Growth from Spores of Nonproteolytic Clostridium botulinum in Heat-Treated Vegetable Juice

Unheated spores of nonproteolytic Clostridium botulinum were able to lead to growth in sterile deoxygenated turnip, spring green, helda bean, broccoli, or potato juice, although the probability of growth was low and the time to growth was longer than the time to growth in culture media. With all five vegetable juices tested, the probability of growth increased when spores were inoculated into the juice and then heated for 2 min in a water bath at 80°C. The probability of growth was greater in bean or broccoli juice than in culture media following 10 min of heat treatment in these media. Growth was prevented by heat treatment of spores in vegetable juices or culture media at 80°C for 100 min. We show for the first time that adding heat-treated vegetable juice to culture media can increase the number of heat-damaged spores of C. botulinum that can lead to colony formation.     

Vegetable juice aids the recovery of heated spores of non-proteolytic Clostridium botulinum

Heating spores of non-proteolytic Clostridium botulinum at 85C for 2 min followed by plating on a standard laboratory medium reduced the count of viable spores by a factor of greater than 10⁴. A similar result was obtained when the plating medium was supplemented with juice from courgette, carrot or mung bean sprout. When plating was on media supplemented with hen egg white lysozyme or juice from turnip, swede, flat bean, cabbage or potato, heating at 85C for 10 min did not reduce the viable count by a factor of 10⁴. Thus these vegetable juices increased the measured heat resistance of spores of non-proteolytic Cl. botulinum . These findings are relevant to the safety of minimally processed (e.g. sous-vide ) foods.     

Thermal destruction of Clostridium botulinum spores suspended in tomato juice in aluminum thermal death time tubes.

The heat destruction characteristics of Clostridium botulinum spores suspended in tomato juice and phosphate buffer were determined by the survivor curve method with aluminum thermal death time tubes. Two type A strains of C. botulinum and a type B strain were evaluated. Strains A16037 and B15580 were implicated in outbreaks of botulism involving home-canned tomato products. Strain A16037 had a higher heat resistance than either 62A or B15580. The mean thermal resistance (D-values) for A16037 in tomato juice (pH 4.2) were: 115.6 degrees C, 0.4 min; 110.0 degrees C, 1.6 min; and 104.4 degrees C, 6.0 min. The mean D-values for A16037 in Sorensen 0.067 M phosphate buffer (pH 7) were: 115.6 degrees C, 1.3 min; 110.0 degrees C, 4.4 min; and 104.4 degrees C, 17.6 min. At each test temperature, the D-values were approximately three times higher in buffer than in tomato juice. The z-value for C. botulinum A16037 spores in tomato juice was 9.4 degrees C, and in buffer the z-value was 9.9 degrees C. The use of aluminum thermal death time tubes in a miniature retort system makes it possible to determine survivor curves for C. botulinum spores at 121.1 degrees C. This is possible because the lag correction factor for the aluminum tubes is only about 0.2 min, making possible heating times as short as 0.5 min.     

Thermal inactivation of nonproteolytic Clostridium botulinum type E spores in model fish media and in vacuum-packaged hot-smoked fish products

Thermal inactivation of nonproteolytic Clostridium botulinum type E spores was investigated in rainbow trout and whitefish media at 75 to 93 degrees C. Lysozyme was applied in the recovery of spores, yielding biphasic thermal destruction curves. Approximately 0.1% of the spores were permeable to lysozyme, showing an increased measured heat resistance. Decimal reduction times for the heat-resistant spore fraction in rainbow trout medium were 255, 98, and 4.2 min at 75, 85, and 93 degrees C, respectively, and those in whitefish medium were 55 and 7.1 min at 81 and 90 degrees C, respectively. The z values were 10.4 degrees C in trout medium and 10.1 degrees C in whitefish medium. Commercial hot-smoking processes employed in five Finnish fish-smoking companies provided reduction in the numbers of spores of nonproteolytic C. botulinum of less than 10(3). An inoculated-pack study revealed that a time-temperature combination of 42 min at 85 degrees C (fish surface temperature) with >70% relative humidity (RH) prevented growth from 10(6) spores in vacuum-packaged hot-smoked rainbow trout fillets and whole whitefish stored for 5 weeks at 8 degrees C. In Finland it is recommended that hot-smoked fish be stored at or below 3 degrees C, further extending product safety. However, heating whitefish for 44 min at 85 degrees C with 10% RH resulted in growth and toxicity in 5 weeks at 8 degrees C. Moist heat thus enhanced spore thermal inactivation and is essential to an effective process. The sensory qualities of safely processed and more lightly processed whitefish were similar, while differences between the sensory qualities of safely processed and lightly processed rainbow trout were observed.     

