Probability of growth and toxin production by nonproteolytic Clostridium botulinum in rockfish fillets stored under modified atmospheres

Whether toxin production by Clostridium botulinum precedes or follows spoilage of fish stored under modified atmospheres (MA), remains unclear. In this factorial design study we inoculated a pool of nonproteolytic C. botulinum spores (5 type E, 4 type B, and 4 type F strains) at 6 levels (10⁴ to 10⁻¹) between two rockfish fillets and then incubated the fillets at 4, 8, 12 and 30°C under vacuum, 100% CO₂ and 70% CO₂+30% air for 21 days. The probability of toxigenesis by one spore was significantly affected (P<0.005) by temperature (T) and storage time (S_t), and not (P>0.1) by MA, MA×T or MA×S_t. At the 10° spore/sample level, the earliest time to detect toxin production at 4,8,12 and 30°C under all MAs was >21, 15–21, 6–9 and 2 days, respectively. No toxin production was detected at 4°C. Only type B toxin was present in the toxic samples. At 30°C storage, spoilage of fillets followed toxigenesis. Using linear and logistic regression models, equations were derived that could estimate the lag phase and predict the probability of one spore initiating growth under a particular storage condition.     

Probability of growth and toxin production by nonproteolytic Clostridium botulinum in rockfish stored under modified atmospheres

The potential risk of C. botulinum growth in fresh fish stored under modified atmospheres remains unclear. Few studies have identified qualitatively certain conditions leading to toxigenesis. This paper is the First of a series attempting to quantify the effect of a variety of parameters on the probability (P) of toxigenesis by one spore in fish. The factorial design experiments included red snapper tissue homogenate inoculated with a pool of nonproteolytic spores (5 type E, 4 type B and 4 type F strains) at 7 levels (10⁴−10⁻² per 3 g sample) and incubated at 4, 8, 12, 17 and 30°C under 3 modified atmospheres (vacuum, 100% CO₂ and 70% CO₂+30% air) for up to 21 days. At the 10⁰ spore/sample level the earliest time to detect toxin production at 4, 8, 12, 17 and 30°C under all modified atmospheres was > 21, 12, 9, 6 and 2 days, respectively. At the 10¹ spores/sample level the earliest times for the same temperatures were > 21, 9, 6, 3–6 and 1–2 days, respectively. The probability of toxigenesis was affected significantly (P < 0.005) by temperature, storage time, atmosphere×temperature, and temperature×time but not by atmosphere (P > 0.1). Using linear and logistic regression models, equations were derived which can predict the P of 1 spore initiating growth and toxigenesis by a particular day and at a particular temperature of storage. Studies involving other fish substrates are in progress.     

The Investigation of the Presence of Clostridium Botulinum Spores in Honey in Serbia

The presence of Clostridium botulinum spores in 59 honey samples originating from different regions of the Republic of Serbia was studied. In addition to microbiological methods, after enrichment, centrifugation and membrane filtration, molecular methods (PCR methods) were utilized. The number of spores in PCR positive samples was estimated by the most probable number (MPN) method. PCR confirmed C. botulinum spores in 5 (8.47%) honey samples. MPN of spores varied from 20/kg to 204/kg honey. PCR was more sensitive than cultural methods. Natural honey contamination with C. botulinum spores is low-level and not homogeneous, and therefore, PCR methods require multiple sub-sampling.     

The effect of redox potential,and its interaction with sodium chloride concentration,on the probability of growth of Clostridium botulinum type E from spore inocula

A most probable number (mpn) count has been used to assess the effect of redox potential and of redox potential combined with increasing concentrations of sodium chloride on the probability of growth of spores of C. botulinum type E strain Beluga. In the majority of cases the maximum count developed within five days. In medium at pH 6·8 – 7·0, containing 0·1% w/v NaCl, incubated at 20°C, the probability of growth of spores in five days was not significantly lower at an E_h of approx +60 mV, adjusted by the introduction of air, than in strictly anaerobic conditions at E_h ?400 mV. Increasing the E_h above +60 mV resulted in a decrease in the probability of growth of spores, and at a redox potential between approx +122 mV and +164 mV the probability of growth was decreased by a factor of over 10⁵ compared with that at the lowest E_h. The inclusion of 3·25% w/v and 4% w/v NaCl in medium at a redox potential of ?400 mV decreased the probability of growth by factors of 10² and 10⁴ respectively, while the combination of 3·25% w/v NaCl and an E_h between +62 mV and +122 mV decreased the probability of growth by a factor of 10⁶.     

