all liquid biocide contact times. Sporicidal activity also was high against G. stearothermophilus and M. thermoacetica/thermoautotrophica spores after 30 min of contact, but we found no population reduction at the 5-min contact time for the highest sporicide concentration tested. The foam biocide effectively inactivated C. sporogenes spores adhered to stainless steel but had a reduced decontamination effect on other species. For G. stearothermophilus spores, sanitization with the foam sporicide was more efficient on horizontal steel than on vertical steel, but foam sanitization was ineffective against M. thermoacetica/thermoautotrophica whatever the position. These results highlight that decontamination efficiency may differ depending on whether spores are suspended in an aqueous solution or adhered to a stainless steel surface. Biocide efficiency must be validated using relevant protocols and bacteria representative of the microbiological challenges and issues affecting each food industry.

Effect of High Pressure Homogenization Process on Bacillus Stearothermophilus and Clostridium Sporogenes Spores in Skim Milk

High pressure homogenization (HPH) is an alternative food processing technique. As product heating is minimum, its characteristics are not affected in a large extend. This characteristic makes the HPH an interesting process to guarantee the safety of thermo-labile food. Inactivation of Bacillus stearothermophilus ATCC 7953 and Clostridium sporogenes PA 3679 spores in skim milk by HPH was studied. Results showed that pressures up to 300 MPa were not able to cause any reduction on spore counts or promote changes on its thermal resistance. The application of heat shock (100°C/15 min) before HPH treatment and the homogenization process realized at mild inlet temperature (45°C) – which results in homogenization temperature of around 84°C at 300 MPa - also did not cause reduction on viable spores counts. A few spores reduction (0.67 logarithmic cycles) were only observed when the milk samples were subjected to homogenization treatment 16 times (multiple passes) at 300 MPa. Therefore, although HPH be recognized as an effective method for milk pasteurization, it was conclude that the HPH process is not able to guarantee the commercial sterility of milk, being necessary the association of the homogenization with another preservative method, as refrigeration.

Heat-sensitizing effects of plant extracts on Clostridium spp. spores

The effects of ethanol-soluble fractions of 12 plant extracts were tested for heat-sensitizing effects on the spores of Clostridium botulinum (type A and type B) and Clostridium sporogenes PA 3679. Licorice, pineapple sage and lemon eucalyptus extracts were respectively found to significantly decrease ( P < 0.05) the thermal resistance of C. botulinum 62A (type A), C. botulinum Okra (type B), and C. sporogenes; reducing initial spore populations at 0.5–1.0% level of addition. Further investigations showed that increasing acidity and licorice extract (0.5%) had an additive heat- sensitizing effect on C. botulinum 62A while increasing NaCl counteracted the effect of the extract on the spores. Exposure of spores to 0.5% licorice extract for 7 d prior to heating significantly reduced the heat resistance of the spores. Finally, a post-heating sporicidal property of licorice extract was also observed, resulting in significantly greater spore population reductions compared to that when licorice extract was removed by washing with Tween 80 prior to enumeration on growth medium. The use of naturally occurring compounds in plants as potential food additives may help address the consumer demands for products with highly acceptable sensory and nutritive qualities, and safety.

Microbial Inactivation Kinetics in Soymilk during Continuous Flow High‐Pressure Throttling

The thermal resistance of Clostridium sporogenes PA 3679 ATCC 7955 was determined in soymilk (pH 7) and 0.1% peptone water (pH 7) by the capillary tube method. In the continuous flow high-pressure throttling, the temperature of soymilk increased due to instantaneous pressure release and the additional heat was supplied by a heat exchanger to achieve the set temperature. The soymilk was immediately cooled after a short preset hold time to below 40 degrees C. A significant increase in the heat resistance was observed in C. sporogenes spores when heated in soymilk in comparison to 0.1% peptone water. The D(121)-value for spores in soymilk was approximately 3-folds higher than peptone water. The z-value was also much higher in soymilk as compared to that in 0.1% peptone water. Continuous flow high-pressure throttling (HPT) from 207 or 276 MPa to atmospheric pressure reduced the microbial populations in inoculated soymilk up to 6 log cycles when the holding times were 10.4, 15.6, and 20.8 s and the process temperatures were 85, 121, 133, and 145 degrees C, respectively. The sporicidal effect increased as the operating pressure, time, and temperature were increased. More injured spores were found at 207 MPa than at 276 MPa, indicating that lower pressure caused cell injury whereas high pressure caused cell death.

