Growth and formation of toxin by Clostridium botulinum in peeled, inoculated, vacuum-packed potatoes after a double pasteurization and storage at 25°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°C–35°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°C. To provide an appropriate reduction in the risk of survival and growth of Cl. botulinum , peeled, vacuum-packed potatoes for storage at ambient temperature should be given a heat treatment equivalent to an F₀3 process. If they are not given such a heat treatment they should be stored at a temperature below 4°C.     

Psychrotrophic clostridia mediated gas and botulinal toxin production in vacuum-packed chilled meat

A cocktail of washed spores from six psychrotrophic Clostridium strains isolated from blown vacuum-packed meats was inoculated onto lamb chumps. A second washed spore cocktail of four toxigenic reference Cl. botulinum strains, types A, B (two strains) and E, and a Cl. butyricum type E strain, was similarly inoculated onto lamb chumps. All inoculated lamb chumps were individually vacuum-packed and placed into storage at various temperatures typical of good to grossly abusive chilled storage (-1 degree C to 15 degrees C). All packs were observed for gas production (pack-'blowing') over a 12 week storage period. On gas production, or after 12 weeks of storage, packs were examined by mouse bioassay for botulinum toxin production. The packs inoculated with the meat isolate cocktail showed evidence of gas production earlier than packs inoculated with reference strains. No botulinum toxin was recovered from the meat isolate inoculated packs, while botulinal toxin was detected in reference strain inoculated packs down to a nominal storage temperature of 2 degrees C.     

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)     

Combined effect of water activity and pH on inhibition of toxin production by Clostridium botulinum in cooked, vacuum-packed potatoes

The effects of water activity (aw, 0.955 to 0.970), pH (4.75 to 5.75), and storage time (up to 60 days) on toxin production by Clostridium botulinum in cooked, vacuum-packed potatoes were studied by using factorial design experiments and most-probable-number methodology. Samples were inoculated with 10(3), 10(4), or 10(5) spores of a mixture of five type A and five proteolytic type B strains, incubated at 25 degrees C, and analyzed for toxin production. Toxin was produced at pH levels of greater than or equal to 4.75 when the aw was greater than or equal to 0.970, pH greater than 5.25 when the aw was 0.965, and pH greater than or equal to 5.75 at an aw of 0.960. No toxin was detected when the aw was 0.955. The probability of toxigenesis was significantly affected (P less than 0.0001) by storage time, aw, pH, and the interactions aw.pH and aw.storage time. The response to a decrease in pH was linear, while the response to a decrease in aw was curvilinear. Using multiple linear regression, equations were derived which could predict the length of time until toxin production and the probability of toxigenesis by a single spore under defined conditions.     

Combined effect of water activity and pH on inhibition of toxin production by Clostridium botulinum in cooked, vacuum-packed potatoes.

The effects of water activity (aw, 0.955 to 0.970), pH (4.75 to 5.75), and storage time (up to 60 days) on toxin production by Clostridium botulinum in cooked, vacuum-packed potatoes were studied by using factorial design experiments and most-probable-number methodology. Samples were inoculated with 10(3), 10(4), or 10(5) spores of a mixture of five type A and five proteolytic type B strains, incubated at 25 degrees C, and analyzed for toxin production. Toxin was produced at pH levels of greater than or equal to 4.75 when the aw was greater than or equal to 0.970, pH greater than 5.25 when the aw was 0.965, and pH greater than or equal to 5.75 at an aw of 0.960. No toxin was detected when the aw was 0.955. The probability of toxigenesis was significantly affected (P less than 0.0001) by storage time, aw, pH, and the interactions aw.pH and aw.storage time. The response to a decrease in pH was linear, while the response to a decrease in aw was curvilinear. Using multiple linear regression, equations were derived which could predict the length of time until toxin production and the probability of toxigenesis by a single spore under defined conditions.     

