Osmotically induced increase in thermal resistance of heat-sensitive, dipicolinic acid-less spores of Bacillus cereus Ht-8.

Thermal resistance in heat-sensitive, dipicolinic acid (DPA)-less spores of Bacillus cereus Ht-8 heated in sucrose solutions increased at and above a concentration of 2 M sucrose. The decimal reduction times at 75 degrees C for spores heated in 0.0, 1.8, 2.2, and 2.6 M sucrose were 2.0, 2.8, 4.5, and 12 min, respectively. Maltose, fructose, and glucose increased heat resistance above that observed in water but did not elevate resistance to the level observed with sucrose at the same osmolality. Cation-induced loss of thermal resistance in chemically sensitized spores was reversed in the presence of sucrose. Spores germinated in brain heart infusion were resistant when heated in sucrose. In the presence of sucrose, spores exhibited an increase in optical density at 700 nm. Electron micrographs of the DPA-less spores suspended in 2.2 M sucrose revealed a shrinkage of outer coats and exosporium membranes. The results suggested that the osmotic property of sugars increased thermal resistance in DPA-less spores. The osmotic pressure exerted by sugars may be similar to the pressure that usually exists within the cortex of normal spores containing DPA and may cause the dehydration of the protoplast and the consequent thermal resistance. The role of dehydration and the nonessential nature of DPA for thermal resistance in spores were confirmed.     

Influence of Temperature and Relative Humidity on Germinability of Mucor piriformis Spores

Studies were conducted to determine the influence of temperature and relative humidity (RH) on germinability and viability of Mucor piriformis spores. Spores did not survive when stored at 35 °C and their survival rate decreased rapidly at 30 °C; however, spores remained viable for more than 1 year at 0 °C. RH also significantly affected spore viability. Spores held at 26 °C and 100% RH no longer germinated after 35 days, while those held at 75 or 90% RH germinated for 65 days. At 20 °C, RH had little effect on spore germinability. The effect of temperature and RH on percentage spore germination also varied. At all temperatures studied, spore viability decreased more rapidly with time at 100% RH than at 75 or 90% RH. The least favorable, temperature-humidity combination, 30 °C and 100% RH, decreased spore germination from 100% to less than 1% in 14 days.     

Osmotically induced increase in thermal resistance of heat-sensitive, dipicolinic acid-less spores of Bacillus cereus Ht-8.

Thermal resistance in heat-sensitive, dipicolinic acid (DPA)-less spores of Bacillus cereus Ht-8 heated in sucrose solutions increased at and above a concentration of 2 M sucrose. The decimal reduction times at 75 degrees C for spores heated in 0.0, 1.8, 2.2, and 2.6 M sucrose were 2.0, 2.8, 4.5, and 12 min, respectively. Maltose, fructose, and glucose increased heat resistance above that observed in water but did not elevate resistance to the level observed with sucrose at the same osmolality. Cation-induced loss of thermal resistance in chemically sensitized spores was reversed in the presence of sucrose. Spores germinated in brain heart infusion were resistant when heated in sucrose. In the presence of sucrose, spores exhibited an increase in optical density at 700 nm. Electron micrographs of the DPA-less spores suspended in 2.2 M sucrose revealed a shrinkage of outer coats and exosporium membranes. The results suggested that the osmotic property of sugars increased thermal resistance in DPA-less spores. The osmotic pressure exerted by sugars may be similar to the pressure that usually exists within the cortex of normal spores containing DPA and may cause the dehydration of the protoplast and the consequent thermal resistance. The role of dehydration and the nonessential nature of DPA for thermal resistance in spores were confirmed.     

Prevention of DNA damage in spores and in vitro by small, acid-soluble proteins from Bacillus species.