High-pressure destruction kinetics of Clostridium sporogenes spores in ground beef at elevated temperatures

High pressure (HP) is an alternative technique for thermal sterilization of foods with minimum quality loss. HP destruction kinetics of bacterial spores is essential to establishing sterilization process, but knowledge in this field is still very limited. In this study, destruction kinetics was investigated using Clostridium sporogenes PA 3679 (ATCC7955) spores in extra-lean ground beef (5 g each sealed in a sterile plastic bag). Duplicated samples were subjected to HP treatments at 700, 800 and 900 MPa in a HP system equipped with a Polyoxymethylene insulator to maintain constant temperatures at 80, 90 and 100 °C during pressure-holding time. The kinetic parameters of the spores ( D- and Z-values) were evaluated at these pressures and temperatures. For the pressure from 700 to 900 MPa, D-values ranged from 15.8 to 7.0 and 1.5 to 0.63 min at 80 and 100 °C, respectively. The pressure resistance of Z T( P) value was 520–563 MPa at 80–100 °C. The temperature resistance of Z P( T) value was 19.1–19.7 °C at 700–900 MPa, much higher than that at atmospheric condition (12.4 °C). A regression model was generated which can be used to predict D-value or the death time of a minimum process under given pressure and temperature conditions. HP treatment with elevated temperatures can destroy bacterial spores with a shorter time or lower temperature than conventional thermal processing. This study provides useful information for the achievement of a safe HP sterilization process.

Microwave sterilization of sliced beef in gravy in 7-oz trays

This research was to investigate the feasibility for developing a short-time sterilization protocol for a highly inhomogeneous food prepackaged in polymeric trays using 915 MHz microwave (MW) energy. A 915-MHz, single-mode, 10-kW pilot-scale MW system developed at Washing State University was used for this study. The inhomogeneous food consisted of sliced beef and gravy packaged in 7-oz polymeric trays. Specially formulated whey protein gel, matching the beef product in their dielectric properties, was chosen as a model food to emulate the real food for determination of heating patterns and cold spots inside food trays. The heating patterns and cold spots were detected using a chemical-marker-assisted computer vision method. Processing schedules to achieve desired levels of F 0 for 7-oz trays of beef in gravy were established based on temperature histories measured at the identified cold spot location. The developed processing schedules were validated by inoculated pack studies using Clostridium sporogenes PA 3679 spores. The results of this study indicate that the 915-MHz single-mode MW sterilization technology is effective for processing of the inhomogeneous food. The procedure established could be used for developing MW sterilization processes for other packaged inhomogeneous foods, such as chicken meat in gravy in trays and salmon in sauce in pouches. The processing data collected could be helpful for industrial scale-up of the MW system.

Performance evaluation of aluminum test cell designed for determining the heat resistance of bacterial spores in foods

Thermal inactivation kinetic studies are necessary to determine heat resistances of spores in the development of new thermal processes for low-acid shelf-stable products. Most currently available sample holders used for solid and semi solid samples in the kinetic studies take long time to reach the target sample temperature, hence fail to provide isothermal condition. In this research, novel aluminum test cells were developed to facilitate easy loading and unloading samples in a hermetically sealed 1 ml cavity to evaluate the heat resistance of bacterial spores when heated at temperatures above 100 °C. Design of the test cell was governed by minimum come-up time. A finite element model based on the commercial software ‘FEMLAB’ was used to simulate transient heat transfer and finalize the test cell dimensions. Performance of the new test cell was evaluated against capillary and aluminum thermal death time tube methods in characterizing the heat resistance of Clostridium sporogenes PA 3679 spores in a phosphate buffer and mashed potato at 121 °C. D 121 values of PA 3679 spores in both the phosphate buffer and mashed potato using the new test cells were not significantly different ( P>0.05) from those by the capillary tube method. The results indicated that the new test cell is appropriate for studying the inactivation kinetics of bacterial spores in microbial validation of conventional and novel thermal processes for low-acid shelf-stable foods.