Predictive model for growth of Clostridium botulinum from spores at temperatures applicable to cooling of cooked ground pork

Cooling deviations and temperature abuse are two main reasons leading to the risk of Clostridium botulinum outgrowth in cooked pork. The aim of this research was to create a model that could be used to estimate C. botulinum growth from spores in cooked pork at temperatures similar to those used to chill cooked pork in processing facilities and food establishments. A cocktail of proteolytic C. botulinum types A and B consisting of five strains per type were used to inoculate pork to a final spore concentration of approximately 2 log CFU/g and cooked to 71 °C to heat shock the spores and kill vegetative microbes. The growth of C. botulinum was established at constant storage temperatures from 10 to 46 °C. C. botulinum growth was also studied under dynamic temperature conditions with cooling set to start at 54.4 °C and end at 4.4 °C or 7.2 °C in monophasic or biphasic cooling profiles, respectively. Growth parameters were estimated using the Baranyi model as a primary model and growth rates were fitted using the modified Ratkowsky secondary model with respect to temperature. The R² values ranged from 0.7653 to 0.9995 indicating that the Baranyi primary model was well suited to the growth data. The modified Ratkowsky secondary model's R² was 0.9653 and its root mean square error (RMSE) was 0.0687. All 11 prediction error values computed were within the limit of acceptable prediction zone (−1.0 to 0.5) suggesting a good fit of the model. The predictive model can provide information for the safety of cooked pork exposed to longer chilling times or for customized process schedule development as cooling of larger diameter products presents a processing challenge in the meat process operations.     

Growth and toxigenesis of C. botulinum type E in fishes packaged under modified atmospheres

Modified atmosphere packaging of fresh fish is used to market high quality products in some European countries. The potential risk of C. botulinum growth in these extended shelf-life foods is still a concern; especially since toxigenesis may precede organoleptic spoilage. This paper will present toxigenic data from rockfish, salmon and sole muscle tissues which were inoculated with a pool of non-proteolytic C. botulinum type E at seven levels (10⁻² −10⁴ spores/sample), and stored under vacuum and 100% CO₂, at incubation temperatures between 30 and 4°C, for up to 60 days. Factorial experimental design allowed predictive formulae to be developed able to describe the lag time prior to C. botulinum toxigenesis and the probability of one spore to initiate toxigenesis based upon the storage conditions. Accurate characterization of the microbial ecology of C. botulinum in modified atmosphere-packaged fish, will support safe exploitation of these packaging systems in the market place, and identify critical control points for potential product or process abuses.     

Effect of Irradiation on the Inhibition of Clostridium botulinum Toxin Production and the Microbial Flora in Bacon

The effect of irradiation (0.5, 1.0, 1.5 Mrad) on the microbiological safety and stability of temperature-abused (27°C, 60 days) bacon cured with' 1.5% NaCl, 0.25% sucrose, 0.3% Na₅P₃O₁₀ 0.055% Na-erythorbate, with or without NaNO₂ (40 μg/g) was determined. Unioculated bacon (120 μG/g NaNO₂/g) had a sour odor within 13 days, and, if inoculated with ca. 2 C. botulinum spores/g, 73% of the samples became toxic within 60 days. Irradiation with 0.5 Mrad prevented spoilage of uninoculated bacon by virtue of reducing the aerobic plate count to <1.0/g; irradiation with 1.0 Mrad sterilized bacon. Pouches of bacon, inoculated with C. botulinum spores (2 spores/g), swelled and became toxic; development of swelling and toxicity was delayed by incorporation of NaNO₂ (40 μg/g) or by irradiation with 0.5 Mrad. Irradiation with 1.0 Mrad decreased the number of swollen and toxic pouches. Irradiation with 1.5 Mrad prevented swelling and toxicity of bacon inoculated with 2 spores/g.     

Primary multiplication of Clostridium botulinum type A in mustard-miso stuffing of ‘karashi-renkon’ (deep-fried mustard-stuffed lotus root)

