Botulinum Toxin Production Research Articles

Effect of Sodium Acid Pyrophosphate in Combination with Sodium Nitrite or Sodium Nitrite/Potassium Sorbate on Clostridium botulinum Growth and Toxin Production in Beef/Pork Frankfurter Emulsions

ABSTRACTCombinations of sodium acid pyrophosphate (SAPP) with added sodium nitrite and/or potassium sorbate were tested at various pH levels to determine effectiveness in delaying Clostridium botulinum growth and toxin production in frankfurter emulsions. Formulations containing sodium nitrite (40 ppm), potassium sorbate (0.26%) and SAPP (0.4%) resulted in a greater delay of toxin production (12–18 days) than other combinations (6–12 days) having similar pH values. Treatments containing 0.4% SAPP appeared to be more inhibitory than their counterparts without SAPP, displaying less numbers of toxic samples during the 53‐day storage period at 27°C. Aerobic mesophilic colony counts and residual nitrite data showed little difference among treatments.

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.

The occurrence ofClostridium botulinumat aquatic sites in and around Auckland and other urban areas of the North Island

Samples of bottom muds from lakes and waterways in the Auckland area were inoculated into bottles of cooked meat medium. After incubation, the media were tested for production of Clostridium botulinum toxin which was detected with samples from 11 of the 20 sites examined. Although toxins produced from all positive samples were neutralized by specific antisera of both type C and D toxins, it is likely that C. botulinum type Calpha is the only type present in the Auckland area. All samples from other urban areas of the North Island gave negative results.

Inhibition of Clostridium botulinum by antioxidants, phenols, and related compounds

A total of 75 compounds, including antioxidants, preservatives, gallic acid and p-hydroxybenzoic acid esters, hydroquinones, hydroxyquinolines, phenol derivatives, and related compounds, were screened for their antibotulinal activity in prereduced Thiotone-yeast extract-glucose broth. The most effective inhibitors of Clostridium botulinum growth and toxin production were long-chain esters of p-hydroxybenzoic acid and gallic acid, antioxidants, and butylphenol derivatives. The antioxidant nordihydroguaiaretic acid at 100 microgram/ml delayed the growth and toxin production for the entire incubation period (7 days). Other antioxidants, such as butylated hydroxytoluene, butylated hydroxyanisole, and tert-butylhydroquinone were also very effective (at 200 to 400 microgram/ml) for the inhibition of C. botulinum growth and toxin production. Toxin was detected, although no detectable growth was found by daily absorbance measurements, in the prereduced medium containing 50 to 400 microgram of 8-hydroxyquinoline, pentylphenol, tert-pentylphenol, 3,5-ditert-butylphenol, 3,5-ditert-butylcatechol, (2-hydroxydiphenyl)methane, or (4-hydroxydiphenyl)methane per ml.

Toxin Production by Clostridium Botulinum in Media at pH Lower Than 4.6

Clostridium botulinum types A and B were able to produce toxin in media containing high concentrations of proteins at pH well below 4.6. A medium containing 15% pork, 0.5% yeast extract, 0.5% glucose and 0.05% cysteine-HC1 at an initial pH between 4.30 and 4.36 produced a toxic sample in as early as one week of incubation at 30 C. Toxin production occurred only when large amounts of precipitated protein were present. The presence of other microorganisms was not required for botulinal toxin production. Media prepared with hydrolyzed protein did not have precipitates and did not support production of botulinal toxin at pH lower than 4.6. Since large amounts of precipitated protein seemed necessary for botulinal toxin production, the possibility of the presence of small, localized pockets of higher pH values within the matrices of precipitated protein could not be ruled out.

Production of Botulinum Toxin in Inoculated Pack Studies of Foil-Wrapped Baked Potatoes

Idaho Russet Burbank potatoes were surface or stab inoculated with 10 to 105 spores of Clostridium botulinum type A strain, overwrapped in aluminum foil, baked at 204 C for 50 min or 96 C for 3 h and then held at 22 or 30 C. The shortest incubations resulting in the first botulinogenic potatoes were inversely related to spore doses and ranged from 3 to 7 days; potatoes inoculated with 10 spores were toxic after 5 to 7 days. Total toxin in individual potatoes incubated 3 to 5 days were 5 × 103 to 5 × 105 mouse mean lethal doses. Toxin was not found at distances greater than 1.6 cm from the spore inoculation site. Results indicate that left-over, foil-wrapped, baked potatoes are a perishable food that must be refrigerated.