HIGH PRESSURE–HIGH TEMPERATURE STERILIZATION: FROM KINETIC ANALYSIS TO PROCESS VERIFICATION†

ABSTRACT The inactivation of Clostridium sporogenes PA 3679 spores by high pressure at high temperatures (HP–HT) in phosphate buffer was investigated in a lab‐scale temperature‐controlled HP system (QFP‐6) with an internal heater to maintain the sample temperature. Some inactivation of spores occurred during the pressurization come‐up time (CUT) and depressurization time. The inactivation of PA 3679 was found to be exponential during the adiabatic holding period of the HP cycle at constant pressures and temperatures. The inactivation rate increased with both pressure and temperature. The kinetic parameters – such as D‐values at tested temperatures and pressures that are necessary for the design of process parameters of HP sterilization process – were determined. Within the pressure range of 600–800 MPa, the calculated D‐values ranged from 270.3 to 357.4 and 49.0 to 67.6 s at 91 and 108C, respectively. These studies provided basic data on the effects of pressure and temperature on the inactivation of PA 3679 spores under conditions applicable to the development of preservation specifications for commercial HP–HT processing of low acid foods. The spore strips of C. sporogenes were used as indicators for microbiological verification of delivered lethality of HP–HT sterilization process at different processing conditions in a pilot scale HP vessel.

Modified Spore Inoculation Method for Thermal‐Process Verification of Pinto Beans and Green Beans Canned in Two Large Reusable Containers

ABSTRACT:As an alternative to standard metal cans, 2 large, reusable containers with volumetric capacities 3.6 to 3.8 times larger than a standard #10 can were constructed of nickel‐plated steel. Process times and cold point determinations were previously determined using mathematical approximations of heat‐penetration data. It was necessary to verify the adequacy of these process times using inoculated containers. Because it was impractical to use traditional inoculated can procedures, a modification was developed using spores of Clostridium sporogenes PA3679 immobilized in alginate beads contained in brass tubes. Adequacy of the calculated thermal processes was demonstrated; no viable anaerobic spores survived in the inoculated packs.

Bacterial Spore Inhibition and Inactivation in Foods by Pressure, Chemical Preservatives, and Mild Heat

Sucrose laurates, sucrose palmitate, sucrose stearates, and monolaurin (Lauricidin) were evaluated for inhibitory effects against spores of Bacillus sp., Clostridium sporogenes PA3679, and Alicyclobacillus sp. in a model agar system. The combined treatment of sucrose laurate, high hydrostatic pressure, and mild heat was evaluated on spores of Bacillus and Alicyclobacillus in foods. The minimum inhibitory concentrations of the sucrose esters were higher than that of Lauricidin for all spores tested in the model agar system, but Lauricidin was not the most readily suspended in the test media. The sucrose laurates and sucrose palmitate were more effective and more readily suspended than the sucrose stearates. A combined treatment of sucrose laurate (≤1.0%), 392 megaPascals (MPa) at 45°C for 10 to 15 min provided 3- to 5.5-log10 CFU/ml reductions from initial populations of 106 CFU/ml for Bacillus subtilis 168 in milk, Bacillus cereus 14579 in beef, Bacillus coagulans 7050 in tomato juice (pH 4.5), Alicyclobacillus sp. N1089 in tomato juice (pH 4.5), and Alicyclobacillus sp. N1098 in apple juice. The most notable change in the appearance of the products was temporary foaming during mixing of the sucrose laurate in the foods. The effect of sucrose laurate appeared to be inhibitory rather than lethal to the spores. The inhibitory effects observed on Bacillus and Alicyclobacillus spores by the combined treatment of pressure, mild heat, and sucrose laurate appear promising for food applications where alternatives to high heat processing are desired.

Sensitivity of spores of Bacillus subtilis and Clostridium sporogenes PA 3679 to combinations of high hydrostatic pressure and other processing parameters

The combined effects of pressure, temperature, pH and the presence of nisin or sucrose laurate on the survival of spores of Bacillus subtilis 168 and Clostridium sporogenes PA 3679 were investigated. Spore populations of PA 3679 were reduced by 2.5-log 10 when exposed to 404 megapascals (MPa) at 25°C, pH 4.0 for 30 min, but the same treatment at pH 7.0 resulted in a <0.5-log 10 reduction in spore counts. Pressurization of B. subtilis spores at 70°C, pH 6.0 or 7.0 for 15 min at 404 MPa resulted in a 5-log 10 reduction as compared to a <0.5-log 10 reduction for the same pressurization treatment at 25°C. For the inactivation of spores of B. subtilis and PA 3679, the addition of nisin to the plating medium appeared to be synergistic in some instances when combined with pressurization at elevated temperatures and reduced pH. B. subtilis 168 was resistant to 0.1% sucrose laurate, but when combined at ≤6.0 pH with a 15-min treatment of 404 MPa at 45°C, a dramatic synergistic effect eliminated spore suspensions of 1×10 6/ml.

