Bacteriocin-mediated inhibition of Clostridium botulinum spores by lactic acid bacteria at refrigeration and abuse temperatures.

The bacteriocinogenicity of Lactococcus lactis ATCC 11454, Pediococcus pentosaceus ATCC 43200, P. pentosaceus ATCC 43201, Lactobacillus plantarum BN, L. plantarum LB592, L. plantarum LB75, and Lactobacillus acidophilus N2 against Clostridium botulinum spores at 4, 10, 15, and 35 degrees C was investigated by modified deferred and simultaneous antagonism methods. All the strains, except L. acidophilus N2, produced inhibition zones on lawns of C. botulinum spores at 30 degrees C. L. plantarum BN, L. lactis ATCC 11454, and P. pentosaceus ATCC 43200 and 43201 were bacteriocinogenic at 4, 10, and 15 degrees C. Supplementation of brain heart infusion agar with 0 to 5% NaCl increased the radii of inhibition zones during simultaneous antagonism assays. Detectable bacteriocin activities were extracted from freeze-thawed agar cultures of L. plantarum BN and L. lactis ATCC 11454 which had been grown at 4 and 10 degrees C. These results suggest that low levels of L. plantarum BN or L. lactis ATCC 11454, in the presence of 3 or 4% NaCl, could be formulated into minimally processed refrigerated food products for protection against possible botulism hazards.     

Effect of heat treatment on survival of, and growth from, spores of nonproteolytic Clostridium botulinum at refrigeration temperatures.

Spores of five type B, five type E, and two type F strains of nonproteolytic Clostridium botulinum were inoculated into tubes of an anaerobic meat medium plus lysozyme to give approximately 10(6) spores per tube. Sets of tubes were then subjected to a heat treatment, cooled, and incubated at 6, 8, 10, 12, and 25 degrees C for up to 60 days. Treatments equivalent to heating at 65 degrees C for 364 min, 70 degrees C for 8 min, and 75 degrees C for 27 min had little effect on growth and toxin formation. After a treatment equivalent to heating at 85 degrees C for 23 min, growth occurred at 6 and 8 degrees C within 28 to 40 days. After a treatment equivalent to heating at 80 degrees C for 19 min, growth occurred in some tubes at 6, 8, 10, or 12 degrees C within 28 to 53 days and at 25 degrees C in all tubes within 15 days. Following a treatment equivalent to heating at 95 degrees C for 15 mine, growth was detected in some tubes incubated at 25 degrees C for fewer than 60 days but not in tubes incubated at 6 to 12 degrees C. The results indicate that heat treatment of processed foods equivalent to maintenance at 85 degrees C for 19 min combined with storage below 12 degrees C and a shelf life of not more than 28 days would reduce the risk of growth from spores of nonproteolytic C. botulinum by a factor of 10(6).(ABSTRACT TRUNCATED AT 250 WORDS)     

Bile acid inhibition of Clostridium botulinum.

Bile acids and their glycine and taurine conjugates were tested in vitro for inhibition of Clostridium botulinum types A and B. Cholic acid inhibited most strains at 2 mg/ml, whereas chenodeoxycholic acid inhibited all strains at 0.4 mg/ml. Deoxycholic acid inhibited one strain at 0.08 mg/ml and other strains at 0.4 and 2 mg/ml. Lithocholic acid inhibited all strains at 0.016 mg/ml. Glycine conjugates also showed considerable inhibition of some strains, whereas taurine conjugates were inactive.     

Bile acid inhibition of Clostridium botulinum.

Bile acids and their glycine and taurine conjugates were tested in vitro for inhibition of Clostridium botulinum types A and B. Cholic acid inhibited most strains at 2 mg/ml, whereas chenodeoxycholic acid inhibited all strains at 0.4 mg/ml. Deoxycholic acid inhibited one strain at 0.08 mg/ml and other strains at 0.4 and 2 mg/ml. Lithocholic acid inhibited all strains at 0.016 mg/ml. Glycine conjugates also showed considerable inhibition of some strains, whereas taurine conjugates were inactive.     

Growth of and toxin production by nonproteolytic Clostridium botulinum in cooked puréed vegetables at refrigeration temperatures.

Seven strains of nonproteolytic Clostridium botulinum (types B, E, and F) were each inoculated into a range of anaerobic cooked puréed vegetables. After incubation at 10 degrees C for 15 to 60 days, all seven strains formed toxin in mushrooms, five did so in broccoli, four did so in cauliflower, three did so in asparagus, and one did so in kale. Growth kinetics of nonproteolytic C. botulinum type B in cooked mushrooms, cauliflower, and potatoes were determined at 16, 10, 8, and 5 degrees C. Growth and toxin production occurred in cooked cauliflower and mushrooms at all temperatures and in potatoes at 16 and 8 degrees C. The C. botulinum neurotoxin was detected within 3 to 5 days at 16 degrees C, 11 to 13 days at 10 degrees C, 10 to 34 days at 8 degrees C, and 17 to 20 days at 5 degrees C.     