EFFECT OF GLYCERYL MONOLAURATE ON TOXIN PRODUCTION BY CLOSTRIDIUM BOTULINUM IN MEAT SLURRY

Glyceryl monolaurate when used in the proportion of 5 g per kg of meat slurry (pH 6.0–6.2) inhibited toxin production by Clostridium botulinum type A (strain 73A), type B (strain OKRA) and type E (strain RIV 2). The concentration of glycerol monolaurate required for inhibition was not reduced by addition of lactic acid to the meat slurry until the pH was lowered to pH 5.2 or below. In meat slurry (pH 6.0–6.2), potassium sorbate inhibited production of type B toxin at 32 g/kg. The addition of butylated hydroxy‐anisole to glyceryl monolaurate had no effect upon the concentration needed for inhibition of botulinum toxin production.

EFFECTS OF VARIOUS CONCENTRATIONS OF SODIUM NITRITE AND POTASSIUM SORBATE ON Clostridium botulinum TOXIN PRODUCTION IN COMMERCIALLY PREPARED BACON

ABSTRACTThe study consisted of five treatments including formulations with or without sodium nitrite (120 ppm) or potassium sorbate (0.26%) or both nitrite (40, 80 ppm) and sorbate (0.26%). Packages (300 per treatment) of commercially prepared bacon were inoculated with Clostridium botulinum spores from 10 strains (5 type A and 5 type B) and temperature abused at 27°C. Uninoculated packages (100 per treatment) were also abused. The packages were visually checked for gas production during a 60‐day incubation period and tested for botulinal toxin. Spore and vegetative cell counts, aerobic total plate counts, product pH, residual nitrite depletion, and sorbate levels were also monitored. Toxic samples frequently occurred without gas, and many samples showing gas were nontoxic. Added sorbate or added nitrite extended the time to detection of first gas‐containing and first toxic samples. A combination of sorbate (0.26%) with reduced nitrite levels (40, 80 ppm) extended this time further. Nitrite or sorbate used singly in the formulation also decreased the rate of toxin production. None of the uninoculated packages was toxic, while the total number of toxic inoculated packages decreased with nitrite or sorbate in the formulations. Ninety percent of the samples from the control treatment became toxic during the 60‐day incubation period; 58.8% from the treatment with 0.26% sorbate; 22.0% from the treatment with 40 ppm nitrite and 0.26% sorbate; none from the treatment with 80 ppm nitrite and 0.26% sorbate; and 0.4% from the treatment with 120 ppm nitrite. Low nitrite‐sorbate combinations were thus effective in delaying botulinal toxin production in temperature abused bacon.

Clostridium botulinum CONTROL BY SODIUM NITRITE AND SORBIC ACID IN VARIOUS MEAT AND SOY PROTEIN FORMULATIONS

ABSTRACTClostridium botulinurn inoculated frankfurter‐type mixtures formulated with different meats (mechanically deboned chicken. meat, beef, and pork) and/or soy proteins (textured, concentrate, and isolate) were incubated at 27°C. The effects of sodium nitrite and/ or sorbic acid on botulinal spore germination, outgrowth, and toxin production were determined at specified time intervals during the incubation period. Also, residual nitrite depletion and in some instances total microbial growth were measured. Sorbic acid alone (0.2%) or in combination with nitrite (80 pg/g) retarded spore germination and outgrowth, and delayed toxin production. These effects were of larger magnitude in mixtures formulated with both nitrite and sorbic acid and were greater in beef, pork, and soy proteins compared to mechanically deboned chicken meat. Nitrite (80 μg/g) alone was only slightly effective in delaying toxin production and of significance only in pork. The highest nitrite level (156 μg/g)) tested in a meat‐soy mixture was significantly inhibitory to toxin development. Botulinal germination, outgrowth, and toxin production were slower in all‐soy, control (nitrite/sorbic acid‐free) formulations than in all‐meat, control samples. Residual nitrite depletion was slower in soy formulations, especially of the textured form, compared to all‐meat treatments. Inclusion of nitrite and/or sorbic acid in the formulation did not significantly change the rate and extent of total microbial growth in meat‐soy formulations.