Measurement of optical absorbance of Clostridium sporogenes PA 3679 spores as a method for determining their thermal inactivation

The loss of optical density of Clostridium sporogenes PA 3679 spores after heating at temperatures of 121, 126, 130 and 135°C was studied, together with the relationship between this parameter and the recovery capacity of the heated spores. The results show that the spores suffered a greater loss of optical density when they were subjected to more severe heating. A linear relationship was observed between the loss of viability and the optical absorbance of the spores. A certain parallel was detected between the heat resistance parameters of the spores DT (decimal reduction time) and z (thermal inactivation coefficient), and the kinetic parameters DT A and zA, which describe the reduction in absorbance.

Extrusion and Low-Dose Irradiation Effects on Destruction of Clostridium sporogenes Spores in a Beef-Based Product

The effects of extrusion cooking alone or in combination with electron beam radiation (3.5 kGy) on vacuum-packaged beef-based snack sticks containing beef cardiac muscle were investigated. During formulation, Clostridium sporogenes PA 3679 spores were added to achieve a concentration of 4 log CFU/g. Twin-screw extrusion cooking at 72°C reduced aerobic plate counts (APCs) by 3.63 log cycles and C. sporogenes viable cell counts by 2.02 log cycles for the inoculated product. After irradiation (3.5 kGy), APCs were decreased to 1 log CFU/g when compared to 0 kGy counterparts receiving 0 kGy. Spores were not detected in irradiated inoculated samples, which contained C. sporogenes PA 3679 at levels of 3.17 to 3.50 log CFU/g after extrusion cooking.

The effect of ethylenediaminetetraacetic acid on heat resistance and recovery of Clostridium sporogenes PA 3679 spores treated in HTST conditions

The effect of ethylenediaminetetraacetic acid (EDTA) on the heat resistance of Clostridium sporogenes PA 3679 spores was studied. EDTA was added to heating substrates and recovery media in order to establish which stage of the heat treatment registered the greatest EDTA activity. The heating substrates assayed were phosphate buffer (pH 7.0) and white asparagus purée, at natural pH (5.8) and acidified with citric acid and glucono-δ-lactone (GDL) to pH 5.5, 5.0 and 4.5. Recovery of survivors was carried out in MPA3679A medium in various conditions of acidification with citric and GDL (250 and 500 ppm), at pH 7.5, 6.5 and 6.0. The results show greater activity of EDTA on spores when it was applied in recovery of heat injured spores, than during heating. The strongest influence of EDTA during heating was found in phosphate buffer (pH 7.0), with the effect being most evident at 121 and 126 °C, and in asparagus purée, at 121 °C and pH 5.8 rather than acidified. In recovery, the inhibiting activity of EDTA was more evident in spores subjected to more severe heat treatment, either by increasing the exposure time or by raising the temperature to 130 or 135 °C. The pH level of the recovery medium also affected the antimicrobial activity of EDTA, which had a greater inhibiting effect at pH 7.5 than at lower pH levels (6.5, 6.0).