Proposed mechanism for sensitization by hypochlorite treatment of Clostridium botulinum spores.

Hypochlorite-treated Clostridium botulinum 12885A spores, but not buffer-treated spores, could be germinated with lysozyme, indicating that their coats are made permeable to lysozyme by hypochlorite treatment so that the cortex is accessible. Hypochlorite-treated spores and spores extracted with 8 M urea-2-mercaptoethanol (pH 3.0) were sensitive to certain components of recovery media, but spores sensitized to lysozyme by other treatments were not. These data indicate that hypochlorite does more than increase coat permeability to lysozyme. Scanning electron microscopy revealed a more open-appearing surface of hypochlorite-treated spores, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that a greater amount of protein was removed from hypochlorite-treated and other lysozyme-sensitized spores than from buffer-treated spores. The data suggest that spore coat proteins may be removed by hypochlorite treatment, and this may be responsible for the sensitivity of spores and for their observed ability to germinate in lysozyme.     

Enumeration of Clostridium botulinum spores in meats by a pour-plate procedure.

Colonies of Clostridium botulinum could be easily distinguished from meat particles by supplementing Wynne agar with 0.4% egg yolk. The pour-plate method was suitable for enumeration of C. botulinum, provided the medium was covered with a layer of agar containing 0.01% dithiothreitol. Viable counts of heat-treated spores were consistently higher in Wynne agar supplemented with egg yolk (Wynne-EY agar) than in Wynne agar alone.     

Hypochlorite injury of Clostridium botulinum spores alters germination responses.

Clostridium botulinum spores were sublethally damaged by exposure to 12 or 28 micrograms of available chlorine per ml for 2 min at 25 degrees C and pH 7.0. The damaging dose was 2.7 x 10(-6) to 3.1 x 10(-6) micrograms of available chlorine per spore. Damage was manifested by a consistent 1.6 to 2.4 log difference between the most probable number enumeration of spores (modified peptone colloid medium) and the colony count (modified peptone yeast extract glucose agar); this did not occur with control spores. Damaged spores could be enumerated by the colony count procedure. Germination responses were measured in several defined and nondefined media. Hypochlorite treatment altered the rate and extent of germination in some of the media. Calcium lactate (9 mM) permitted L-alanine (4.5 mM) germination of hypochlorite-treated spores in a medium containing 12 or 55 mM sodium bicarbonate, 0.8 mM sodium thiosulfate, and 100 mM Tris-hydrochloride (pH 7.0) buffer. Tryptose inhibited L-alanine germination of the spores. Treatments with hypochlorite and with hydrogen peroxide (7%, 25 degrees C, 2 min) caused similar enumeration and germination responses, indicating that the effect was due to a general oxidation phenomenon.     

Germination of spores from Clostridium botulinum B-aphis and Ba410.

The germination of spores from Clostridium botulinum B-aphis and Ba410 was examined. In a complex medium, heat activation of spores from both strains doubled the germination rates and was required for germination in the presence of 2% NaCl. In a defined medium (CTB [D. B. Rowley and F. Feeherry, J. Bacteriol. 104:1151-1157, 1970]), the parent strain B-aphis germinated at a rate of 0.77% min-1 in the absence of NaCl and was not affected by 2% NaCl. A salt-tolerant derivative, strain Ba410, germinated at rates of 0.16% min-1 in CTB and 0.04% min-1 in CTB containing 2% NaCl. L-Alanine-triggered spores germinated faster than did L-cysteine-triggered spores from both strains. When both amino acids were present, B-aphis germinated rapidly in the absence of NaCl and had biphasic kinetics in the presence of NaCl. Strain Ba410 had biphasic kinetics in the absence of NaCl and germinated slowly with single-phase kinetics in the presence of NaCl. L-Alanine- and L-cysteine-triggered germinations were each inhibited by both D-alanine and D-cysteine, indicating a common germinant-binding site for both alanine and cysteine. Attempts to select for variants with amino acid-specific germinant-binding sites were unsuccessful. Differences in the germination kinetics of both strains could not be explained by ultrastructural differences. Transmission electron micrographs revealed striking similarities between the strains.     

Effect of acid and salt concentration in fresh-pack pickles on the growth of Clostridium botulinum spores.

The addition of various amounts of acetic acid to pureed cucumbers inoculated with Clostridium botulinum spores has shown that outgrowth is inhibited at pH 4.8 but not at pH 5.0. Inoculation experiments with whole cucumbers showed that as little as 0.9% acetic acid in the brine was sufficient to prevent outgrowth from spore inocula as high as 10(6)/cucumber. It was further shown that the rapid rate of acetic acid penetration into fresh-pack pickles prevents the growth of any C. botulinum spores that may be present.     

