Effect of sporulation conditions on the resistance of Bacillus subtilis spores to heat and high pressure

Bacillus subtilis(B. subtilis) cells were placed in various environmental conditions to study the effects of aeration, water activity of the medium, temperature, pH, and calcium content on spore formation and the resulting properties. Modification of the sporulation conditions lengthened the growth period of B. subtilis and its sporulation. In some cases, it reduced the final spore concentration. The sporulation conditions significantly affected the spore properties, including germination capacity and resistance to heat treatment in water (30 min at 97°C) or to high pressure (60 min at 350 MPa and 40°C). The relationship between the modifications of these spore properties and the change in the spore structure induced by different sporulation conditions is also considered. According to this study, sporulation conditions must be carefully taken into account during settling sterilization processes applied in the food industry.     

The effect of recovery conditions on the apparent heat resistance of Bacillus cereus spores

The effect of recovery media and incubation temperature on the apparent heat resistance of three ATCC strains (4342, 7004 and 9818) of Bacillus cereus spores were studied. Nutrient Agar (NA), Tryptic Soy Agar (TSA), Plate Count Agar (PCA) and Milk Agar (MA) as the media and temperatures in the range of 15–40°C were used to recover heated spores. Higher counts of heat injured spores were obtained on PCA and NA. The optimum subculture temperature was about 5°C below the optimum temperature for unheated spores. No significant differences in heat resistance were observed with the different recovery conditions except for strains 4342 and 9818 when MA was used as plating medium.
Large differences in D -values were found among the strains ( D ₁₀₀=0·28 min for 7004; D ₁₀₀=0·99 min for 4342; D ₁₀₀= 4·57 min for 9818). The 7004 strain showed a sub-population with a greater heat resistance. The z values obtained for the three strains studied under the different recovery conditions were similar (7·64°C 0·25).     

Emergence and development of resistance to antimicrobial chemicals and heat in spores of Bacillus subtilis

The emergence and development of chemical and thermal resistance in spores of Bacillus subtilis was examined. The chemicals studied were of the disinfectant type: glutaraldehyde, hypochlorite, hypochlorite-methanol and povidone-iodine. Growth and sporulation were followed by electron microscopy and resistance assigned to specific stages in relation to 45Ca and DPA accumulation. A sequential development of resistance was observed with thermal resistance appearing first at early Stage V corresponding to maturation of cortex and deposition of rudimentary spore coat material. Chemical resistance coincided with middle to late Stage V dependent on the chemical concerned. A progressive development of resistance was observed on prolonged incubation in sporulation medium and was affected by inclusion of lysozyme in the spore washing sequence.     

Emergence and development of resistance to antimicrobial chemicals and heat in spores of Bacillus subtilis

The emergence and development of chemical and thermal resistance in spores of Bacillus subtilis was examined. The chemicals studied were of the disinfectant type: glutaraldehyde, hypochlorite, hypochlorite-methanol and povidone-iodine. Growth and sporulation were followed by electron microscopy and resistance assigned to specific stages in relation to ⁴⁵Ca and DPA accumulation. A sequential development of resistance was observed with thermal resistance appearing first at early Stage V corresponding to maturation of cortex and deposition of rudimentary spore coat material. Chemical resistance coincided with middle to late Stage V dependent on the chemical concerned. A progressive development of resistance was observed on prolonged incubation in sporulation medium and was affected by inclusion of lysozyme in the spore washing sequence.     

Heat shock applied early in sporulation affects heat resistance of Bacillus megaterium spores.

Cells of Bacillus megaterium 27 were challenged by a 30-min heat shock at 45 degrees C during various sporulation stages and then shifted back to a temperature permissive for sporulation (27 degrees C), at which they developed spores. Heat shock applied at 120 min after the end of the exponential phase induced synthesis of heat shock proteins (HSPs) in the sporangia and delayed the inactivation of spores at 85 degrees C. Several HSPs, mainly HSP 70, could be detected in the cytoplasm of these spores. An analogous HSP, the main HSP induced by increased temperature during growth, belongs to the GroEL group according to its N-terminal sequence. The identity of this protein was confirmed by Western blot (immunoblot) analysis with polyclonal antibodies against B. subtilis GroEL. Sporangia treated by heat shock immediately or 240 min after exponential phase also synthesized HSPs, but none of them could be detected in the spores in an appreciable amount. These spores showed only a slightly increased heat resistance.     

