Factors controlling the growth of Clostridium botulinum types A and B in pasteurized, cured meats

The effect of combinations of sodium chloride (2.5, 3.5, 4.5% w/v on water), sodium nitrite (100, 200, 300 μg/g), sodium nitrate (0, 500 μg/g), sodium isoascorbate (0,1000 μg/g, or equimolar with nitrite level) and polyphosphate (Curaphos 700; 0, 0.3% w/v), on the growth of Clostridium botulinum types A and B was studied in an experimental pork slurry system, without heating and after two heat treatments (80°C for 7 min and 80°C for 7 min plus 70°C for 1 hr) followed by storage at: 15, 17.5, 20 or 35°C for up to 6 months.
Statistical analyses showed that increasing salt or nitrite levels, adding isoascorbate or nitrate, using the highest heat treatment or decreasing the storage temperature all significantly reduced toxin production by Cl. botulinum . The addition of 0.3% polyphosphate (Curaphos 700) significantly increased toxin production. There were many significant two-factor interactions; the effect of increasing nitrite was relatively less if isoascorbate was present, at 4.5% salt, or at low storage temperature. The presence of isoascorbate also counteracted the increase in toxin production attributed to the presence of polyphosphate.     

Factors controlling the growth of Clostridium botulinum types A and B in pasteurized, cured meats III. The effect of potassium sorbate

The growth of Clostridium botulinum types A and B spores, at 10¹ or 10³ per container, was studied in a pork slurry system containing nitrite (40 μg/g), sodium chloride (2.5, 3.5, 4.5% w/v) sodium isoascorbate (550 μg/g) at varying pH levels, with or without potassium sorbate (0.26% w/v), without heating and after two heat treatments (80°C for 7 min, and 80°C for 7 min + 70°C for 1 hr) followed by storage at 15, 17.5, 20 or 35°C for up to 6 months. At a given spore inoculum, potassium sorbate significantly decreased toxin production, as did increasing NaCl, decreasing pH or decreasing storage temperature. Heat treatment did not significantly affect spoilage or toxin production overall, but interacted significantly with some factors. The effect of sorbate was greater at 3.5% NaCl than at 2.5%, at pH values below 6.0, and at low storage temperature.     

Factors controlling the growth of Clostridium botulinum types A and B in pasteurized cured meats IV. The effect of pig breed, cut and batch of pork

Pork slurries were prepared from leg or shoulder muscle from three animals from each of three breeds of pig (Pietrain, Gloucester Old Spot and Large White × Landrace cross). Slurries (pork:water, 1:1.5) contained NaNO₂ (100 μ/g), NaCl (2.5, 3.5 or 4.5% w/v on the water), were subjected to one of three heat treatments (unheated, 80°C for 7 min, 80°C for 7 min plus 70°C for 1 hr) and stored at 35, 20, 17.5 or 15°C for up to 6 months to determine the relative effects of the above factors on growth (spoilage) and toxin production by Clostridium botulinum types A and B at 10³ spores per bottle.
Increasing salt or heat treatment, or decreasing storage temperature or inoculum level all reduced spoilage and toxin production. Both 'animal' and 'cut' significantly affected spoilage and toxin production. More spoilage and toxin production occurred in meat from the shoulder cut than from the leg cut. In both cases there was considerable variation between animals within breed, but there was no systematic difference between breeds. There is no obvious explanation for the variation in meat between animals, but it should be borne in mind when planning and assessing results of large multifactor experiments. Although there was more spoilage and toxin production after 6 months' than 3 months' storage, the statistical analyses yielded essentially similar conclusions.     

Factors controlling the growth of Clostridium botulinum types A and B in pasteurized, cured meats V. Prediction of toxin production: Non-linear effects of storage temperature and salt concentration

While investigating the effects of potassium sorbate and pig breed, cut and batch of pork in a pork slurry system, non-linear effects of storage temperature and salt concentration on toxin production by Clostridium botulinum were detected. Predicted probabilities of toxin production after analysis by logistic regression, published previously, were re-examined and similar effects detected. Improved formulae for the probability of toxin production in a model pork slurry system are given and the implications of the non-linearity of storage temperature and salt concentration on the predicted probability of toxin production are discussed.     

Nitrite, nitrite alternatives, and the control of Clostridium botulinum in cured meats

Historically, nitrite has been a component of meat-curing additives for several centuries. In recent years the safety of nitrite as an additive in cured meats has been questioned mainly because of the possible formation of carcinogenic nitrosamines. Nitrite has many important functions in meat curing including its role in color development, flavor, antioxidant properties, and antimicrobial activity. The inhibition of Clostridium botulinum growth and toxin production is an especially important antimicrobial property of nitrite. This review discusses the effects of processing, curing ingredients (especially nitrite), and storage of cured meats in relation to the control of C. botulinum. If nitrite is eliminated from cured meats or the level of usage decreased, then alternatives for the antibotulinal function of nitrite need to be considered. Several potential alternatives including sorbates, parabens, and biological acidulants are discussed.     