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)     

Effect of water activity and pH on growth and toxin production by Clostridium botulinum type G.

The combined effect of water activity (aw) and pH on growth and toxin production by Clostridium botulinum type G strain 89 was investigated. The minimum aw at which growth and toxin formation occurred was 0.965, for media in which the pH was adjusted with either sodium chloride or sucrose. The minimum pH (at the optimum aw) for growth and toxin production of C. botulinum type G was found to be 5.6. Optimum conditions for toxin activation were a trypsin concentration of 0.1%, a pH of the medium of 6.5, and an incubation for 45 min at 37 degrees C. These data did not show evidence of heat-labile spores, since a heat shock of 75 degrees C for 10 min did not significantly decrease the spore count of strain 89G in media at pH 7.0 or 5.6. It was frequently observed that cells grown at reduced aw or pH experienced severe morphological changes.     

Effect of water activity and pH on growth and toxin production by Clostridium botulinum type G

The combined effect of water activity (aw) and pH on growth and toxin production by Clostridium botulinum type G strain 89 was investigated. The minimum aw at which growth and toxin formation occurred was 0.965, for media in which the pH was adjusted with either sodium chloride or sucrose. The minimum pH (at the optimum aw) for growth and toxin production of C. botulinum type G was found to be 5.6. Optimum conditions for toxin activation were a trypsin concentration of 0.1%, a pH of the medium of 6.5, and an incubation for 45 min at 37 degrees C. These data did not show evidence of heat-labile spores, since a heat shock of 75 degrees C for 10 min did not significantly decrease the spore count of strain 89G in media at pH 7.0 or 5.6. It was frequently observed that cells grown at reduced aw or pH experienced severe morphological changes.     

Combining heat treatment and subsequent incubation temperature to prevent growth from spores of non-proteolytic Clostridium botulinum

Refrigerated processed foods of extended durability rely on a mild heat treatment combined with refrigerated storage to ensure microbiological safety and quality. The principal microbiological safety risk in foods of this type is non-proteolytic Clostridium botulinum. In this article the combined effect of mild heat treatment and refrigerated storage on the time to growth and probability of growth from spores of non-proteolytic Cl. botulinum is described. Spores of non-proteolytic Cl. botulinum (two strains each of type B, E and F) were heated at 90°C for between 0 and 60 min and subsequently incubated at 5°, 10° or 30°C in PYGS broth in the presence or absence of lysozyme. The number of spores that resulted in turbidity depended on the combination of heat treatment, incubation time and incubation temperature they received. Heating at 90°C for 1 or more min ensured a 10⁶ reduction when spores were subsequently incubated at 5°C for up to 23 weeks. Heating at 90°C for 60 min ensured a 10⁶ reduction over 23 weeks when subsequent incubation was at 10°C in the presence of added lysozyme. The same treatment did not reduce the spore population by 10⁶ when subsequent incubation was at 30°C.     

Characterization of spore germination of a thermoacidophilic spore-forming bacterium, Alicyclobacillus acidoterrestris

The germination behaviors of spores of Alicyclobacillus acidoterrestris, which has been considered to be a causative microorganism of flat sour type spoilage in acidic beverages, were investigated. The spores of A. acidoterrestris showed efficient germination and outgrowth after heat activation (80 degrees C, 20 min) in Potato dextrose medium (pH 4.0). Further, the spores treated with heat activation germinated in McIlvaine buffer (pH 4.0) in the presence of a germinative substance (L-alanine) and commercial fruit juices, although not in phosphate buffer (pH 7.0). Heat activation was necessary for germination. The spores of A. acidoterrestris, which easily survived the heat treatment in acidic conditions, lost their resistance to heat during germination. Our results suggest that the models obtained from spore germination of A. acidoterrestris might be beneficial to determine adequate thermal process in preventing the growth of potential spoilage bacteria in acidic beverages.     