EFFECTS OF SODIUM NITRITE ON Clostridium botulinum TOXIN PRODUCTION IN FRANKFURTER EMULSIONS FORMULATED WITH MEAT AND SOY PROTEINS

ABSTRACTBotulinal toxin production, residual nitrite depletion, and total microbial growth were measured in meat and/or soy‐containing frankfurter formulations during 27° C temperature abuse. Botulinal toxin production in formulations containing soy proteins was equal to or less rapid than in all‐meat samples, both in the absence and presence of nitrite. Of the nine soy proteins examined, nitrite did not delay C. botulinurn toxin production in products made from the isolate form, whereas it was an effective control in textured, flours and concentrates. In all‐meat formulations the effectiveness of nitrite decreased with increasing spore load. Residual nitrite depletion in meat wasmore rapid than in soy‐containing treatments. Among the soy proteins, nitrite depletion was more rapid in the isolate than in the textured form tested. Total microbial growth was similar in both meat and soy‐containing samples. Gas production was an inadequate index of toxin development.

Botulism Control by Nitrite and Sorbate in Cured Meats: A Review

Nitrite plays a major role in the botulinal safety of cured meat products. When used at appropriate levels, it retards Clostridium botulinum growth and delays production of its deadly neurotoxin. Even though the incidence of botulinal spores in meat is very low, factors such as the tonnage of cured meats consumed, the potential for mishandling such products, and the nature of the disease necessitate the use of nitrite or other equally effective compound(s) for extra safety. Residual nitrite and nitrosamine levels in cured meat products have been decreasing in recent years through control and research conducted by the meat industry and related institutions. Such levels are minimal compared to total nitrate and nitrite amounts ingested or formed in the human body. Sorbate, especially in combination with nitrite at concentrations adequate only for cured meat color and flavor development, is at least as effective as currently used nitrite levels in delaying C. botulinum growth and toxin production. The mechanism(s) through which nitrite and/or sorbate perform their actions upon C. botulinum remains to be found. A review of the pertinent research completed to date should be helpful in the search for the mechanism(s).

SODIUM NITRITE AND SORBIC ACID EFFECTS ON Clostridium botulinum TOXIN FORMATION IN CHICKEN FRANKFURTER‐TYPE EMULSIONS

ABSTRACT C. botulinum growth and toxin production and residual nitrite depletion were studied in a mechanically deboned chicken meat (MDCM) frankfurter‐type product, during 27°C temperature abuse. A series of three trials were conducted in order to determine the effects of sodium nitrite and sorbic acid on the above parameters. Low nitrite concentrations (20 and 40 μg/g), did not influence C botulinum growth and toxin production. The addition of sorbic acid (0.2%) to these nitrite levels resulted in a significant extention of the time necessary for toxin to develop. The 0.1% sorbic acid level was ineffective. Nitrite concentrations of 156 μg/g doubled the time necessary, for botulinal toxin production, as did 0.2% sorbic acid alone. The magnitude of toxin production delay was increased five fold when 156 μg/g nitrite and 0.2% sorbic acid were combined. Nitrite depletion, was rapid during processing and 27°C incubation. Low residual nitrite concentrations were present for a longer time in nitrite‐sorbic acid combination treatments, indicating that the presence of sorbic acid may delay depletion of residual nitrite. Gas production and spoilage of the product followed the same rate of development as toxin.

Clostridium botulinum growth and toxin production in tomato juice containing Aspergillus gracilis.

The ability of spores of one type A and one type B strain of Clostridium botulinum to grow and produce toxin in tomato juice was investigated. The type A strain grew at pH 4.9, but not at pH 4.8; the type B strain grew at pH 5.1, but not at pH 5.0. Aspergillus gracilis was inoculated along with C. botulinum spores into pH 4.2 tomato juice; in a nonhermetic unit, a pH gradient developed under the mycelial mat, resulting in C. botulinum growth and toxin production. In a hermetic unit, mold growth was reduced, and no pH gradient was detected; however, C. botulinum growth and low levels of toxin production (less than 10 50% lethal doses per ml) still occurred and were associated with the mycelial mat. The results of tests to find filterable or dialyzable growth factors were negative. It was demonstrated that for toxin production C. botulinum and the mold had to occupy the same environment.