Inhibitory effect of combinations of heat treatment, pH, and sodium chloride on a growth from spores of nonproteolytic Clostridium botulinum at refrigeration temperature.

Nonproteolytic strains of Clostridium botulinum will grow at refrigeration temperatures and thus pose a potential hazard in minimally processed foods. Spores of types B, E, and F strains were used to inoculate an anaerobic meat medium. The effects of various combinations of pH, NaCl concentration, addition of lysozyme, heat treatment (85 to 95 degrees C), and incubation temperature (5 to 16 degrees C) on time until growth were determined. No growth occurred after spores were heated at 95 degrees C, but lysozyme improved recovery from spores heated at 85 and 90 degrees C.     

Chemical manipulation of the heat resistance of Clostridium botulinum spores.

The chemical forms of Clostridium botulinum 62A and 213B were prepared, and their heat resistances were determined in several heating media, including some low-acid foods. The heat resistance of C. botulinum spores can be manipulated up and down by changing chemical forms between the resistant calcium form and the sensitive hydrogen form. The resistant chemical form of type B spores has about three times the classical PO4 resistance at 235 F (112.8 C). As measured in peas and asparagus, both types of C. botulinum spores came directly from the culture at only a small fraction of the potential heat resistance shown by the same spores when chemically converted to the resistant form. The resistant spore form of both types (62A and 213B), when present in a low-acid food, can be sensitized to heating at the normal pH of the food.     

Effect of storage time and temperature on the survival of Clostridium botulinum spores in acid media.

Clostridium-botulinum type A and type B spores were stored in tomato juice (pH 4.2) and citric acid-phosphate buffer (pH 4.2) at 4, 22, and 32 degrees C for 180 days. The spore count was determined at different intervals over the 180-day storage period. There was no significant decrease in the number of type A spores in either the tomato juice or citric acid-phosphate buffer stored for 180 days at 4, 22, and 32 degrees C. The number of type B spores did not decrease when storage was at 4 degrees C, but there was an approximately 30% decrease in the number of spores after 180 days of storage at 22 and 32 degrees C.     

Effect of storage time and temperature on the survival of Clostridium botulinum spores in acid media.

Clostridium-botulinum type A and type B spores were stored in tomato juice (pH 4.2) and citric acid-phosphate buffer (pH 4.2) at 4, 22, and 32 degrees C for 180 days. The spore count was determined at different intervals over the 180-day storage period. There was no significant decrease in the number of type A spores in either the tomato juice or citric acid-phosphate buffer stored for 180 days at 4, 22, and 32 degrees C. The number of type B spores did not decrease when storage was at 4 degrees C, but there was an approximately 30% decrease in the number of spores after 180 days of storage at 22 and 32 degrees C.     

Effect of acid and salt concentration in fresh-pack pickles on the growth of Clostridium botulinum spores.

The addition of various amounts of acetic acid to pureed cucumbers inoculated with Clostridium botulinum spores has shown that outgrowth is inhibited at pH 4.8 but not at pH 5.0. Inoculation experiments with whole cucumbers showed that as little as 0.9% acetic acid in the brine was sufficient to prevent outgrowth from spore inocula as high as 10(6)/cucumber. It was further shown that the rapid rate of acetic acid penetration into fresh-pack pickles prevents the growth of any C. botulinum spores that may be present.     

Recovery of Clostridium botulinum from mud samples incubated at different temperatures

Summary In mud samples naturally contaminated with Clostridium botulinum type C, other types cannot be recovered when incubated at 37°C. At incubation temperatures equal to or lower than 30°C and in the presence of type E, Clostridium botulinum type C cannot always be detected. The use of two incubation temperatures to increase the probability of detecting all types of Clostridium botulinum can therefore be recommended.     

Hypochlorite injury of Clostridium botulinum spores alters germination responses

Clostridium botulinum spores were sublethally damaged by exposure to 12 or 28 micrograms of available chlorine per ml for 2 min at 25 degrees C and pH 7.0. The damaging dose was 2.7 x 10(-6) to 3.1 x 10(-6) micrograms of available chlorine per spore. Damage was manifested by a consistent 1.6 to 2.4 log difference between the most probable number enumeration of spores (modified peptone colloid medium) and the colony count (modified peptone yeast extract glucose agar); this did not occur with control spores. Damaged spores could be enumerated by the colony count procedure. Germination responses were measured in several defined and nondefined media. Hypochlorite treatment altered the rate and extent of germination in some of the media. Calcium lactate (9 mM) permitted L-alanine (4.5 mM) germination of hypochlorite-treated spores in a medium containing 12 or 55 mM sodium bicarbonate, 0.8 mM sodium thiosulfate, and 100 mM Tris-hydrochloride (pH 7.0) buffer. Tryptose inhibited L-alanine germination of the spores. Treatments with hypochlorite and with hydrogen peroxide (7%, 25 degrees C, 2 min) caused similar enumeration and germination responses, indicating that the effect was due to a general oxidation phenomenon.