In June, 1984, a large-scale outbreak of type A botulism occurred in 14 different prefectures in Japan, involving 36 cases and 11 deaths, due to consumption of vacuum-packed ‘karashi-renkon’ (deep-fried mustard-stuffed lotus root). The onset of symptoms ranged from June 9th through July 28th and many batches of ‘karashi-renkon’, having been manufactured by a factory during a period of three weeks from June 5th to June 25th, were found to be contaminated with type A spores. Type A spores (1.0 × 10⁴ cfu/g) experimentally inoculated into the mustard-miso stuffing [water activity (a_w) 0.92, pH 5.0, NaCl 5.7%] did not multiply. By adjusting the stuffing to a_w 0.98 (pH 5.4) growth of type A was still inhibited. When the stuffing with elevated a_w and pH was heat-treated for 60 min at 80°C or autoclaved, it supported growth of Clostridium botulinum type A as indicated by the achievement of toxin levels higher than 10⁵ LD₅₀/g. When mustard-miso was stuffed into the holes of lotus roots and refrigerated overnight or a longer period, both a_w and pH of the stuffing increased to 0.98 and 5.3, respectively. The changes were apparently due to dialysis between the stuffing and the lotus root. This refrigeration process was actually performed at the plant, where the incriminated foodstuff was manufactured. The dialyzed mustard-miso stuffing, when heat-treated, supported the growth of type A organisms. Thus, we substantiated the possibility of primary multiplication of C. botulinum type A in mustard-miso stuffing subjected to refrigeration in lotus roots and then heat-treated.     

Proteolytic Clostridium botulinum growth at 12–48°C simulating the cooling of cooked meat: development of a predictive model

The objective of this study was to develop a model to predict the germination, outgrowth and lag (GOL), and exponential growth rates of Clostridium botulinum from spores at temperatures (12–48°C) applicable to the cooling of cooked meat products. The growth medium, Reinforced Clostridial medium (RCM) supplemented with oxyrase enzyme to create suitable anaerobic conditions, was inoculated with approximately 4 log₁₀spores ml⁻¹. Clostridium botulinum populations were determined at appropriate intervals by plating onto RCM. Clostridium botulinum growth from spores was not observed at temperatures <12°C or >48°C for up to 3 weeks. Growth curves were determined by fitting Gompertz functions to the data. From the parameters of the Gompertz function the growth characteristics, GOL times and exponential growth rates were calculated. These growth characteristics were subsequently described by Ratkowsky functions using temperature as the independent variable. Closed form equations were developed that allow for predicting relative growth for a general cooling scenario. By applying multivariate statistical procedures, the standard errors and confidence intervals were computed on the predictions of the amount of relative growth for a cooling scenario. The predictive model is capable of predicting spore outgrowth and multiplication for general cooling scenarios, for suitable but un-verified mathematical assumptions, and should aid in evaluating the safety of cooked products after cooling.     

Selective recovery of proteolytic strains of Clostridium botulinum types A and B from model cured meat systems

A selective medium (SBM) containing a combination of antimicrobials was developed which allowed the quantitative isolation of inoculated proteolytic Clostridium botulinum types A, B and F from a simulated cured meat product (cured pork slurry) containing natural spoilage organisms. The medium effectively suppressed the growth of Cl. perfringens, Cl. butyricum, Cl. histolyticum and non-proteolytic strains of Cl. botulinum.Other proteolytic clostridia including putrefactive anaerobes and Cl. bifermentans were capable of growth in SBM but were rarely isolated from the inoculated cured pork slurry. From unheated slurries, inoculated with Cl. botulinum type A spores and stored at 27° or 35°C 356 of 384 (92·7%) colonies picked from SBM were confirmed as Cl. botulinum type A. Selectivity was greater at 27° or 35°C than 15° or 20°C but with experience presumptive Cl. botulinum colonies can be differentiated from other resistant organisms at the lower temperatures. When heated slurries were studied all 441 colonies picked from SBM were confirmed as Cl. botulinum type A.     

The sensory and microbiological quality of fresh sliced mushroom (Agaricus bisporus L.) packaged in modified atmospheres

The effect of modified atmospheres, generated by using different packaging films, on the quality of sliced mushrooms was evaluated. The carbon dioxide and oxygen content inside the packages as well as the colour, texture, weight loss, sensory attributes, mesophiles, psychrotrophs, Pseudomonas fluorescens, faecal coliforms, Escherichia coli and anaerobic spores were determined. Modified atmospheres containing 2.5% CO₂ and 10–20% O₂ reduced the microbial counts and improved the mushrooms’ appearance when compared with an air atmosphere. Modified atmospheres containing 15% CO₂ and <0.1% O₂ inhibited mushroom development and toughening and reduced microbial growth. Although these atmospheres had no effect on colour, they did allow the development of off odours and anaerobic spores were detected. No differences in microbial growth or mushroom spoilage were observed under the different moisture contents generated in the packages at 4 °C. Aerobic bacteria counts were considered very high even at the beginning.     