Prediction of toxin production by Clostridium botulinum in pasteurized pork slurry

A model pork slurry system was used to study factors controlling the growth of Clostridium botulinum types A & B (Roberts, Gibson & Robinson, 1981a,b). The following factors were studied in combination: sodium chloride (2.5, 3.5, 4.5% w/v on water); sodium nitrite (100, 200, 300 μg/g); sodium nitrate (0, 500 μg/g); sodium isoascorbate (0, 1000 μg/g); polyphosphate (Curaphos 700, 0, 0.3% w/v); heat treatment (none, 8°C/7 min, 80°C/7 min + 70°C/1 hr); at two pH levels and stored at: 15, 17.5, 20 or 35°C.
Analyses of results yielded a statistical model providing two formulae (for 'low' and 'high' pH slurries) which estimate the probability of toxin production in the pork slurry system within the limits defined above.     

Nitrite,nitrite alternatives,and the control of clostridium botulinum in cured meats

Historically, nitrite has been a component of meat‐curing additives for several centuries. In recent years the safety of nitrite as an additive in cured meats has been questioned mainly because of the possible formation of carcinogenic nitrosamines. Nitrite has many important functions in meat curing including its role in color development, flavor, antioxidant properties, and antimicrobial activity. The inhibition of Clostridium botulinum growth and toxin production is an especially important antimicrobial property of nitrite. This review discusses the effects of processing, curing ingredients (especially nitrite), and storage of cured meats in relation to the control of C. botulinum. If nitrite is eliminated from cured meats or the level of usage decreased, then alternatives for the antibotulinal function of nitrite need to be considered. Several potential alternatives including sorbates, parabens, and biological acidulants are discussed.     

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.     

Predictive model for growth of Clostridium perfringens in cooked cured pork

Mathematical models have been developed and used for predicting growth of foodborne pathogens in various food matrices. However, these early models either used microbiological media or other model systems to develop the predictive models. Some of these models have been shown to be inaccurate for applications in meat and specific food matrices, especially under dynamic conditions, such as constantly changing temperatures that are encountered during food processing. The objective of this investigation was to develop a model for predicting growth of Clostridium perfringens from spore inocula in cured pork ham. Isothermal growth of C. perfringens at various temperatures from 10 to 48.9 degrees C were evaluated using a methodology that employed a numerical technique to solve a set of differential equations. The estimated theoretical minimum and maximum growth temperatures of C. perfringens in cooked cured pork were 13.5 and 50.6 degrees C, respectively. The kinetic and growth parameters obtained from this study can be used in evaluating growth of C. perfringens from spore populations during dynamically changing temperature conditions such as those encountered in meat processing. Further, this model can be successfully used to design microbiologically "safe" cooling regimes for cured pork hams and similar products.     

Quantitative duplex PCR of Clostridium botulinum types A and B neurotoxin genes

A duplex quantitative polymerase chain reaction (PCR) assay for Clostridium botulinum types A and B was developed. The sensitivity and specificity of the assay were verified by using 6 strains of type A, 7 strains of type B, and 14 genera of 42 non-C. botulinum types A and B strains, including C. botulinum types C, D, E, F, and G. In pure culture, the detection limit was 10(2) CFU/ mL for type A and 10(3) CFU/mL for type B. In mushroom broth, increases in the amounts of C. botulinum types A and B could be monitored separately (the quantifiable range was 10(2) to 10(6) for type A and 10(2) to 10(7) for type B) from each sample that contained a large number of background bacteria, and toxin could be detected much earlier than with mouse assay. These results suggest that duplex quantitative PCR methods are useful to detect and quantify C. botulinum types A and/ or B toxin genes.     

In-vitro heat denaturation of Clostridium botulinum toxins types A, B and C

Under standard conditions the effects of heat on Clostridium botulinum toxins types A, B and C were essentially similar. Heat treatments based on existing extensive data for types A and B toxins should be effective against type C toxin for which there is no such information.     

Evaluation of survival of Staphylococcus aureus and Clostridium botulinum in charqui meats

Charqui meats were prepared in laboratory conditions in order to carry out experiments to observe the possibility of development of enterotoxigenic Staphylococcus aureus and Clostridium botulinum proteolytic type B spores and their toxins. Results demonstrated that the harsh processing conditions, high salt concentration, relative high temperature, a(w) values, inhibited the growth of both bacteria. Under our experimental conditions, S. aureus would survive throughout the sequence of salting steps i.e. brine followed by rock salting and the sunshine drying step. However, at final a(w) value of 0.70-0.75 would create conditions to inhibit its development. The other experiment revealed that C. botulinum spores germination also was impaired because of these low a(w) values. Under these conditions, charqui meats revealed to be safe products in relation to toxins from both enterotoxigenic S. aureus and C. botulinum.     