Effects of temperature and desiccation on ex situ conservation of nongreen fern spores

? Premise of the study: Fern spores are unicellular and haploid, making them a potential model system to study factors that regulate lifespan and mechanisms of aging. Aging rates of nongreen spores were measured to compare longevity characteristics among diverse fern species and test for orthodox response to storage temperature and moisture. ? Methods: Aging of spores from 10 fern species was quantified by changes in germination and growth parameters. Storage temperature ranged from ambient room to -196°C (liquid nitrogen); spores were dried to ambient relative humidity (RH) or using silica gel. ? Key results: Survival of spores varied under ambient storage conditions, with one species dying within a year and two species having greater than 50% survival after 3 years. Few changes in germination or growth were observed in spores stored at either -80°C or -196°C over the same 3-yr study period. Spores stored at -25°C aged anomalously quickly, especially those dried to ambient RH or subjected to repeated freeze-thaw cycles. ? Conclusions: Spore longevity is comparable to orthodox seed longevity under ambient storage conditions, with wide variation among species and shelflife extended by drying or cooling. However, faster aging during freezer storage may indicate a similar syndrome of damage experienced by seeds categorized as "intermediate". The damage is avoided by storage at -80°C or liquid nitrogen temperatures, making cryoconservation an effective and broadly applicable tool to extend fern spore longevity. The study demonstrates that spore banks are a feasible approach for ex situ conservation of this important plant group.     

Effect of Iysozyme concentration, heating at 90°C, and then incubation at chilled temperatures on growth from spores of non-proteolytic Clostridium botulinum

The heat treatment necessary to inactivate spores of non-proteolytic Clostridium botulinum in refrigerated, processed foods may be influenced by the occurrence of lysozyme in these foods. Spores of six strains of non-proteolytic Cl. botulinum were inoculated into tubes of an anaerobic meat medium, to give 10⁶ spores per tube. Hen egg white lysozyme (0–50 μg ml^-1) was added, and the tubes were given a heat treatment equivalent to 19·8 min at 90°C, cooled, and incubated at 8°, 12°, 16° and 25°C for up to 93 d. In the absence of added lysozyme, neither growth nor toxin formation were observed. A 6–D inactivation was therefore achieved. In tubes to which lysozyme (5–50 μg ml^-1) had been added prior to heating, growth and toxin formation were observed. With lysozyme added at 50 μg ml^-1, growth was first observed after 68 d at 8°C, 31 d at 12°C, 24 d at 16°C, and 9 d at 25°C. Thus, in these circumstances, a heat treatment equivalent to 19·8 min at 90°C was not sufficient, on its own, to give a 6–D inactivation. A combination of the heat treatment, maintenance at less than 12°C, and a shelf-life not more than 4 weeks reduced the risk of growth of non-proteolytic Cl. botulinum by a factor of 10⁶.     

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.     

Use of a novel method to characterize the response of spores of non-proteolytic Clostridium botulinum types B, E and F to a wide range of germinants and conditions

AIMS: Limited information is available on the germination triggers for spores of non-proteolytic Clostridium botulinum. An automated system was used to study the effect of a large number of potential germinants, of temperature and pH, and aerobic and anaerobic conditions, on germination of spores of non-proteolytic Cl. botulinum types B, E and F. METHODS AND RESULTS: A Bioscreen analyser was used to measure germination by decrease in optical density. Results were confirmed by phase-contrast light microscopy. Spores of strains producing type B, E and F toxin gave similar results. Optimum germination occurred in L-alanine/L-lactate, L-cysteine/L-lactate and L-serine/L-lactate (50 mmol l(-1) of each). A further 12 combinations of factors induced germination. Sodium bicarbonate, sodium thioglycollate and heat shock each enhanced germination, but were not essential. Germination was similar in aerobic and anaerobic conditions. The optimum pH range was 5.5-8.0, germination occurred at 1-40 degrees C, but not at 50 degrees C, and was optimal at 20-25 degrees C. CONCLUSIONS: The automated system enabled a systematic study of germination requirements, and provided an insight into germination in spores of non-proteolytic Cl. botulinum. SIGNIFICANCE AND IMPACT OF THE STUDY: The results extend understanding of germination of non-proteolytic Cl. botulinum spores, and provide a basis for improving detection of viable spores.     

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.     