Inhibition of Bacterial Spores and Vegetative Cells by Glycerides

Monolaurin, monomyristin, monolinolein, and monolinolenin inhibited cell growth from Bacillus cereus T, Clostridium botulinum 62A, and Clostridium sporogenes PA3679 spores and vegetative cells. The inhibitory action increased in the order monolinolein monomyristin, monolaurin, monolinolenin. Monopalmitin, monostearin, and monolein had a partial effect and diglycerides and triglycerides had no effect.B. cereus T spores were more sensitive to monolaurin, monomyristin, monolinolein, and monolinolenin than vegetative cells. Spores and vegetative cells of C. botulinum 62A were similarly affected by monolaurin, whereas vegetative cells were more resistant to monomyristin, monolinolein, and monolinolenin than spores.In general, C. botulinum 62A and C. sporogenes PA 3679 were more resistant than B. cereus T, except for monolinolein and monomyristin, in which Bacillus spores were more resistant than PA3679 spores. C. botulinum 62A spores were equally (monomyristin) or less resistant (monolaurin, monolinolenin) than PA3679. Monomyristin had the same activity on the three strains. Monolaurin (0.073 to 0.109 mM), monomyristin (0.331 to 0.497 mM), and monolinolenin (0.085 to 0.182 mM) affected germination and outgrowth, whereas monolinolein (0.564 to 0.848 mM) inhibited cell multiplication from B. cereus T spores. Monolaurin (0.145 to 0.182 mM) inhibited outgrowth, but monomyristin (0.331 to 0.497 mM) and monolinolenin (0.142 to 0.182 mM) affected germination of 62A spores. Cell multiplication was inhibited by monolinolein (0.564 to 0.848 mM). C. sporogenes PA3679 spore germination and outgrowth were inhibited by monomyristin (0.331 to 0.497 mM) and monolinolenin (0.142 to 0.282 mM), or monolaurin (0.182 to 0.255 mM), monomyristin (0.165 to 0.248 mM), and monolinolenin (0.085 to 0.113 mM) inhibited cell multiplication from PA3679.Starch neutralized the inhibitory activity of monolaurin, monomyristin, monolinolein, and monolinolenin against 62A spores. The starch antagonism decreased as the concentration of glyceride increased.

Evaluation of citric acid and GDL in the recovery at different pH levels of Clostridium sporogenes PA 3679 spores subjected to HTST treatment conditions

Spores of Clostridium sporogenes PA 3679 were treated at different temperatures (121, 126, 130 and 135 °C) in white asparagus purée (pH 5.8) and acidified with glucono-δ-lactone (GDL) and citric acid to pH levels of 5.5, 5.0 and 4.5. Afterwards, the spores were recovered in MPA3679 medium in various conditions: unacidified (pH 7.5), acidified with GDL (500 ppm) and acidified with citric acid (500 and 250 ppm) to pH levels of 6.5, 6.0 and 5.0. The results indicated that the pH levels, concentration and type of acid used act synergistically rather than independently. Citric acid has a stronger inhibiting effect than GDL on the recovery of C. sporogenes PA 3679 spores. At the higher heat treatments (130 and 135 °C) the major injury on the spores sensitize more than against the acids and low pH values.

Growth and Heat Resistance of Clostridium SporogenesPA 3679 Spores Heated and Recovered in Acidified Media

The effect of subculture medium composition (pH, type of acidulant, presence of EDTA) on the germination of spores of Clostridium sporogenes PA 3679, both with and without heat treatment in phosphate buffer (30 s at l25°C), was studied. Additionally, the effect of the heating medium substrate and pH and subculture medium pH and composition on the heat resistance of the spores was evaluated. The results indicate that the pH of the recovery medium and the type of acidulant have significant effects (P ≤ 0.05) on counts of PA 3679. Adding EDTA only increases inhibition of this microorganism when the pH is near neutral. For a given pH level, citric acid provided greater inhibiting power than glucono-δ-lactone, although the percentage of undissociated acid was less for citric acid. No growth was observed at pH 5.7, although in the case of glucono-δ-lactone sporadic colony development was seen in some plates. The pH of the heating medium acidified with glucono-δ-lactone did not affect the heat-resistance parameters (D and z) of spores heated to temperatures between 121 and 140°C in relation to the unacidified extract. Nor did we see any significant influence of the composition and pH of the recovery medium on these heat-resistance parameters when comparing the results with those obtained using the usual recovery medium.

Glucono-δ-lactone and citric acid as acidulants for lowering the heat resistance of Clostridium sporogenes PA 3679 in HTST working conditions

The heat resistance of Clostridium sporogenes PA 3679 spores has been studied to establish the influence of acidification with glucono-δ-lactone (GDL) and citric acid on the thermal resistance parameters ( D T and z) of this microorganism and to compare their effect with phosphate buffer and natural asparagus as reference substrates. A reduction in D T values was observed in asparagus purée as the acidification level increased with both acidulants although this effect was more evident at the lower treatment temperatures studied (121–127 °C). Citric acid was more effective for reducing the heat resistance of spores than GDL at all of the temperatures. The reduction in pH diminished the value of the z parameter, although it was necessary to lower the pH to 4.5 to obtain a significant reduction.