High prevalence of Clostridium botulinum types A and B in honey samples detected by polymerase chain reaction

A test protocol for reliable detection of Clostridium botulinum types A and B spores in honey by polymerase chain reaction (PCR) was developed and used for a prevalence survey of C. botulinum spores in 190 honey samples. The inhibiting effects of honey on microbial growth and PCR analysis were overcome by using a method of supernatant filtration (SF) in the preparation of the samples before enrichment and PCR. By using this method, an inoculum of 0.1 spore of C. botulinum/g honey could be detected. In the prevalence survey, spores of C. botulinum were detected in 8 (7%) of the 114 Finnish and in 12 (16%) of the 76 imported honey samples. The quantity of spores in PCR-positive samples varied from less than 18 to 140 spores/kg. Neurotoxin gene sequences corresponding to C. botulinum type A were detected in 17 samples and proteolytic type B in 12 samples by PCR analysis. Both types A and B were detected in nine samples. Strains of C. botulinum type A were isolated from 14 and type B from 2 of the 20 PCR-positive samples. This is the first report of type A spores of C. botulinum being detected and isolated in Fennoscandia.     

Prevalence and diversity of Clostridium botulinum types A, B, E and F in honey produced in the Nordic countries

A total of 294 honey samples produced in Denmark, Norway and Sweden were studied for the presence of Clostridium botulinum types A, B, E and F by using a multiplex-PCR method. The samples consisted of honeycombs taken directly from beehives, and extracted honey representing several hives or apiaries. The prevalence of C. botulinum showed a significant variation between Denmark, Norway and Sweden, the proportions of positive samples being 26%, 10% and 2%, respectively. The major serotype detected was type B. When analysed with pulsed-field gel electrophoresis (PFGE) using restriction enzyme SacII, the 24 strains isolated produced eight different PFGE patterns. At a similarity level of 95%, four clusters were produced, three of which contained 20 of the 24 analysed strains. One of the clusters included isolates from both Denmark and Norway.     

Food safety objective approach for controlling Clostridium botulinum growth and toxin production in commercially sterile foods

As existing technologies are refined and novel microbial inactivation technologies are developed, there is a growing need for a metric that can be used to judge equivalent levels of hazard control stringency to ensure food safety of commercially sterile foods. A food safety objective (FSO) is an output-oriented metric that designates the maximum level of a hazard (e.g., the pathogenic microorganism or toxin) tolerated in a food at the end of the food supply chain at the moment of consumption without specifying by which measures the hazard level is controlled. Using a risk-based approach, when the total outcome of controlling initial levels (H(0)), reducing levels (ΣR), and preventing an increase in levels (ΣI) is less than or equal to the target FSO, the product is considered safe. A cross-disciplinary international consortium of specialists from industry, academia, and government was organized with the objective of developing a document to illustrate the FSO approach for controlling Clostridium botulinum toxin in commercially sterile foods. This article outlines the general principles of an FSO risk management framework for controlling C. botulinum growth and toxin production in commercially sterile foods. Topics include historical approaches to establishing commercial sterility; a perspective on the establishment of an appropriate target FSO; a discussion of control of initial levels, reduction of levels, and prevention of an increase in levels of the hazard; and deterministic and stochastic examples that illustrate the impact that various control measure combinations have on the safety of well-established commercially sterile products and the ways in which variability all levels of control can heavily influence estimates in the FSO risk management framework. This risk-based framework should encourage development of innovative technologies that result in microbial safety levels equivalent to those achieved with traditional processing methods.     

Prevalence of Clostridium botulinum types B,E and F in faecal samples from Swedish cattle

Faeces were collected from 60 cows at three slaughterhouses situated in southern and central Sweden. The faecal samples were collected during two sampling periods over the year, summer and winter. All samples were analysed for the presence of Clostridium botulinum spores, according to a combined selection and enrichment PCR procedure. One PCR assay was specific for part of the type B neurotoxin gene, while the other assay was specific for both type E and F neurotoxin genes. The prevalence of C. botulinum in Swedish cattle was established to be 73% for non-proteolytic type B and less than 5% for types E and F. Twenty-eight (64%) of the positive faecal samples had a spore load of less than 4 spores/g. Statistical analysis (ANOVA) showed that seasonal variation (summer and winter) had a significant effect on the prevalence of C. botulinum type B in cattle, whereas the effect of geographical location of rearing of the cattle (southern and central Sweden) was less significant.     