Optimization and evaluation of heat-shock condition for spore enumeration being used in thermal-process verification: Differential responses of spores and vegetative cells of Clostridium sporogenes to heat shock

To evaluate a heat-shock condition for the enumeration of Clostridium sporogenes spores, a surrogate for C. botulinum spores, we examined the heat tolerance of C. sporogenes spores and vegetative cells exposed to a heat shock at 90°C. From the D values of the spores determined in the temperature range of 113–121°C, z value (±SD) and D_90°C value were estimated to be 10.16±0.90°C and 1,071.52 min, respectively, and the inactivation rates were predicted to be only approximately 2% at 90°C for up to 10 min. Meanwhile, the viable count of spores was significantly higher when activated under a heat-shock condition of 90°C for over 9 min than those activated for shorter time periods. The heat tolerance of vegetative cells was extremely low, showing a D_90°C value (±SD) of 0.21±0.01 min. Finally, 3 different heat-shock conditions were compared: 70°C for 30 min, 80°C for 20 min, and 90°C for 10 min, and the experimental comparative data showed no significant differences in viable spore counts. Consequently, these results support that the heat-shock treatment at 90°C for 10 min is suitable to activate spores and to inactivate vegetative cells of C. sporogenes.     

The effect of oxygen and redox potential on growth of Clostridium botulinum type E from a spore inoculum

Small volumes of oxygen introduced into vials of medium at pH 7 prepared under anaerobic conditions reacted with the medium during sterilization by heating, and also raised the redox potential. The partial pressure of oxygen (pO₂) in the medium, and the redox potential (E_h) were measured and their effect on the number of spores of Clostridium botulinum type E required to produce growth, and hence on the probability of growth from single spore inocula, was determined.In the presence of a pO₂ of up to 4·5 × 10⁻³ atm resulting in an E_h of c. + 217 mV, the probability of growth from single spores within 5 days at 20°C was equal to that in strictly anaerobic conditions at an E_h of − 400 mV. At pO₂ values of between 5·3 × 10⁻³ atm and 8·4 × 10⁻³ atm corresponding to E_h values of between + 226 mV and + 254 mV an inoculum of between 10 and 1000 spores was required to produce growth and at pO₂ values of between 1·12 × 10⁻² atm,and 1·6 × 10⁻² atm, corresponding to redox potentials of between + 271 mV and + 294 mV, the number of spores required to produce growth was between 2 × 10⁴ and > 2 × 10⁵. The relationship between pO₂ and E_h depends on the chemical nature of a culture medium or food, and in order to assess the probable influence of these parameters on growth of C. botulinum in a medium or a food it is necessary to determine both the redox potential and the partial pressure of oxygen.     

Effects of vacuum,modified atmospheres and storage temperature on the microbial flora of packaged beef

The flora growing on beef stored in vacuum and 100% CO₂at 2 or 6°C (experiment 1) and in vacuum and different mixtures of CO₂and N₂at −1 or 2°C (experiment 2) was determined in two separate experiments. Both a high concentration of CO₂and a low storage temperature inhibited bacterial growth, especially of the spoilage bacteria pseudomonads andBrochothrix thermosphacta. No packaging conditions gave growth of coliforms. High numbers (log₈5–6) of lactic acid bacteria after storage in vacuum or gas packs, inhibited growth of pseudomonads andB. thermosphactaduring subsequent storage under retail conditions. Samples of the lactic acid bacteria obtained in experiment 2 were identified by genus-specific rRNA probes. Leuconostocs dominated in vacuum and CO₂packs at both temperatures, whereas carnobacteria dominated in N₂at −1°C.     

Microbial quality and lipid oxidation of Manchega breed suckling lamb meat: Effect of stunning method and modified atmosphere packaging

The effect of CO₂ concentration and exposure time at stunning [80% CO₂ for 90 s (G1); 90% CO₂ for 90 s (G2); 90% CO₂ for 60 s (G3); 80% CO₂ for 60 s (G4)] plus an electrically stunned control group (G5) was assessed for lipid oxidation (LO) and microbial levels, [total viable counts, lactic acid bacteria, Enterobacteriaceae and Pseudomonas spp.] in Manchega breed suckling lamb meat at 24 h and 7 days post-mortem. Differences in LO were found at 7 days post-mortem (P < 0.05) with the highest value for G4. In general, values of all microorganisms studied were higher in G5.     