High-pressure-mediated survival of Clostridium botulinum and Bacillus amyloliquefaciens endospores at high temperature

Endospores of proteolytic type B Clostridium botulinum TMW 2.357 and Bacillus amyloliquefaciens TMW 2.479 are currently described as the most high-pressure-resistant bacterial spores relevant to food intoxication and spoilage in combined pressure-temperature applications. The effects of combined pressure (0.1 to 1,400 MPa) and temperature (70 to 120 degrees C) treatments were determined for these spores. A process employing isothermal holding times was established to distinguish pressure from temperature effects. An increase in pressure (600 to 1,400 MPa) and an increase in temperature (90 to 110 degrees C) accelerated the inactivation of C. botulinum spores. However, incubation at 100 degrees C, 110 degrees C, or 120 degrees C with ambient pressure resulted in faster spore reduction than treatment with 600 or 800 MPa at the same temperature. This pressure-mediated spore protection was also observed at 120 degrees C and 800, 1,000, or 1,200 MPa with the more heat-tolerant B. amyloliquefaciens TMW 2.479 spores. Inactivation curves for both strains showed a pronounced pressure-dependent tailing, which indicates that a small fraction of the spore populations survives conditions of up to 120 degrees C and 1.4 GPa in isothermal treatments. Because of this tailing and the fact that pressure-temperature combinations stabilizing bacterial endospores vary from strain to strain, food safety must be ensured in case-by-case studies demonstrating inactivation or nongrowth of C. botulinum with realistic contamination rates in the respective pressurized food and equipment.     

Challenge testing of the lactoperoxidase system in pasteurized milk

AIMS: To determine the role of lactoperoxidase (LP) in inhibiting the growth of micro-organisms in pasteurised milk. METHODS AND RESULTS: Four micro-organisms of importance in the spoilage of pasteurized milk were challenged in lactoperoxidase (LP)-enriched ultra-heat treated (UHT) milk after subsequent pasteurization. Milk samples were stored at the optimum temperatures for growth of the individual bacteria. Pasteurization was carried out at 72 degrees C/15 s and 80 degrees C/15 s to determine the effect of the LP system on the micro-organisms. An active LP system was found to greatly increase the keeping quality (KQ) of milks inoculated with Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus thermophilus and pasteurized at 72 degrees C, but had little or no effect in milks heated at 80 degrees C, presumably due to virtual inactivation of LP at 80 degrees C. However, pasteurization temperature had no effect on the KQ of milks challenged with Bacillus cereus spores. CONCLUSIONS: This study suggests that the LP system, rather than heat-shocking of spores, is responsible for the greater KQ of milk pasteurized at 72 degrees C/15 s compared with 80 degrees C/15 s. SIGNIFICANCE AND IMPACT OF THE STUDY: The study emphasizes the care required in selecting pasteurization temperatures in commercial practice and to avoid the temptation to compensate for inferior quality of raw milk by increasing pasteurization temperature.     

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.     

Effect of temperature on spore germination and vegetative cell growth of Clostridium botulinum.

Spore germination and vegetative growth of Clostridium botulinum type E strain VH at 2 to 50 degrees C were studied. At all of these temperatures, germination began immediately after the addition of the spores to the germination medium. Microscopic observations during germination revealed three types of spores: phase bright (ungerminated), phase variable (partially germinated), and phase dark (fully germinated). At all temperatures except 50 degrees C, there was a pronounced lag between the initial appearance of phase-variable spores and their eventual conversion to phase-dark spores. The number of partially germinated spores increased steadily, reaching 40 to 60% by 18 to 21 h of incubation. During this time, phase-dark, fully germinated spores developed slowly and did not exceed 28% in any of the samples. At 18 to 26 h of incubation, the rate of full germination increased abruptly four-fold. There was extensive and relatively rapid germination at 2 degrees C, the lowest temperature tested, yielding about 60% phase-variable spores by 18 h, which became phase-dark by 26 h of incubation. The optimum temperature for partial and full germination was consistently 9 degrees C. Germination at 50 degrees C was exceptionally rapid and was completed within 1 to 2 h, although 40% remained phase bright. Vegetative cells showed detectable growth at 6 to 41 degrees C, with a distinct optimum at 32.5 degrees C. No growth occurred at 50 degrees C, and only marginal growth was observed at 6 to 14 degrees C. The psychrophilic nature of the germination process coupled with the cold tolerance of vegetative growth appears to give C. botulinum type E an advantage in cold climates as well as in cold-stored foods.     