Influence of water activity and storage conditions on survival and growth of proteolytic Clostridium botulinum in peanut spread

Outgrowth of Clostridium botulinum spores followed by toxin production in peanut spread at A_w0·98, 0·96, 0·94 and 0·92 stored at 30°C under anaerobic or aerobic conditions for 0, 3, 7 and 16 weeks or 0, 1, 9 and 16 weeks, respectively, was investigated. Botulinal toxin was not detected in peanut spreads stored under anaerobic conditions for 16 weeks. Peanut spreads at A_w0·98 and two of three samples at A_w0·96 stored aerobically became toxic after 9 and 16 weeks, respectively. Clostridium botulinum in peanut spread at A_w0·98 and 0·96 grew to populations of 10⁶and 10⁵cfu g⁻¹, respectively, within 16 weeks. Lactic acid bacteria grew within 3 days in peanut spread at A_w0·98 and 0·96 stored under aerobic or anaerobic conditions. Regardless of A_w, populations of aerobic and anaerobic micro-organisms decreased in peanut spread stored under anaerobic conditions. Only slight decreases occurred in samples stored under aerobic conditions. The pH of inoculated and uninoculated peanut spread at A_w0·98 and 0·96 increased from 4·8 to 7·0 within 16 weeks and was attributed to growth of Penicillium andMucor spp. Similarly, redox potential (Eh) of peanut spread stored under anaerobic conditions for 3 weeks, decreased as the A_wwas increased. Significantly lower Eh values in peanut spread samples at A_w0·98 or 0·96 stored under aerobic conditions occurred within 1 week and/or 9 weeks compared to peanut spread at A_w0·92 or 0·94. Peanut spreads were judged inedible due to growth of lactic acid bacteria and molds which resulted in ‘off’ aromas before toxicity developed, thus greatly minimizing the likelihood of consumption.     

Thermochemical Factors Influencing the Death Kinetics of Spores of Clostridium botulinum 62 A

SUMMARY: Death kinetics of spores of Clostridium botulinum, Type A, strain 62, were studied at sterilant gas temperatures in the range of 40°C (104°F) to 70°C (158°F). Hygroscopic carriers in the form of small discs of filter paper were used. The sterilant gas used was a mixture of dichlorodifluoromethane and ethylene oxide (88 and 12% by weight respectively). Pressures in the exposure atmosphere were adjusted to obtain an ethylene oxide concentration of 700 mg per liter at all temperatures. It was shown by gas-chromatographic analysis that an actual concentration of 700 ± 20 mg per liter was maintained at all exposure temperatures during all exposure periods. The effect of various moisture levels on spore death kinetics was also studied. The relative humidities employed in both the preconditioning and exposure of spores were 3, 23, 33, 53 and 73%, respectively. Thermochemical resistance parameters, D and z, were calculated as the reciprocals of the slopes of the survivor curves and thermochemical destruction time curves, respectively. Of the various environmental moisture levels studied, a preconditioning and exposure relative humidity of 3% for destruction of C. botulinum spores taken from an aqueous suspension, was found to be most effective. This effect held for all temperatures studied. Over the range studied, temperature was seen to have the greatest effect at 3% relative humidity (RH), second greatest at 33% and least effect at 73%.     

Control of nonproteolytic Clostridium botulinum types B and E in crab analogs by combinations of heat pasteurization and water phase salt.

Water phase sodium chloride (WPS) levels of 1.8 to 3.0% in combination with heat pasteurization for 15 min at temperatures of 75, 80, 85, and 90 degrees C were evaluated as methods for the inactivation or inhibition of nonproteolytic, psychrotrophic Clostridium botulinum types B and E in crab analogs (imitation crab legs) subsequently stored at 10 and 25 degrees C. Samples inoculated with 10(2) type B or E spores per g prior to pasteurization remained nontoxic for 120 days at 10 degrees C and for 15 days at 25 degrees C. With 10(4) type E spores per g and 80 degrees C pasteurization, > or = 2.4 and 2.7% WPS was required for inhibition at 10 and 25 degrees C storage, respectively. Pasteurization at 85 degrees C decreased the inhibitory level of WPS to 2.1% at 10 degrees C and to 2.4% at 25 degrees C. When the inoculum was 10(4) type B spores per g, samples with 2.7% WPS were toxic after 80 days of storage at 10 degrees C. Samples inoculated with 10(3) type B spores per g and processed at 85 degrees C remained nontoxic for 15 days at 25 degrees C with a WPS of > or = 2.4%. When pasteurization was carried out before inoculation and packaging, 1.8% WPS prevented toxin production by 10(2) and 10(4) type E spores per g for 30 days at 10 degrees C, and this time period increased as the WPS concentrations increased. Three percent WPS prevented toxin production by 10(4) type E spores per g in vacuum-packaged analogs stored 110 days at 10 degrees C. Pasteurization processes used in these experiments, however, do not inactivate the heat-resistant proteolytic types of Clostridium botulinum. Therefore, the most important factor controlling the growth of this bacterium is continuous refrigeration below 3.0 degrees C or frozen storage of the finished product.