EFFECT OF SORBIC ACID AND SODIUM NITRITE ON Clostridium botulinum OUTGROWTH AND TOXIN PRODUCTION IN CANNED COMMINUTED PORK

ABSTRACTExperiments to determine the effect of sorbic acid alone and in combination with low nitrite and phosphate on botulinal toxin production in inoculated perishable, canned, comminuted, cured pork were conducted. Forty‐eight test lots of product were inoculated with approximately 100 spores per gram of five strains each of type A and B Clostridium botulinum. Twenty‐five cans of each test lot were incubated at 27° C for up to 110 days. The time of swelling was recorded for each can and the first 10 swells per test lot were tested for toxin. Multiple regression analysis of the time to first swell showed nitrite concentration had a significant linear effect on delaying outgrowth and toxin production of C. botulinum. Sorbic acid concentration was also significantly related to inhibition but through the fourth power of sorbic acid concentration. The use of either sodium acid pyrophosphate or sodium hexametaphosphate was synergistic with sorbic acid as indicated by their statistically significant positive interactions. Several test lots containing 0.2% sorbic acid and either phosphate had no toxic swells after 110 days of incubation with or without 50 ppm NaNO2. These data indicate that sorbic acid may be a potential alternative preservative to high levels of nitrite in canned, comminuted pork products.

Clostridium botulinum and Acid Foods

Outbreaks of botulism involving acid foods are rare. Of the 722 total botulism outbreaks reported from 1899 to 1975, only 34 (4.7%) involved acid foods. Home-canned acid foods were implicated in 34 of the 35 acid food outbreaks. Clostridium botulinum cannot grow at a pH of ⩽ 4.6; therefore, for a botulism hazard to exist in an acid food, a contamination with other microorganisms due to a process delivery failure and/or post-process contamination, (c) favorable composition of the food and storage conditions which are particularly conducive to C. botulinum growth and toxin production, and (d) metabiosis. The way each factor affects the botulism hazard in acid foods is discussed in this report. An acid food is safe from C. botulinum if the heat process kills all organisms capable of growth at a pH of ⩽4.6 and there is no post-process contamination.

INTESTINAL INFECTION AND TOXIN PRODUCTION BY CLOSTRIDIUM BOTULINUM AS ONE CAUSE OF SUDDEN INFANT DEATH SYNDROME

The spontaneous production of botulinum toxin in the infant gut by ingested Clostridium botulinum organisms is the underlying cause of infant botulism, recognised as an infectious disease only in late 1976. Because of the recognition of the pathophysiology of this disease and because the known potency and action of botulinum toxin can lead to rapid respiratory arrest, it appeared possible that the in-vivo production of botulinum toxin could cause the sudden death of some infants. To test this hypothesis, serum, selected tissues, and bowel contents from 280 dead infants were examined for the presence of C. botulinumtoxin and/or organisms. We found C. botulinumorganisms in 10 infants, all of whom died suddenly and unexpectedly. 9 of these deaths were classified by the forensic pathologist as sudden infant death syndrome (S.I.D.S. or crib death). In 2 of these 10 sudden deaths both C. botulinum organisms and botulinum toxin were identified, and from the spleen of 1, C. botulinumorganisms were isolated. Fæcal specimens from 160 age-matched healthy infants who served as controls in studies of inpatient infant botulism cases were negative for both C. botulinum organisms and toxin, except for one specimen that contained only C. botulinum type A organisms. The 9 S.I.D.S. cases with evidence of C. botulinum infection comprised 4·3% of the 211 S.I.D.S. cases examined over 12 months. These findings suggest that intestinal production of botulinum toxin by C. botulinum is one cause of S.I.D.S. The strikingly similar age-distribution of 62 inpatient infant botulism cases and the 211 S.I.D.S. cases is also consistent with this concept. The possibility that in-vivo production of botulinum toxin may account for a larger proportion of S.I.D.S. cases is discussed.

EXPERIMENTAL BOTULISM IN CHICKENS: THE CECUM AS THE SITE OF PRODUCTION AND ABSORPTION OF BOTULINUM TOXIN

Highly purified preparations of Clostridium botulinum toxins were administered to chickens by various routes. Chickens were highly susceptible to type A toxin, but relatively resistant to toxins of other types. Type C toxin (12S) at a dose of 1 X 10(7) mouse ip LD50 failed to kill the chicken by the oral route. Oral administration of 10 or more of type A, C, or D spores killed normal chickens, whereas cecoligated chickens were insusceptible to oral administration of 10(6) spores. These results show that the site of production and absorption of botulinum toxin in chickens is the cecum. Peroral administration of spores of a type C strain cured of its prophages and producing the C2 factor only also killed normal chickens. Chickens appeared to the more susceptible to the C2 factor than to the C1 toxin. The C2 factor, therefore, may play more important role in chicken deaths from toxico-infection with type C organisms. The optimum temperature for growth of C. botulinum types C and D was found to be 40-42 C. Type C and D toxins were significantly more stable than type A toxin in the cecum contents with pH above 7. These characteristics and the high density of distribution of type C spores in the environment may explain prevailing cases of type C botulism among broiler chickens.

