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Bacterial spores are usually more heat resistant when sporulated at higher temperatures (6, 10, 19). Higher sporulation temperatures have been correlated with higher levels of mineralization of spores (20). However, published data on the relationship between mineralization and heat resistance are not always in agreement (16, 22, 28).

Minerals in the spore protoplast are thought to increase the degree of immobilization of molecules and structures, making them less heat sensitive (15). Calcium is usually the major mineral component of bacterial spores, and it has been related most often to heat resistance (22). The types of minerals and the overall content of spores can be modified somewhat by changing the mineral composition of the sporulation medium (26). Furthermore, spore mineral content can be modified dramatically through acid extraction of dormant spores and remineralization (4, 7, 21).

Demineralization markedly reduces heat resistance of bacterial spores (4, 21) and also affects their germination systems (5, 14, 23). Several authors have proposed that there are different pools of calcium, retained with different affinities by spores that play different roles in heat resistance and germination (22, 23, 28). The presence of these different pools of divalent cations could be responsible for the above-mentioned low correlation between results of biochemical analysis of mineral contents of bacterial spores and heat resistance data (16, 22, 28). Therefore, it is difficult to draw a clear relationship among sporulation temperature, mineralization degree, and heat resistance. If higher sporulation temperatures lead to higher heat resistance by increasing mineralization, then after demineralization, these differences should decrease.

Sporulation temperature has also been implicated in the influence of other factors on heat resistance, such as the pH of the heating menstruum (24). Heat resistance of demineralized spores in acidic media has not been studied yet. The decrease in heat resistance of native, fully mineralized spores when heated in acidic media has been attributed to an acid wash of the minerals of the spores (3). It has also been proposed that a protonization of the cortex could be responsible for the decrease in heat resistance in acidic media (17). If the lower heat resistance in acidic media were due to a release of spore cations, demineralized spores, with their mineral content already reduced, should show smaller differences in heat resistance in acidic or in neutral-pH media.

The objective of this work was to investigate the heat resistance of native and demineralized spores of a strain of Bacillus subtilis (STCC 4524; Spanish Type Culture Collection) sporulated at 32 and 52°C in citrate-phosphate buffer of pH 7 and 4.

Spore suspensions. The strain of B. subtilis used in this investigation (STCC 4524) was isolated during a routine check of the sterility of canned asparagus. Growth and sporulation were carried out in Roux bottles of nutrient agar (Biolife, Milan, Italy) containing 500 mg of Bacto Dextrose (Difco, Detroit, Mich.) liter¹ and 3 mg of manganese sulfate (Probus, Barcelona, Spain) liter¹. Roux bottles were inoculated with a young culture (for 24 h at 37°C) in nutrient broth (Biolife) and incubated at 32 or 52°C. After 80 to 90% sporulation was obtained, spores were harvested and washed five times by centrifugation with sterile distilled water (ca. 10⁹ spores ml¹).

Demineralization. Spores were demineralized by acid titration with HCl (0.033 N) and subsequent incubation for 14 h (22) at 60°C, as suggested by other authors (21). After this incubation, spores were washed three times, by centrifugation and resuspension. The first wash was with sterile citrate-phosphate McIlvaine buffer (pH 7) (12), to restore the neutral pH, and the others were with sterile distilled water. Demineralized suspensions were stored at 0 to 5°C. No changes in heat resistance were observed during the time this investigation was carried out.

Heat treatments. Heat resistance determinations were carried out in a thermoresistometer (TR-SC) as already described (9). For each survival curve, 8 to 15 samples were taken at different heating times. Heat resistance was determined at least at seven temperatures for each spore suspension in each heating medium. Incubation of plates for survivor counting was carried out at 35°C for 24 h. Longer incubation times did not increase counts. Survivors on plates were counted with an improved Image Analyzer Automatic Counter (Protos, Synoptics, Cambridge, England) as described by Condón et al. (11). The decimal reduction times (D_t values [time in minutes at temperature t for a 10-fold reduction in survival]) obtained by this method always showed coefficients of variation of <20% (9). The z values (change in temperature [degrees Celsius] necessary for a 10-fold change in D_t) were determined from regression lines obtained by plotting log D_t versus heating temperatures (decimal reduction time curves [DRTC]). Correlation coefficients (r₀) of DRTC obtained in this investigation were always >0.99. Comparison of slopes of survival curves and DRTC were carried out as described by Steel and Torrie (27). r₀ and 95% confidence limits were calculated by use of the appropriate statistical package (StatView SE+Graphics; Abacus Concepts, Inc., Berkeley, Calif.).

