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 VI. Nitrite monitoring during storage of pasteurized pork slurries

Residual nitrite levels were monitored during storage for up to 6 months, in a model pork slurry system used to study the relative effects of curing ingredients and additives used in pasteurized cured meats to control the growth of Clostridium botulinum.
In 'low' pH slurries the rate of loss of nitrite fell with reducing storage temperature. Less residual nitrite remained after HIGH heat treatment but the rate of loss of that residual nitrite was slower during storage than nitrite remainly after LOW heat treatment. Inclusion of nitrate resulted in higher residual nitrite levels, particularly after HIGH heat and if stored below 20°C. If isoascorbate was added nitrite became undetectable within circa 30 days, even when nitrate had been added. The rate of loss of nitrite was slower in 'high' pH slurries (pH 6.3–6.8).
Monitoring levels of nitrite in the product soon after production would detect its accidental overuse but monitoring nitrite in the product during distribution or at retail, without knowledge of the composition and prior history of the product, gives little indication of the amount used at manufacture. The level of residual nitrite was not directly related to the ability of the curing mixture to control the growth of CI. botulinum types A and B. Some slurries in which C1. botulinum grew least during 6 months' storage contained no residual nitrite because isoascorbate was also present.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Evaluation of the effects of sodium chloride,potassium sorbate,nisin and lysozyme on the probability of growth of Clostridium sporogenes

The aim of this study was to evaluate the combined effect of salt (sodium chloride, 0–8% w/v), sorbate (potassium sorbate, 0–4.5% w/v), nisin (0–500 ppm) and lysozyme (0–500 ppm) on the survival of Clostridium sporogenes as a non‐toxigenic surrogate of Clostridium botulinum in terms of the probability of growth by using a central composite rotatable design. The results indicated that salt and sorbate were the most effective factors in preventing the growth of Cl. sporogenes in high‐moisture (>95%) and low‐acid conditions. The probability of growth of Cl. sporogenes in broth was reduced by combinations of salt and sorbate. Nisin and lysozyme had insignificant effects on the probability of growth of Cl. sporogenes (P > 0.05). Lysozyme individually and in combination with nisin had no inhibitory effect on Cl. sporogenes. Overall, the addition of sorbate and lysozyme may allow the salt concentration to be reduced while preventing growth.     

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.     

Mycoflora of two types of Portuguese dry-smoked sausages and inhibitory effect of sodium benzoate, potassium sorbate, and methyl p-hydroxybenzoate on mold growth rate

The mycoflora of chouriqo types Alentejano and Ribatejano, two varieties of Portuguese dry-smoked sausages, have been investigated after a producer-defined shelf life period (120 days at 20 +/- 5 degrees C) in modified atmosphere packaging (55% N2 and 45% CO2). On the basis of morphological and physiological characteristics, the isolates were identified as Penicillium, Aspergillus, Fusarium, Rhizopus, Monilia, Absidia, and Cephalosporium. The species identified were as follows: Penicillium terrestres (43.4%), Penicillium sp. (13.3%), Fusarium sp. (10%), Aspergillus glaucus (10%), Aspergillus versicolor (6.8%), Monilia fruticola (3.3%), Absidia sp. (3.3%), Cephalosporium sp. (3.3%), Rhizopus stolonifer (3.3%), and Fusarium tricinctum (3.3%). Additionally, the effects of three preservatives (potassium sorbate [PS], sodium benzoate [SB], and methyl p-hydroxybenzoate [MHB]) were studied on the growth rate of mold representative isolates. MHB showed a greater inhibitory effect than SB and PS in all fungi isolates, except in A. glaucus [Tm30(A)], in which the inhibitory effect of MHB was similar to PS. At 0.05% (wt/vol), all fungi were inhibited with MHB, except for R. stolonifer [Tm25(A)], which started to decrease the growth rate only at a concentration higher than 0.1%. PS was more effective at inhibiting mold growth than SB, except in Absidia sp. [Tm16(R)], in which both showed a similar inhibitory effect. MHB showed great promise as an application to the surface at 0.1% (wt/vol) to improve the stability and safety of the product through the inhibition of potential spoilage and toxigenic molds.     

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.     

Evaluation of the effect of acetylsalicylic acid on Clostridium botulinum growth and toxin production

The Republic of Georgia (ROG) has the highest incidence of botulism among all countries in the world, with most cases attributed to home-preserved vegetables. Based on epidemiologic data, the occurrence of botulism in ROG is lower in areas where aspirin (active ingredient, acetylsalicylic acid [ASA]) is added to home-canned vegetables. The objective of this study was to evaluate, with a broth medium, the antibotulinal activity of ASA to determine the possible role of ASA in preventing botulinum toxin production in home-canned vegetables. Trypticase-peptone-glucose-yeast (TPGY) broth (pH 7.0) with 0, 0.3, and 0.6 mg of ASA per ml was inoculated with a 10-strain mixture of proteolytic Clostridium botulinum type A and B spores at ca. 10(3) spores per ml. The inoculated broths were incubated at 31 degrees C under anaerobic conditions, and C. botulinum growth and botulinum toxin production were determined for up to 36 h. Results showed ASA in broth delayed (time to initial detectable toxin produced and amount of toxin produced), but did not prevent, both growth and toxin production by C. botulinum. These results would not provide a definitive explanation for differences in toxin production in canned vegetables prepared with and without aspirin.     

Influence of potassium sorbate on the growth of yeasts and moulds in yogurt

Yeasts and moulds are little affected by low pH and may cause spoilage of yogurt during storage. In the present study, potassium sorbate was added as a preservative in concentrations of 0.05%, 0.1% and 0.2%. The unpreserved yogurt showed unfavourable characteristics: yeast and mould counts and acidity increased. The addition of potassium sorbate, however, inhibited yeast and mould levels, with normal characteristic properties extending more than 14 days. Potassium sorbate is thus seen as valuable for preserving yogurt.     

苯甲酸钠和山梨酸钾的复配抑菌效果研究

采用液体培养法和比浊法研究苯甲酸钠和山梨酸钾的3种复合配伍(1∶1∶2∶3∶3∶2)对大肠杆菌和金黄色葡萄球菌的抑菌效果,运用SPSS16.0统计软件对所测数据进行统计分析,从中筛选最佳抑菌配伍及最佳抑菌浓度.结果表明,这两种防腐剂的3种复合配伍对两种菌的生长都有显著的抑制作用,且随质量浓度增加抑菌作用逐渐增强.苯甲酸钠和山梨酸钾复合配比为3∶2时,对大肠杆菌的抑菌效果较好;复合配比为2∶3时,对金黄色葡萄球菌的抑菌效果较好;3种复合配伍的最佳抑菌浓度均为1.0 g/L.