The DNA in dormant spores of Bacillus species is saturated with a group of nonspecific DNA-binding proteins, termed alpha/beta-type small, acid-soluble spore proteins (SASP). These proteins alter DNA structure in vivo and in vitro, providing spore resistance to UV light. In addition, heat treatments (e.g., 85 degrees C for 30 min) which give little killing of wild-type spores of B. subtilis kill > 99% of spores which lack most alpha/beta-type SASP (termed alpha - beta - spores). Similar large differences in survival of wild-type and alpha - beta - spores were found at 90, 80, 65, 22, and 10 degrees C. After heat treatment (85 degrees C for 30 min) or prolonged storage (22 degrees C for 6 months) that gave > 99% killing of alpha - beta - spores, 10 to 20% of the survivors contained auxotrophic or asporogenous mutations. However, alpha - beta - spores heated for 30 min at 85 degrees C released no more dipicolinic acid than similarly heated wild-type spores (< 20% of the total dipicolinic acid) and triggered germination normally. In contrast, after a heat treatment (93 degrees C for 30 min) that gave > or = 99% killing of wild-type spores, < 1% of the survivors had acquired new obvious mutations, > 85% of the spore's dipicolinic acid had been released, and < 1% of the surviving spores could initiate spore germination. Analysis of DNA extracted from heated (85 degrees C, 30 min) and unheated wild-type spores and unheated alpha - beta - spores revealed very few single-strand breaks (< 1 per 20 kb) in the DNA. In contrast, the DNA from heated alpha- beta- spores had more than 10 single-strand breaks per 20 kb. These data suggest that binding of alpha/beta-type SASP to spore DNA in vivo greatly reduces DNA damage caused by heating, increasing spore heat resistance and long-term survival. While the precise nature of the initial DNA damage after heating of alpha- beta- spores that results in the single-strand breaks is not clear, a likely possibility is DNA depurination. A role for alpha/beta-type SASP in protecting DNA against depurination (and thus promoting spore survival) was further suggested by the demonstration that these proteins reduce the rate of DNA depurination in vitro at least 20-fold.     

A rapid method for the determination of bacterial fatty acid composition

Heat treatment of spores of non-proteolytic strains of Clostridium botulinum at 75–90°C, and enumeration of survivors on a nutrient medium containing lysozyme gave biphasic survival curves. A majority of spores were inactivated rapidly by heating, and the apparent heat-resistance of these spores was similar to that observed by enumeration on medium without lysozyme. A minority of spores showed much greater heat-resistance, due to the fact that the spore coat was permeable to lysozyme, which diffused into the spore from the medium and replaced the heat-inactivated germination system. The proportion of heated spores permeable to lysozyme was between 0.2 and 1.4% for spores of strains 17B (type B) and Beluga (type E), but was about 20% for spores of strain Foster B96 (type E). After treatment of heated spores with alkaline thioglycolate, all were permeable to lysozyme. D-values for heated spores that were permeable to lysozyme (naturally and after treatment with thioglycolate) were: for strain 17B, D₈₅°C, 100 min; D₉₀°C, 18.7 min; D₉₅°C, 4.4 min; for strain Beluga, D₈₅°C, 46 min; D₉₀°C, 11.8 min; D₉₅°C, 2.8 min. The z-values for these spores of strains 17B and Beluga were 7.6°C and 8.3°C.     

Changes in spores of Bacillus megaterium treated with thioglycolate at a low pH and restoration of germinability and heat resistance by cations

Spores of Bacillus megaterium QM B1551 treated with thioglycolate (0.4 m, pH 2.6) at 50 C for 30 min remained refractile, but they became stainable, lysozymesensitive, and nonviable, and they lost dipicolinic acid (DPA). The loss of DPA and of viability were functions of the time and temperature of exposure to thioglycolate. Spores treated with thioglycolate at a lower temperature and for a shorter time (30 C, 5 min) retained DPA, viability, and nonstainability. Although these spores also retained their resistance to gamma radiation and to lysozyme, they lost thermo-resistance. Their percentage of germination over a 2-hr period in glucose was markedly reduced. Germinability and heat resistance were restored by exogenous cations, suggesting that the thioglycolate treatment (30 C, 5 min) resulted in the loss of spore ions essential for normal germination in glucose and for heat resistance.     