Increased numbers of heat-resistnat spores produced by two strains of Clostridium perfringens bearing temperate phage s9.

Sporulation kinetics and spore heat resistance data were compared for a lysogenic strain of Clostridium perfringens, s9, before and after curing with ultraviolet irradiation. The cured strain showed the same growth rate in broth media as the lysogenic strain but took 6 h longer to form refractile spores. For lysogenized and cured strains the percentages of refractile spores produced that were heat-resistant (80 degrees C for 15 min) were 50 and 0.2, respectively. When reinfected with the temperature phage, the cured strain produced spores in 2 to 3 h, like the original lysogenic culture, and 10% of the spores produced were heat-resistnat.     

Evaluation of different growth media for the recovery of the species of Alicyclobacillus

AIMS: Five different isolation media, namely potato dextrose agar (PDA), orange serum agar (OSA), K agar, yeast-starch-glucose agar and Bacillus acidocaldarius medium were evaluated for the recovery of Alicyclobacillus spp. from inoculated diluted and undiluted fruit-juice concentrates. METHODS AND RESULTS: Plates of PDA (pH 3.7), spread with vegetative cells (3.9 x 10(6) CFU ml(-1)) of Alicyclobacillus acidoterrestris from single-strength pear juice, recovered 2.9 x 10(6 )CFU ml(-1) after 5 days at 50 degrees C (74% recovery). The recovery of endospores from single-strength pear juice, after a heat treatment at 80 degrees C for 10 min, was higher on spread plates of OSA (pH 5.5) at 50 degrees C for 5 days (97% recovery). CONCLUSIONS: PDA (pH 3.7) and OSA (pH 5.5) at 50 degrees C for 3-5 days recovered the highest numbers of vegative cells and endospores of Alicyclobacillus spp. from sterilized fruit juices and concentrates. SIGNIFICANCE AND IMPACT OF THE STUDY: The most appropriate synthetic media for the recovery of Alicyclobacillus species from inoculated fruit juices and concentrates are shown.     

Sensitivity of an Enrichment Culture Procedure for Detection of Clostridium botulinum Type E in Raw and Smoked Whitefish Chubs

The sensitivity of an enrichment culture procedure for detecting Clostridium botulinum type E in whitefish chubs (Leucichthys sp.) was assayed. Data demonstrated that fish inoculated with 10 or more viable C. botulinum spores regularly develop specifically neutralizable enrichment cultures. Mild heat treatment (60 C, 15 min) substantially reduced the sensitivity of enrichment culturing. This effect was particularly noticeable in the culturing of fish which harbored fewer than 10 spores each. Evidence is presented which indicates that sensitivity of enrichment, without heat, approaches the level of one spore per fish. Smoked whitefish chubs, containing from one to several hundred spores each, were examined for toxin content after storage at 5, 10, 15, and 28 C for as long as 32 days. The lowest temperature at which detectable toxin was produced was 15 C. This occurred in 1 of 10 fish incubated for 14 days. C. botulinum was regularly recovered, by enrichment culture, from fish inoculated with small numbers of spores, even though toxin was not detected by direct extraction of incubated fish. Persistence of C. botulinum type E spores was observed to decline with an increase in the temperature and time at which inoculated fish were stored.     

Heat activation/shock temperatures for Bacillus anthracis spores and the issue of spore plate counts versus true numbers of spores

Assessing true numbers of viable anthrax spores is complex. Optimal heat activation conditions vary with species, media and germinants. Published time/temperature combinations for Bacillus anthracis spores range from 60 degrees C for 1, post-heating counts were less than their pre-heating counterparts on between 71% and 88% of occasions. A high probability was found of viable spore counts differing significantly from counts determined microscopically, with differences of almost 1 log possible. Viable counts were lower than microscopic counts in 15 of 18 tests.     

