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 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 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.     

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.     

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.     

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.     

The effect of 100% CO2 on the growth of nonproteolytic Clostridium botulinum at chill temperatures

The growth of a cocktail of spores from six nonproteolytic Clostridium botulinum type B and E isolates at 5 and 10 degrees C was used to assess the combined effect of NaCl (0.5-4.5% w/v), pH (5.5-6.5) and atmosphere (10% H2:90% N2, 5% CO2:10% H2:85% N2, or 100% CO2) in buffered peptone, yeast, glucose, starch broth with an Eh of approximately -350 mV. Under all atmospheres growth tended to be slower as the concentration of NaCl increased and with NaCl combined with pH levels below 6.0. Of the atmospheres tested, growth occurred at a slower rate and over a narrower range of conditions when C. botulinum was exposed to 100% CO2. This effect was enhanced when the incubation temperature was 5 degrees C. The results indicate that while CO2 decreased C. botulinum growth at chill temperatures, prevention of growth also depended on the NaCl concentration and the pH of the medium.     

The effect of selected functional additives and heat treatment on nitrosamine content in pasteurized pork ham

The influence of pasteurization and sodium chloride, sodium ascorbate, polyphosphates and sodium nitrite coupled with pasteurization on nitrosamine contents in pork was studied. Nitrosamines: dimethylonitrosamine (DMNA) and diethylonitrosamine (DENA) were extracted from raw material, distilled, condensed in an evaporator under lowered pressure and analyzed chromatographically. An inhibitory effect of NaCl and sodium ascorbate on volatile nitrosamines (DMNA and DENA) was seen. Adding solutions of polyphosphates to the meat caused a slight increase in nitrosamine contents, higher than noted with sodium chloride. The effect of these compounds on nitrosamine formation depended on the presence of polyphosphates and sodium nitrite in the brine. If the brine contained nitrites, the adverse effect of sodium ascorbate and NaCl on nitrosamine formation was weaker. Moreover, a strong inhibitory effect of pasteurization on DMNA and DENA formation was observed.     

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.     

The influence of breed on the carcass and eating quality of pork.

Twelve gilts from four breeds were reared from 30 to 65 kg liveweight and assessed for carcass, muscle and eating quality. The breeds were Large White (LW), Gloucester Old Spot (GOS) Crossbreed 1 (C1) and Crossbreed 2 (C2). The latter two were commercial products from breeding companies. There were small differences in quality between the breeds. C1 had about 2% more lean than the others. GOS had shorter carcasses, a higher proportion of total lean in the fore limb and thicker backfat but were not fatter overall. C1 and C2 had lower pH₁ measurements than LW or GOS but similar colour and drip loss measurements in M. longissimus lumborum. There were positive but weak relationships between pH₁ and percentage fat (r= +0.1 to +0.3). C1 had the more tender meat. When all breeds were pooled, about 9 % of the variation in toughness was accounted for by variation in percentage lean or fat. There was no suggestion that the pure breeds or fatter pigs had meat of better eating quality.     

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.     

Iron and zinc compounds in the muscle meats of beef,lamb, pork and chicken

Beef, lamb, pork and chicken leg muscles were extracted with distilled water and the soluble iron and zinc compounds separated by gel filtration and dialysis. Iron was distributed between five main components: an insoluble fraction, ferritin, haemoglobin, myoglobin and a low molecular weight fraction. In beef and lamb, myoglobin was the predominant iron compound but in pork and chicken, most of the iron was associated with the insoluble fraction. Whereas more than 70% of beef iron was associated with the haemoproteins, haemoglobin and myoglobin, less than 30% of chicken iron was in this form. Even so, in all meats most of the soluble iron was associated with the haemoproteins. Zinc was present mainly in the insoluble fraction. The soluble zinc was distributed between five main components. Over 70% of soluble zinc was associated with two components having molecular weights of 65 000 and 35 000. The nature and availability of zinc and iron in the various meat fractions is discussed.