Recovery of Viability and Radiation Resistance by Heat-Injured Conidia of Penicillium expansum Lk. ex Thom

Spores heated in water at 54 C for up to 1 hr were plated on nutrient agar immediately or held for 3 days in aerated water at 23 C and then plated. Under these conditions, holding was optimal for recovery, increasing survival percentage up to 20-fold over values for immediate plating. Recovery was prevented partially or completely, however, when spores were held in any of the following solutions: glucose, potassium phosphate, ammonium or sodium acetate, sodium azide, or 2,4-dinitrophenol, or in the sodium or potassium salts of pyruvate, and tricarboxylic acid cycle acids. Both anaerobiosis and incubation at 0 C prevented recovery. Survivors of a heat treatment were more sensitive to gamma radiation than were unheated spores. Conditions which affected the recovery of viability had the same effect on restoration of radiation resistance. Thus, many of the processes for restoration of radiation resistance seem involved also in recovery of viability after heating. After a 99% inactivating treatment (about 30 min at 54 C), heated spores respired as fast as unheated spores, or faster. Malate, citrate, succinate, and acetate stimulated respiration in unheated spores and inhibited it in heated spores.     

Moist-heat resistance, spore aging, and superdormancy in Clostridium difficile

Clostridium difficile spores can survive extended heating at 71°C (160°F), a minimum temperature commonly recommended for adequate cooking of meats. To determine the extent to which higher temperatures would be more effective at killing C. difficile, we quantified (D values) the effect of moist heat at 85°C (145°F, for 0 to 30 min) on C. difficile spores and compared it to the effects at 71 and 63°C. Fresh (1-week-old) and aged (≥20-week-old) C. difficile spores from food and food animals were tested in multiple experiments. Heating at 85°C markedly reduced spore recovery in all experiments (5 to 6 log(10) within 15 min of heating; P < 0.001), regardless of spore age. In ground beef, the inhibitory effect of 85°C was also reproducible (P < 0.001), but heating at 96°C reduced 6 log(10) within 1 to 2 min. Mechanistically, optical density and enumeration experiments indicated that 85°C inhibits cell division but not germination, but the inhibitory effect was reversible in some spores. Heating at 63°C reduced counts for fresh spores (1 log(10), 30 min; P < 0.04) but increased counts of 20-week-old spores by 30% (15 min; P < 0.02), indicating that sublethal heat treatment reactivates superdormant spores. Superdormancy is an increasingly recognized characteristic in Bacillus spp., and it is likely to occur in C. difficile as spores age. The potential for reactivation of (super)dormant spores with sublethal temperatures may be a food safety concern, but it also has potential diagnostic value. Ensuring that food is heated to >85°C would be a simple and important intervention to reduce the risk of inadvertent ingestion of C. difficile spores.     

Inactivation of Clostridium perfringens Type A Spores at Ultrahigh Temperatures

The inactivation of Clostridium perfringens type A spores (three strains of different heat resistances) at ultrahigh temperatures was studied. Aqueous spore suspensions were heated at 85 to 135 C by the capillary tube method. When survivors were enumerated on the standard plating medium, the spores appeared to have been rapidly inactivated at temperatures above 100 C. The addition of lysozyme to the plating medium did not affect the recovery of spores surviving the early stages of heating, but lysozyme was required for maximal recovery of spores surviving extended heat treatments. The percentage of survivors requiring lysozyme for colony formation increased greatly with longer exposure times or increasing treatment temperature. Time-survivor curves indicated that each spore suspension was heterogeneous with respect to the heat resistance of spore outgrowth system or in the sensitivity of the spores to lysozyme. Recovery of survivors on the lysozyme containing medium revealed greater heat resistance for one strain than has been reported for spores of many mesophilic aerobes and anaerobes. The spores of all three strains were more resistant to heat inactivation when suspended in phosphate buffer, but a greater percentage of the survivors required lysozyme for colony formation.     

Heat sensitization of bacterial spores after exposure to ethidium bromide, acriflavine, or daunomycin.