Heat resistance at pH 7. Survival curves corresponding to native spores produced at 32 and 52°C showed shoulders (lag phases before killing begins) at every heating temperature tested (Fig. 1). The ratio between the duration of these lag phases and D_t values remained constant regardless the temperature of treatment (data not shown). Survival curves of demineralized spores did not show shoulders (Fig. 1). Shoulders have been related to the activation of dormant spores (1, 25). Several authors have observed that acid shocks, such as demineralization treatments, induce spore activation (8, 14). The absence of shoulders in survival curves corresponding to our demineralized spores could indicate that these spores had been activated during demineralization. However, shoulders have also been related to heat damage repair mechanisms (11). Therefore, the possibility of a lower heat damage repair capacity of demineralized spores should not be disregarded.

View larger version (11K): [in this window] [in a new window]   FIG. 1.   Survival curves of native () and demineralized () spores in McIlvaine buffer (pH 7). (A) Survival curves at 102°C of spores sporulated at 52°C; (B) survival curves at 103°C of spores sporulated at 32°C.

View larger version (17K): [in this window] [in a new window]   FIG. 2.   DRTC of native (solid lines) and demineralized (dashed lines) spores of B. subtilis sporulated at 32°C (circles) and 52°C (diamonds) in McIlvaine buffer (pH 7).

Heat resistance at pH 4. The acidification of the heating menstruum from pH 7 to 4 reduced the D_99°C of demineralized spores sporulated at 52°C from 11 min to 1.6 min, but the effect was lower at higher temperatures, being negligible at temperatures greater than 110°C (Fig. 3A and Table 1). The same acidification decreased the D_99°C of demineralized spores sporulated at 32°C from 2.8 to 0.43 min, and in this case, the effect was constant regardless of the temperature of treatment (P > 0.05) (Table 1). Heat treatment at acid pH reduced the heat resistance not only of native but also of demineralized spores (Fig. 3). It has been postulated that the lower heat resistance of native spores in acidic media could be due to a demineralization process taking place in spores during heating (3). However, our results showed that the magnitude of the effect of the pH of the heating menstruum on the thermotolerance of demineralized spores sporulated at different temperatures (Fig. 3a) was similar to that observed for native spores (Fig. 3b). This seemed to indicate that other mechanisms besides demineralization during heat treatment could be responsible for the loss of heat resistance of demineralized spores in acidic media. In the opinion of Gould and Dring (17) the lower heat resistance of spores in acidic media could be due to a protonization of the carboxyl groups of the cortex, which would lead to the protoplast rehydration and thus to heat sensitization. This hypothesis could explain why the behavior of demineralized spores under acidic conditions is similar to that shown by their corresponding native spores (Fig. 3); i.e., the z value in acidic media increased largely for spores sporulated at 52°C and remained approximately constant for those sporulated at 32°C, for both native and demineralized spores (Table 1). Whatever the mechanism by which spores lost heat resistance in acidic media, the process is fast for spores sporulated at 32°C (it was observed even at higher temperatures, when spores were heat killed in fractions of seconds) and slow for spores sporulated at 52°C (at high temperatures there was no effect of acidic pH).

View larger version (17K): [in this window] [in a new window]   FIG. 3.   DRTC of B. subtilis sporulated at 32°C (circles) and 52°C (diamonds) in McIlvaine buffer at pH 7 (solid lines) and pH 4 (dashed lines). (A) Demineralized spores; (B) native spores.