Effects of sporulation pH on the heat resistance and the sporulation of Bacillus cereus

Spores of Bacillus cereus ATCC 7004, 4342 and 9818 were obtained in nutrient agar at several pH from 5·9 to 8·3. The optimum pH for sporulation was around 7, but good production of spores was obtained in the range 6·5–8·3. With all three strains, D -values clearly dropped with sporulation pH, decreasing by about 65% per pH unit. z -Values were not significantly modified ( P > 0·05) by this factor. Mean z -values of 7·13 °C ± 0·16 for strain 7004, 7·67 °C ± 0·04 for 4342 and 8·80 °C ± 0·64 for 9818 were obtained.     

The effect of the pH of the sporulation environment on the heat resistance ofClostridium perfringens spores

The inactivation ofClostridium perfringens NCTC 8239 spores at 95° and 105° C, as determined by colony formation on an agar base containing lysozyme (BASE + lysozyme), was influenced by the initial pH of the sporulation medium. In the pH range of 7.0–8.5, established by the addition of each of several biological buffers or carbonate buffer to Duncan-Strong (DS) medium, increased pH resulted in formation of spores with greater resistance to inactivation at elevated temperatures. An increase of pH from 8.5 to 9.0 resulted in increased resistance of spores formed in DS-carbonate but not DS-TAPS (N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid) medium. Resistance to spore injury, as determined by reduced recovery on BASE compared with BASE + lysozyme, was not increased for spores formed in media with higher pH's. As the pH of the medium increased, cell growth and number of spores formed were decreased, but the percentage of sporulation was apparently not affected.     

Maturation of released spores is necessary for acquisition of full spore heat resistance during Bacillus subtilis sporulation

The first ～10% of spores released from sporangia (early spores) during Bacillus subtilis sporulation were isolated, and their properties were compared to those of the total spores produced from the same culture. The early spores had significantly lower resistance to wet heat and hypochlorite than the total spores but identical resistance to dry heat and UV radiation. Early and total spores also had the same levels of core water, dipicolinic acid, and Ca and germinated similarly with several nutrient germinants. The wet heat resistance of the early spores could be increased to that of total spores if early spores were incubated in conditioned sporulation medium for ～24 h at 37°C (maturation), and some hypochlorite resistance was also restored. The maturation of early spores took place in pH 8 buffer with Ca(2+) but was blocked by EDTA; maturation was also seen with early spores of strains lacking the CotE protein or the coat-associated transglutaminase, both of which are needed for normal coat structure. Nonetheless, it appears to be most likely that it is changes in coat structure that are responsible for the increased resistance to wet heat and hypochlorite upon early spore maturation.     

Perturbation of the heat resistance of bacterial spores by sporulation temperature and ethanol

Spores ofBacillus megaterium, B. subtilis, andB. stearothermophilus, harvested from cultures grown and sporulated at different temperatures or in the presence of ethanol, had different thermal resistance. There was a direct relationship between the sporulation temperature and the spore-killing temperature. The spores were more temperature-sensitive when formed in ethanol-supplemented media. Temperature and ethanol are known to perturb the degree of order within membranes and to alter membrane functions. Thus, alteration of spore membranes is an additional factor in the multifactorial nature of heat resistance. Another interpretation may be that heat shock proteins, known to be induced by heat, are formed during sporulation and may increase the thermostability of the spores.     

A note on the effect of storage on the chemical resistance of spores of Bacillus subtilis SA22 and Bacillus subtilis globigii B17

Spores of Bacillus subtilis SA22, harvested from nutrient agar after 9 d at 30°C and stored in distilled water at 4°C, were unaltered in their resistance to 17.7% hydrogen peroxide or 0.04% peracetic acid after storage for up to 134 weeks. Three spore crops of B. subtilis globigii were unaffected by storage for up to 134 weeks with respect to 17.7% hydrogen peroxide resistance but were significantly more resistant to 0.04% peracetic acid following storage.     