A 20-min exposure of 10(7) unmodified spores of either Bacillus subtilis NCTC 3610 (harvested from potato-dextrose agar plus manganese) or Bacillus megaterium ATCC 19213 (harvested from nutrient agar plus manganese) per ml to 5 microgram of ethidium bromide per ml did not kill the spores (recovered on TAM [thermoacidurans agar modified]-plus thymidine medium). However, in both cases, the ability to survive various heat treatments was reduced after exposure of the spores to ethidium bromide. With B. subtilis, a 10-min heat treatment at 85 degrees C of unexposed spores resulted in an 85% survival rate, whereas only 50% of the ethidium bromide-exposed spores survived. With B. megaterium similar results were obtained at 75 degrees C; 77% of the unexposed spores survived, whereas only 31% of the ethidium bromide-exposed spores survived. Similarly, a 10-min exposure of B. subtilis spores to 0.005 microgram of acriflavine per ml did not kill unheated spores; however, the ability of the spores to survive exposure at 85 degrees C for 10 min was reduced to 40%. After exposure to 10 microgram of daunomycin per ml, the survival rate was 35%. Binding studies with ethidium bromide showed strong binding to spores, but as yet, the site of binding is unknown.     

Role of dipicolinic acid in resistance and stability of spores of Bacillus subtilis with or without DNA-protective alpha/beta-type small acid-soluble proteins

Dipicolinic acid (DPA) comprises approximately 10% of the dry weight of spores of Bacillus species. Although DPA has long been implicated in spore resistance to wet heat and spore stability, definitive evidence on the role of this abundant molecule in spore properties has generally been lacking. Bacillus subtilis strain FB122 (sleB spoVF) produced very stable spores that lacked DPA, and sporulation of this strain with DPA yielded spores with nearly normal DPA levels. DPA-replete and DPA-less FB122 spores had similar levels of the DNA protective alpha/beta-type small acid-soluble spore proteins (SASP), but the DPA-less spores lacked SASP-gamma. The DPA-less FB122 spores exhibited similar UV resistance to the DPA-replete spores but had lower resistance to wet heat, dry heat, hydrogen peroxide, and desiccation. Neither wet heat nor hydrogen peroxide killed the DPA-less spores by DNA damage, but desiccation did. The inability to synthesize both DPA and most alpha/beta-type SASP in strain PS3664 (sspA sspB sleB spoVF) resulted in spores that lost viability during sporulation, at least in part due to DNA damage. DPA-less PS3664 spores were more sensitive to wet heat than either DPA-less FB122 spores or DPA-replete PS3664 spores, and the latter also retained viability during sporulation. These and previous results indicate that, in addition to alpha/beta-type SASP, DPA also is extremely important in spore resistance and stability and, further, that DPA has some specific role(s) in protecting spore DNA from damage. Specific roles for DPA in protecting spore DNA against damage may well have been a major driving force for the spore's accumulation of the high levels of this small molecule.     

Proceedings: Preservation of rust fungi in liquid nitrogen

Spores of rust fungi can be expected to retain viability without loss of infectivity for at least several years when stored in liquid nitrogen (?196 °C). Addition of liquid suspending media is harmful and not necessary. Some rust fungi experience cold-induced dormancy when exposed to less than 0 °C for a minute or longer but germinability is dependably restored on applying a heat shock by heating the spores to 40 °C for at least 15 sec during or after thawing. Most rust fungi are not sensitive to moisture content at the time of freezing but Puccinia striiformis must be vacuum dried before freezimg. The need for heat shock may not show up until several days after thawing. All of the rust strains tested to date have retained their properties to the extent tested and for the duration of storage. Data are available for up to 11 years. Preliminary experiments to preserve saprophytic mycelial cultures of P. graminis have so far failed, with and without use of 10% glycerol and 5% DMSO. The successful preservation of rust spores has made feasible the development of a collection of living rust fungi at ATCC beginning in 1965 and which now has over 80 strains in 20 species in 7 genera.     

Characterization of Clostridium perfringens spores that lack SpoVA proteins and dipicolinic acid