Breaking the spores of the fungus Ganoderma lucidum by supercritical CO2

The hard sporoderm of Ganoderma lucidum spores prevents the release of bioactive components such as polysaccharides which have significant anti-tumour activity. In the present study, supercritical carbon dioxide (SC–CO₂) was used for the sporoderm breaking of G. lucidum spores, and polysaccharides were subsequently extracted and determined for evaluating the performances of SC–CO₂. The operating parameters were optimized by orthogonal array design (OAD), and the morphological status of sporoderm was observed by scanning electron microscope (SEM). The optimum operating conditions for SC–CO₂ breaking of sporoderm were as follows: pressure 35 MPa, temperature 25 °C, time 4 h, and CO₂ flow rate 10 kg/h. After SC–CO₂ processing, the extraction yield of polysaccharides reached 2.98%, which was 3-fold to that of the intact ones (0.94%). This method is fast, efficient and advanced enough to break the hard sporoderm of G. lucidum, which may provide a scientific reference for the large-scale processing of spores in the pharmaceutical and food industries.     

Antibacterial action on pathogenic bacterial spore by green tea catechins

Antibacterial effects of catechins, the major green tea polyphenols, were studied using Clostridium and Bacillus spores. Incubation with crude catechins decreased the number of C botulinum and C butyricum spores but not B cereus spores. Furthermore, the effects of six catechin derivatives on spores were investigated. (−)‐Epicatechin gallate (ECg), (−)‐epigallocatechin (EGC), (−)‐epigallocatechin gallate (EGCg) and (+)‐gallocatechin gallate (GCg) were more effective in decreasing C botulinum and C butyricum spore numbers than (+)‐catechin (C) and (−)‐epicatechin (EC). The vegetative growth of C botulinum and B cereus was inhibited by crude extracts of the catechins. Specifically, purified GCg and EGCg inhibited the vegetative growth of C botulinum and B cereus. The inhibitory effect of ECg on B cereus was similar to that of GCg. However, toxin‐production by B cereus was not inhibited by catechin. Damage to the membrane of C butyricum spores by catechin derivatives was shown using fluorescent microscopy. This study shows that low concentrations of catechins, although requiring a long exposure time, inhibited the growth of bacterial spores. However, the effects of the purified derivatives of the catechins were not the same and GCg and EGCg were found to be the most potent. Spores that are generally resistant to many disinfectants were sensitive to catechins. Copyright © 2005 Society of Chemical Industry     

F-value Calculator – A Tool for Calculation of Acceptable F-value in Canned Luncheon Meat Reduced in NaCl

Canned meat products are usually protected against C. botulinum by combinations of heat, NaCl and NaNO₂. When meat products are reduced in NaCl for health reasons, they need a higher heat treatment to maintain same level of protection against C. botulinum. We describe a new tool for calculating the F-value, necessary to obtain equivalent safety for canned meat when reduced in NaCl, compared to the original combination of aqueous salt and F–value. The tool is valid for combinations of F–values between 0.51 and 3.25 and aqueous salt between 1.66 and 3.54%. The tool is available at http://dmripredict.dk.     

Effect of Modified Atmosphere Storage on C. Botulinum Toxigenesis and the Spoilage Microflora of Salmon Fillets

Modified atmosphere (MA) storage in conjunction with refrigeration has been shown to significantly increase the shelf life of fresh fish and other products, but the effects, if any on the outgrowth and toxigenesis of C. botulinum are unknown. A commercial system was duplicated in the laboratory and the effects of modified atmosphere on the outgrowth of C. botulinum types A, B, and E were observed in inoculated salmon fillets and sandwiches stored at 4.4°C and 22.2°C. Inoculated samples stored in air at the same temperatures were used as controls. No toxigensis was observed in either the air or modified samples stored at 4.4°C, but all inoculated samples held at 22.2°C were toxic within 2–3 days. Spoilage generally preceded toxigenesis. In a concurrent study, the tendency of CO₂ environments to repress the growth of gram negative bacteria to a greater extent than gram positive bacteria was also noted.     

Lag time variability in individual spores of Clostridium botulinum

Quantifying lag times from individual spores and the associated variability is an important part of understanding the hazard associated with spore-forming pathogens such as Clostridium botulinum. Knowledge of the underlying distribution would allow greater refinement of risk assessments. To date most studies have either examined lag time indirectly by measuring time to growth or have only examined the first stage of lag, germination. Recent studies have attempted to quantify the variability of spores during the different stages of lag phase and to examine the relationships between these stages. The effect of incubation temperature (22 °C, 15 °C, 10 °C or 8 °C), heat treatment (unheated or 80 °C for 20 s) and sodium chloride concentration in both the sporulation medium (0 or 3% w/v) or growth medium (0 or 2% w/v) on growth from individual spores has been examined. These studies found spores within a single population are very heterogeneous with large variability in all stages of lag. The duration and variability of times for germination, outgrowth and first doubling depended on both the historic treatment of the spores and the prevailing growth conditions, and the stage of lag most affected was treatment dependant.