Effect of the osmotic conditions during sporulation on the subsequent resistance of bacterial spores

The causes of Bacillus spore resistance remain unclear. Many structures including a highly compact envelope, low hydration of the protoplast, high concentrations of Ca-chelated dipicolinic acid, and the presence of small acid-soluble spore proteins seem to contribute to resistance. To evaluate the role of internal protoplast composition and hydration, spores of Bacillus subtilis were produced at different osmotic pressures corresponding to water activities of 0.993 (standard), 0.970, and 0.950, using the two depressors (glycerol or NaCl). Sporulation of Bacillus subtilis was slower and reduced in quantity when the water activity was low, taking 4, 10, and 17 days for 0.993, 0.970, and 0.950 water activity, respectively. The spores produced at lower water activity were smaller and could germinate on agar medium at lower water activity than on standard spores. They were also more sensitive to heat (97 degrees C for 5-60 min) than the standard spores but their resistance to high hydrostatic pressure (350 MPa at 40 degrees C for 20 min to 4 h) was not altered. Our results showed that the water activity of the sporulation medium significantly affects spore properties including size, germination capacity, and resistance to heat but has no role in bacterial spore resistance to high hydrostatic pressure.     

Influence of sporulation medium and divalent ions on the heat resistance of Alicyclobacillus acidoterrestris spores

The influence of divalent cations on the heat resistance of spores of the thermoacidophilic spoilage bacterium Alicyclobacillus acidoterrestris was studied. The heat resistance of A. acidoterrestris spores was not affected by the presence of the different divalent cations (Ca²⁺, Mg²⁺, Ba²⁺, Mn²⁺ and Sr²⁺) in the sporulation medium, and by the demineralization or remineralization. And the Ca and Mn contents in A. acidoterrestris spores were scarcely changed by these treatments. However, the heat resistance of Bacillus subtilis spores was affected with the changes of Ca content in the spores. The Ca contents in A. acidoterrestris spores of the different forms were greater than the DPA content. In contrast, the DPA content in the B. subtilis spores was greater than the Ca content. These findings suggest that the presence of constant amount of Ca-DPA and a stronger binding characteristic of divalent ions, especially Ca and Mn, is reflected in the specific heat resistance of A. acidoterrestris spores.     

Relationship between Bacillus sphaericus spore heat resistance and sporulation temperature

Bacillus sphaericus 9602 was grown in batch culture at various temperatures. At 10°C and 12°C the maximum sporulation yield was <10%, while at 15°C, 20°C and 30°C, a sporulation yield of >95% was achieved. However at 40°C B. sphaericus grew only vegetatively. The heat resistances (D values at 90°C) of spores grown at 15°C and 20°C were significantly higher than those grown at 30°C.     

Effects of minerals on resistance of Bacillus subtilis spores to heat and hydrostatic pressure

Among Bacillus subtilis IFO13722 spores sporulated at 30, 37, and 44 degrees C, those sporulated at 30 degrees C had the highest resistance to treatments with high hydrostatic pressure (100 to 300 MPa, 55 degrees C, 30 min). Pressure resistance increased after demineralization of the spores and decreased after remineralization of the spores with Ca(2+) or Mg(2+), whereas the resistance did not change when spores were remineralized with Mn(2+) or K(+), suggesting that former two divalent ions were involved in the activation of cortex-lytic enzymes during germination.     

Effect of ultrasonic waves on the heat resistance of Bacillus spores.

The effect of ultrasonic treatment on the heat resistance of Bacillus spores differs widely both with the species and the strain tested. Ultrasonic waves do not affect the heat resistance of some strains, whereas they greatly reduce that of other strains of the same species. The heat-sensitizing effect of ultrasonic waves is more pronounced when short heating periods are used. Ultrasonic treatment does not seem to affect the "Z value".