Spores of Clostridium perfringens possess high heat resistance, and when these spores germinate and return to active growth, they can cause gastrointestinal disease. Work with Bacillus subtilis has shown that the spore's dipicolinic acid (DPA) level can markedly influence both spore germination and resistance and that the proteins encoded by the spoVA operon are essential for DPA uptake by the developing spore during sporulation. We now find that proteins encoded by the spoVA operon are also essential for the uptake of Ca(2+) and DPA into the developing spore during C. perfringens sporulation. Spores of a spoVA mutant had little, if any, Ca(2+) and DPA, and their core water content was approximately twofold higher than that of wild-type spores. These DPA-less spores did not germinate spontaneously, as DPA-less B. subtilis spores do. Indeed, wild-type and spoVA C. perfringens spores germinated similarly with a mixture of l-asparagine and KCl (AK), KCl alone, or a 1:1 chelate of Ca(2+) and DPA (Ca-DPA). However, the viability of C. perfringens spoVA spores was 20-fold lower than the viability of wild-type spores. Decoated wild-type and spoVA spores exhibited little, if any, germination with AK, KCl, or exogenous Ca-DPA, and their colony-forming efficiency was 10(3)- to 10(4)-fold lower than that of intact spores. However, lysozyme treatment rescued these decoated spores. Although the levels of DNA-protective alpha/beta-type, small, acid-soluble spore proteins in spoVA spores were similar to those in wild-type spores, spoVA spores exhibited markedly lower resistance to moist heat, formaldehyde, HCl, hydrogen peroxide, nitrous acid, and UV radiation than wild-type spores did. In sum, these results suggest the following. (i) SpoVA proteins are essential for Ca-DPA uptake by developing spores during C. perfringens sporulation. (ii) SpoVA proteins and Ca-DPA release are not required for C. perfringens spore germination. (iii) A low spore core water content is essential for full resistance of C. perfringens spores to moist heat, UV radiation, and chemicals.     

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.     

Analysis of the Loss in Heat and Acid Resistance during Germination of Spores of Bacillus Species

A major event in the nutrient germination of spores of Bacillus species is release of the spores'' large depot of dipicolinic acid (DPA). This event is preceded by both commitment, in which spores continue through germination even if germinants are removed, and loss of spore heat resistance. The latter event is puzzling, since spore heat resistance is due largely to core water content, which does not change until DPA is released during germination. We now find that for spores of two Bacillus species, the early loss in heat resistance during germination is most likely due to release of committed spores'' DPA at temperatures not lethal for dormant spores. Loss in spore acid resistance during germination also paralleled commitment and was also associated with the release of DPA from committed spores at acid concentrations not lethal for dormant spores. These observations plus previous findings that DPA release during germination is preceded by a significant release of spore core cations suggest that there is a significant change in spore inner membrane permeability at commitment. Presumably, this altered membrane cannot retain DPA during heat or acid treatments innocuous for dormant spores, resulting in DPA-less spores that are rapidly killed.     

Effect of microwave radiation on Bacillus subtilis spores

AIMS: To compare the killing efficacy and the effects exerted by microwaves and conventional heating on structural and molecular components of Bacillus subtilis spores. METHODS AND RESULTS: A microwave waveguide applicator was developed to generate a uniform and measurable distribution of the microwave electric-field amplitude. The applicator enabled the killing efficacy exerted by microwaves on B. subtilis spores to be evaluated in comparison with conventional heating at the same temperature value. The two treatments produced a similar kinetics of spore survival, while remarkably different effects on spore structures were seen. The cortex layer of the spores subjected to conductive heating was 10 times wider than that of the untreated spores; in contrast, the cortex of irradiated spores did not change. In addition, the heated spores were found to release appreciable amounts of dipicolinic acid (DPA) upon treatment, while extracellular DPA was completely undetectable in supernatants of the irradiated spores. These observations suggest that microwave radiation may promote the formation of stable complexes between DPA and other spore components (i.e. calcium ions); thus, making any release of DPA from irradiated spores undetectable. Indeed, while a decrease in measurable DPA concentrations was not produced by microwave radiation on pure DPA solutions, a significant lowering in DPA concentration was detected when this molecule was exposed to microwaves in the presence of either calcium ions or spore suspensions. CONCLUSIONS: Microwaves are as effective as conductive heating in killing B. subtilis spores, but the microwave E-field induces changes in the structural and/or molecular components of spores that differ from those attributable only to heat. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides information on the effect of microwaves on B. subtilis spore components.     