VARIATION IN INHIBITION OF C. botulinum BY NITRITE IN PERISHABLE CANNED COMMINUTED CURED MEAT

ABSTRACTA series of seven tests was conducted to establish a base line for the inhibition of C. botulinum by nitrite in a perishable canned meat product. The product was subjected to abusive storage at 27°C. The degree of variation in swell times and rates among the tests was determined. Predicted average times to first swell were 6.7, 29.8, 82.6 and 94.3 days when 0, 50, 100 and 156 μg/g of sodium nitrite was added to the meat. The primary effect of nitrite appears to have been in determining the length of the lag phase. Once swelling commenced, the rate at which the cans swelled was not significantly different at 50, 100, and 156 μg/g of sodium nitrite. This information will be used to evaluate additives for controlling botulinal growth and toxin production under the conditions described.

Effect of sodium nitrite and sodium nitrate on botulinal toxin production and nitrosamine formation in wieners.

Wieners were formulated and processed approximating commercial conditions as closely as possible. Twenty-four batches of product were made with the addition of six levels of sodium nitrite (0, 50, 100, 150, 200, and 300 mug/g), four levels of sodium nitrate (0, 50, 150, and 450 mug/g), and two levels of Clostridium botulinum (0 and 620 spores/g). After formulation, processing, and vacuum packaging, portions of each batch were incubated at 27 C or held for 21 days at 7 C followed by incubation at 27 C for 56 days. The latter storage condition approximated distribution of product through commercial channels and potential temperature abuse at the consumer level. Samples were analyzed for botulinal toxin, nitrite, and nitrate levels after 3, 7, 14, 21, 28, and 56 days of incubation. When nitrite was not added, toxic samples were detected after 14 days of incubation at 27 C. At the lowest level of nitrite added (50 mug/g), no toxic samples were observed until 56 days of incubation. Higher levels of nitrite completely inhibited toxin production throughout the incubation period. Nine uninoculated samples, representing various levels and combinations of nitrite and nitrate, were evaluated organoleptically. The flavor quality of wieners made with nitrite was judged significantly higher (P = 0.05) than of wieners made without nitrite. The nine samples were negative for 14 volatile nitrosamines at a sensitivity level of 10 ng/g. The results indicated that nitrite effectively inhibited botulinal toxin formation at commercially employed levels in wieners and that detectable quantities of nitrosamines were not produced during preparation and processing of the product for consumption.

Effect of Sodium Nitrite and Sodium Nitrate on Botulinal Toxin Production and Nitrosamine Formation in Wieners

Wieners were formulated and processed approximating commercial conditions as closely as possible. Twenty-four batches of product were made with the addition of six levels of sodium nitrite (0, 50, 100, 150, 200, and 300 mug/g), four levels of sodium nitrate (0, 50, 150, and 450 mug/g), and two levels of Clostridium botulinum (0 and 620 spores/g). After formulation, processing, and vacuum packaging, portions of each batch were incubated at 27 C or held for 21 days at 7 C followed by incubation at 27 C for 56 days. The latter storage condition approximated distribution of product through commercial channels and potential temperature abuse at the consumer level. Samples were analyzed for botulinal toxin, nitrite, and nitrate levels after 3, 7, 14, 21, 28, and 56 days of incubation. When nitrite was not added, toxic samples were detected after 14 days of incubation at 27 C. At the lowest level of nitrite added (50 mug/g), no toxic samples were observed until 56 days of incubation. Higher levels of nitrite completely inhibited toxin production throughout the incubation period. Nine uninoculated samples, representing various levels and combinations of nitrite and nitrate, were evaluated organoleptically. The flavor quality of wieners made with nitrite was judged significantly higher (P = 0.05) than of wieners made without nitrite. The nine samples were negative for 14 volatile nitrosamines at a sensitivity level of 10 ng/g. The results indicated that nitrite effectively inhibited botulinal toxin formation at commercially employed levels in wieners and that detectable quantities of nitrosamines were not produced during preparation and processing of the product for consumption.