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 degrees 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 degrees 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.     

A Model System for Testing the Microbiological Stability of Foods Processed in Laminated Flexible Pouches

A suitable time temperature process for packaging small potatoes in flexible pouches is described. Spores of Bacillus stearothermophilus were inoculated onto the surface of peeled potatoes in flexible pouches. These were evacuated, heat sealed and heated in a steam retort modified to allow a water cooking process with an air overpressure of ca. 68·95 kPa (10 lbf/in²). The D and z values determined in the retort were confirmed by parallel heat-resistance tests with spores in glass ampoules held in an oil bath. Heat treatment at 121·1 °C for 20 min in the retort satisfactorily killed test inocula of spores without overcooking the potatoes. Uninoculated peeled potatoes with a natural level of contamination ( ca. 4 x 10³ spores/sealed pouch) were treated in the retort for different times at 121·1 °C and then incubated at 30 °C for 6 months. No pouches heated for 17–25 min showed microbial growth. This procedure may be applied to any type of food if the numbers of naturally occurring heat-resistant contaminants can be related to the number and heat resistance of a suitable test micro-organism.     

Influence of environmental storage relative humidity on biological indicator resistance, viability, and moisture content.

The effect of environmental storage relative humidity (RH) on the moisture content, viability, and moist heat and gaseous ethylene oxide (EO) resistance of biological indicators (BIs) was evaluated. No statistically significant difference was observed between the initial Bacillus stearothermophilus spore population and the spore population of BIs stored at 20 degrees C and 0, 20, 44, of 55% RH or under ambient, 4 degrees C, or -20 degrees C conditions after 12 months. A statistically significant decrease in moist heat resistance from initial starting levels was found for BIs stored at 20 degrees C and either 0 or 20% RH. There was a statistically significant decrease in the B. subtilis BI spore population, compared with initial levels, when the BIs were stored at 20 degrees C and 0% RH concomitant with a significant increase in their EO resistance. BI storage at 20 degrees C and 20 or 44% RH, or under ambient, 4 degrees C, or -20 degrees C conditions, had no significant effect on EO resistance. BIs stored at 20 degrees C and 66% RH demonstrated a significantly lower EO resistance compared with starting levels.     

Water properties in fern spores: sorption characteristics relating to water affinity, glassy states, and storage stability

Ex situ conservation of ferns may be accomplished by maintaining the viability of stored spores for many years. Storage conditions that maximize spore longevity can be inferred from an understanding of the behaviour of water within fern spores. Water sorption properties were measured in spores of five homosporeous species of ferns and compared with properties of pollen, seeds, and fern leaf tissue. Isotherms were constructed at 5, 25, and 45 degrees C and analysed using different physicochemical models in order to quantify chemical affinity and heat (enthalpy) of sorption of water in fern spores. Fern spores hydrate slowly but dry rapidly at ambient relative humidity. Low Brunauer-Emmet-Teller monolayer values, few water-binding sites according to the D'Arcy-Watt model, and limited solute-solvent compatibility according to the Flory-Huggins model suggest that fern spores have low affinity for water. Despite the low water affinity, fern spores demonstrate relatively high values of sorption enthalpy (DeltaH(sorp)). Parameters associated with binding sites and DeltaH(sorp) decrease with increasing temperature, suggesting temperature- and hydration-dependent changes in volume of spore macromolecules. Collectively, these data may relate to the degree to which cellular structures within fern spores are stabilized during drying and cooling. Water sorption properties within fern spores suggest that storage at subfreezing temperatures will give longevities comparable with those achieved with seeds. However, the window of optimum water contents for fern spores is very narrow and much lower than that measured in seeds, making precise manipulation of water content imperative for achieving maximum longevity.