Resistance of Bacillus subtilis Spores to Inactivation by Gamma Irradiation and Heating in the Presence of a Bactericide: I. Suitability of Viable Count Procedures

A statistical evaluation of viable count procedures utilized for obtaining treatment survival curve data for Bacillus subtilis NCTC 8236 spores is described. Within the various recovery conditions tested, incubation on nutrient agar containing 1% dextrose for 48 hr at 37 C was found to promote the highest count of viable spores surviving a variety of bactericidal treatments involving gamma irradiation, heat, and chlorocresol. The count of viable spores on the medium was not significantly altered when the dextrose was added to the nutrient agar either before autoclaving or aseptically at 50 to 55 C from a solution sterilized by filtration. The volume of medium which promoted the highest count of viable spores was 20 ml per 85 mm of diameter in disposable plastic plates. Counts of viable spores were reproducible on successive batches of media. The carry-over of variable concentrations of chlorocresol into the medium from serial dilutions affected the count of viable spores. Spores in the aqueous stock suspension used for all experiments were uniformly distributed after shaking and did not diminish significantly in viability after 16 months of storage at 5 C. Grouping of indexes of dispersion, calculated from quintuplicate plate colony counts, indicated that the suitability of the viable count procedures, employed for the enumeration of spores surviving the various bactericidal treatments, tended to diminish as the level of spore inactivation exceeded 95%.     

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.     

Pressure inactivation of Bacillus endospores

The inactivation of bacterial endospores by hydrostatic pressure requires the combined application of heat and pressure. We have determined the resistance of spores of 14 food isolates and 5 laboratory strains of Bacillus subtilis, B. amyloliquefaciens, and B. licheniformis to treatments with pressure and temperature (200 to 800 MPa and 60 to 80 degrees C) in mashed carrots. A large variation in the pressure resistance of spores was observed, and their reduction by treatments with 800 MPa and 70 degrees C for 4 min ranged from more than 6 log units to no reduction. The sporulation conditions further influenced their pressure resistance. The loss of dipicolinic acid (DPA) from spores that varied in their pressure resistance was determined, and spore sublethal injury was assessed by determination of the detection times for individual spores. Treatment of spores with pressure and temperature resulted in DPA-free, phase-bright spores. These spores were sensitive to moderate heat and exhibited strongly increased detection times as judged by the time required for single spores to grow to visible turbidity of the growth medium. The role of DPA in heat and pressure resistance was further substantiated by the use of the DPA-deficient mutant strain B. subtilis CIP 76.26. Taken together, these results indicate that inactivation of spores by combined pressure and temperature processing is achieved by a two-stage mechanism that does not involve germination. At a pressure between 600 and 800 MPa and a temperature greater than 60 degrees C, DPA is released predominantly by a physicochemical rather than a physiological process, and the DPA-free spores are inactivated by moderate heat independent of the pressure level. Relevant target organisms for pressure and temperature treatment of foods are proposed, namely, strains of B. amyloliquefaciens, which form highly pressure-resistant spores.     

The effect of peroxyacetic acid-based sanitizer, heat and ultrasonic waves on the survival of Clostridium estertheticum spores in vitro

AIM: To determine the effect of selected physical and chemical treatments on the survival of 'blown pack'-causing Clostridium estertheticum. METHODS AND RESULTS: The study investigated the survival of the spores of 'blown pack'-causing C. estertheticum following the four treatments, which include: heat alone, ultrasound followed by heat treatment, peroxyacetic acid (POAA)-based sanitizer followed by heat treatment and POAA sanitizer followed by heat treatment in the presence of 20% animal fat. No C. estertheticum survivors were recovered in spore preparations that underwent either of the two treatments with the sanitizer, resulting in the inactivation of 4 to 5 log CFU ml(-1) of spores. Similarly, no survivors were detected in spore preparations that were treated with the sanitizer for 5 min at room temperature without further heat treatment. When using heat alone and ultrasound followed by heat treatment, complete spore inactivation did not occur for spores heated at times and temperature combinations other than 240 s at 100 degrees C. CONCLUSIONS: POAA sanitizer used with or without heat is capable of in vitro inactivation of at least 4 log CFU ml(-1)C. estertheticum spores. SIGNIFICANCE AND IMPACT OF THE STUDY: The data generated in the study provide background information for controlling 'blown pack'-causing clostridia on dressed carcasses and in meat plant environment.