Response of Micromonospora echinospora (NCIMB 12744) spores to heat treatment with evidence of a heat activation phenomenon

The effects of heat treatment on spores of the actinomycete Micromonospora echinospora were investigated. The percentage of culturable spores in untreated spore stocks was found to be approximately 20%. A 60 degrees C treatment of spores in phosphate buffer for 10 min led to an approximately five-fold increase in the number of culturable units. This indicated that a large proportion of the spores were constitutively dormant. Within 10 min and in the absence of an external energy-yielding substrate, the heat treatment was found to stimulate spore respiration suggesting that endogenous storage compounds were being utilized. Heating spores at 70 degrees C shortened the time period required for activation; holding times greater than 10 min, however, resulted in a reduction of culturable cells. Classic thermal death characteristics were seen at temperatures of 80 degrees C and above with D-values of 21.43, 2.67, 0.45 and 0.09 min being recorded at 70, 80, 90 and 100 degrees C, respectively. Spores of this organism, while being weakly heat resistant in comparison with bacterial endospores, are significantly more resistant than vegetative cells.     

The combined effect of sub-optimal temperature and sub-optimal pH on growth and toxin formation from spores of Clostridium botulinum

Low-acid foods (pH greater than or equal to 4.5) are not sufficiently acidic to prevent growth of Clostridium botulinum in otherwise optimal conditions. The combination of sub-optimal pH and sub-optimal temperature may, however, result in a very significant reduction in the risk of growth of this bacterium compared with the risk in optimal conditions. The combined effect of incubation temperatures of 12 degrees and 16 degrees C and pH values between 5.2 and 5.5 on growth and toxin production from spores of Cl. botulinum during incubation for 28 d has been investigated. Growth and formation of toxin (type B) were detected only in medium at pH 5.5 and incubated at 16 degrees C, corresponding to a probability of growth from a single spore within 14 d of 1.6 x 10(-5). The probability of growth in 28 d in the remaining conditions was less than 9 x 10(-6). After transfer of inoculated media from 12 degrees to 30 degrees C growth occurred at pH 5.2-5.5 within 19 d. After transfer of inoculated media from 12 degrees to 20 degrees C growth occurred at pH 5.5 and 5.4 but not at pH 5.3 or 5.2 in 40 d. Growth at pH 5.2-5.5 was accompanied by formation of toxin, in most cases of types A or B. In addition to the effect of sub-optimal temperature and pH, chelation of divalent metal ions by citrate may have contributed to inhibition.     

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.     

Novel strains of Moorella thermoacetica form unusually heat-resistant spores

Two strains of Moorella thermoacetica, JW/B-2 and JW/DB-4, isolated as contaminants from autoclaved media for chemolithoautotrophic growth containing 0.1% (wt/vol) yeast extract, formed unusually heat-resistant spores. Spores of the two strains required heat activation at 100 degrees C of more than 2 min and up to 90 min for maximal percentage of germination. Kinetic analysis indicated the presence of two distinct subpopulations of heat-resistant spores. The decimal reduction time (D10-time=time of exposure to reduce viable spore counts by 90%) at 121 degrees C was determined for each strain using spores obtained under different conditions. For strains JW/DB-2 and JW/ DB-4, respectively, spores obtained at approximately 25 degrees C from cells grown chemolithoautotrophically had D10-times of 43 min and 23 min; spores obtained at 60 degrees C from cells grown chemoorganoheterotrophically had D10-times of 44 min and 38 min; spores obtained at 60 degrees C from cells grown chemolithoautotrophically had D10-times of 83 min and 111 min. The thickness of the cortex varied between 0.10 and 0.29 microm and the radius of the cytoplasm from 0.14 to 0.46 microm. These spores are amongst the most heat-resistant noted to date. Electron microscopy revealed structures within the exosporia of spores prior to full maturity that were assumed to be layers of the outer spore coat.     

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.     

Growth and formation of toxin by Clostridium botulinum in peeled, inoculated, vacuum-packed potatoes after a double pasteurization and storage at 25 degrees C

A process that claims to use a double pasteurization to produce vacuum-packed potatoes for storage at ambient temperature has been evaluated. After the first pasteurization, potatoes are vacuum-packed and stored at 25 degrees-35 degrees C for up to 24 h, which is intended to allow germination of bacterial spores, and are then pasteurized again. When potatoes were inoculated with spores of Clostridium botulinum and subjected to this double-pasteurization process a high proportion of spores remained viable and resulted in growth and formation of toxin within 5-9 d at 25 degrees C. To provide an appropriate reduction in the risk o survival and growth of Cl. botulinum, peeled, vacuum-packed potatoes for storage at ambient temperature should be given a heat treatment equivalent to an F(0)3 process. If they are not given such a heat treatment they should be stored at a temperature below 4 degrees C.