Everything anyone wanted to know about botulinum toxin
Botulinum neurotoxin (BoNT) is the most toxic substance known to man and maybe its wise to understand it thoroughly.
There are 7 types of botulinum toxin (A through G). Types A, B, E and, in rare cases, F cause disease in humans. Types C and D cause disease in birds and mammals. Type G has not yet been confirmed as a cause of illness in humans or animals.
Until the early 1960s nearly all recognized outbreaks of botulism in which toxin types were determined were caused by type A or B toxins and were usually associated with with ingesting home-canned vegetables, fruits and meat products.
Virtually all cases of botulism type E are from contaminated aquatic (i.e. originating either in sea or fresh water) products (fish or aquatic mammals) with several cases attributed to beavers. A limited number of outbreaks of type F botulism have been reported in this country with one outbreak traced to home-prepared venison jelly.
From 1950 through 1996 time period, 65% of botulism outbreaks have been traced to home-processed foods, while 7% have been linked to commercially processed foods, including foods served in restaurants.
Vegetables have been the most important vehicle for the botulism toxin in the US from 1950 through 1996. Beef, milk products, pork and poultry have caused fewer outbreaks.
Thermal Inactivation of Botulinum Toxins in Canned Salmon
Canned salmon is a low acid food. If all C. botulinum spores are not destroyed by proper processing in home canning, a potential hazard of botulism exists. Boiling will destroy the toxin, but will also affect the palatability and appearance of the salmon. To determine the minimum temperature and heating time to destroy toxin, type A, B, or E toxin was added to canned salmon. Two-gram portions in small glass vials were heated at 68, 71, 74, 79 and 85°C for various lengths of time up to 20 minutes. To establish a heating procedure for cans and jars of salmon, spores of types A, B, and E C. botulinum were added to commercially canned salmon and to glass jars of home-canned salmon and incubated for 10 days. Open cans and jars were oven-heated at 350°F (177°C) to the same end-point temperatures. Assays for toxin were carried out with mice. Inactivation of toxin in salmon heated in vials showed an initial rapid drop in toxicity followed by a leveling-off of the rate so that 20-minute heating at 68 or 71°C did not destroy all of the toxin. At 79 and 85°C, the inactivation was much more rapid and all detectable toxin was destroyed within 2 to 5 minutes. For the oven-heating method for two sizes of cans and jars, an internal temperature of 85°C followed by a 30-minute holding period at room temperature was effective in inactivating botulinum toxin.
HEAT INACTIVATION RATES OF BOTULINUM TOXINS A, B, E AND F IN SOME FOODS AND BUFFERS
Inactivation of botulinum toxins was determined in selected acid and low acid foods and buffer systems. Heating at 74°C and 79°C gave a biphasic curve when the log of the inactivation of the toxins was plotted against the time of heating. At 74°C, the time for inactivation of 103 LD50 of type A toxin per gram of an acid food such as tomato soup to no detectable toxin by mouse assay was an hr. or more. At 85°C the inactivation was very rapid and approached exponential decrease with inactivation to no detectable toxin within 5 min. In general, the toxins were more stable in acid foods such as tomato soup at pH 4.2 than in low acid foods, such as canned corn at pH 6.2. Twenty minutes at 79°C or 5 min at 85°C is recommended as the minimum heat treatment for inactivation of 103 LD50 botulinum toxins per gram of the foods tested.
HEAT INACTIVATION OF BOTULINUM TOXIN TYPE A IN SOME CONVENIENCE FOODS AFTER FROZEN STORAGE
Crystalline type A toxin from the Hall Strain of Clostridium botulinum was added to beef pie fillings (pH 5.9), 0.05M phosphate buffer (pH 5.9), cream of mushroom soup (pH 6.2) or tomato soup (pH 4.1) and 1 ml placed in 2-ml thin glass ampules. These were frozen and stored at -20°C for 180 days. At timed intervals a few ampules were thawed and the contents tested for toxicity and for the rate of heat inactivation of the toxin. The toxicity of type A in the contents remained the same throughout the frozen storage. Although the literature reports show a decrease in the heat stability of type E toxin after frozen storage, the heat inactivation rates for type A remained the same. pH is one of the important variables affecting the heat stability of type A toxin dissolved in various buffers.
The Case of Botulinum Toxin in Milk
We demonstrated that standard pasteurization at 72°C for 15s inactivates at least 99.99% of BoNT/A and BoNT/B and at least 99.5% of their respective complexes. Our results suggest that if BoNTs or their complexes were deliberately released into the milk supply chain, standard pasteurization conditions would reduce their activity much more dramatically than originally anticipated and thus lower the threat level of the widely discussed “BoNT in milk” scenario.
For the current study, it was important to consider the food matrix used for the experiments, in this case raw milk, since it has a major impact on the heat inactivation rate of BoNT.
Early work by Scott and Stewart (published in 1950) demonstrated that vegetable juice increased the heat stability of BoNT/A and BoNT/B due to their being protected by bivalent cations and organic acid anions present in the juice.
Later, Bradshaw et al. (in 1979) showed that BoNT/A and BoNT/B were more heat stable in beef and mushroom patties than in a phosphate buffer at the same pH.
Woodburn et al. (in 1979) also observed increased heat stability of BoNT/A when 1% gelatin was added to a phosphate buffer.
Recently, it has been shown that the molten-globule-like character of BoNT and its interaction with NAPs are responsible for variations in physical stability at different pH values.
heat resistance of C. botulinum type E toxin
The heat resistance of C. botulinum type E toxin depends on the pH; the toxin is destroyed by moderate heating at neutral pH and is more resistant at lower pH values (pH 4.0 - 5.0). Thus, the toxin was destroyed after 5 min at 60 °C (140 °F) in a cooked meat medium (pH 7.5) (cited from Huss 1981) but at 62 °C (144 °F) and 65 °C (149 °F) in meat broth (pH 6.2) (Abrahamsson and others 1965). Woodburn and others (1979) investigated the heat inactivation of several botulinum toxins and found that in canned corn at pH 6.2, a 3D reduction occurred in 2 min at 74 °C (165 °F). In phosphate buffer, a similar reduction occurred in 1 min at pH 6.8 but took 6 min if 1% gelatin was added to the buffer (Woodburn and others 1979).
Inactivation by ph and type at 74C
- Type A in tomato soup at ph 4.2 takes 25 min
- Type A in string beans at ph 5.1 takes 4 min
Type A in canned corn at ph 6.2 takes 2 min
Type B in tomato soup at ph 4.2 takes 20 min
- Type B in string beans at ph 5.1 takes 15 min
Type B in canned corn at ph 6.2 takes 1 min
Type E in tomato soup at ph 4.2 takes 2 min
- Type E in string beans at ph 5.1 takes 1 min
- Type E in canned corn at ph 6.2 takes 2 min
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