Heat Resistance of Native and Demineralized Spores of Bacillus subtilis Sporulated at Different Temperatures

Abstract

Demineralization reduced heat resistance of B. subtilis spores, but the pattern and magnitude of the reduction depended on sporulation temperature and on heating menstruum pH. The differences in heat resistance of native spores caused by sporulation temperature almost disappeared after demineralization. Demineralized spores were still susceptible to the heat-sensitizing effect of acidic pH.

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.) liter⁻¹ and 3 mg of manganese sulfate (Probus, Barcelona, Spain) liter⁻¹. 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 ml⁻¹).

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.

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.

The effect of demineralization on D_t values was strongly dependent on sporulation temperature. The D_102°C of spores formed at 52°C was decreased from 15 min for native to 3.8 min for demineralized spores (Fig. 1a). The effects of demineralization on heat resistance were even greater at higher temperatures (Fig. 2 and Table 1). Demineralization had less effect on spores produced at 32°C. As shown by Fig. 1b, the D_103°C was 1.1 min for native spores and 0.94 min for demineralized spores. No statistically significant differences (P > 0.05) could be found among D_t values of native and demineralized spores at heating temperatures in the range 95 to 110°C, and they only were reduced to one-third at temperatures around 120°C (Fig. 2 and Table 1). Higher sporulation temperatures usually lead to more-resistant spore crops (6, 10, 19), and a relation between sporulation temperature and mineralization has been proposed (6, 20). However, the presence of different pools of minerals in the spore that are not always related to heat resistance (23, 28) reduces the significance of this hypothetical relationship. Our results (Fig. 2) demonstrated that differences in heat resistance between spores obtained at 32 and 52°C decreased after demineralization. These results point out that sporulation temperature increases heat resistance by increasing the mineralization of bacterial spores. Still, heat resistance at pH 7 of demineralized spores sporulated at 52°C was slightly higher than that of demineralized spores sporulated at 32°C (Fig. 2). Other mechanisms besides mineralization, such as protoplast dehydration level (6) and the possible formation of heat shock proteins in spores sporulated at higher temperatures (18), could be related to the higher heat resistance of demineralized spores sporulated at higher temperatures.

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).

TABLE 1.

Influence of demineralization and of the pH of the heating menstruum upon z values of B. subtilis spores

Suspensiona	pHb	z (°C)	95% CLc	r0
Native 52°C	7	8.7	8.3–9.2	0.999
Native 32°C	7	8.6	8.2–9.0	0.999
Native 52°C	4	10.3	9.6–11.0	0.998
Native 32°C	4	8.8	8.1–9.8	0.996
Demineralized 52°C	7	7.0	6.8–7.3	0.999
Demineralized 32°C	7	7.2	6.8–7.6	0.998
Demineralized 52°C	4	11.8	11.0–12.7	0.997
Demineralized 32°C	4	6.4	5.5–7.6	0.991

^aSpore type and temperature of sporulation. 

^bpH of the heating menstruum. 

^c95% confidence limits. 

Demineralization also reduced z values in citrate-phosphate McIlvaine buffer (pH 7) in a similar way for both spore suspensions (Fig. 2 and Table 1). Bender and Marquis (7) found an increase on z values as a consequence of demineralization, while according to Alderton et al. (2) z values of demineralized spores may also decrease, depending on other heating conditions. Native spores can be demineralized to some extent during heat treatment (7). The phosphates present in the citrate-phosphate McIlvaine buffer (pH 7) we used as heating menstruum can also lead to a mild demineralization process by chelating the cations released from the native spores during heating (13). This demineralization process of native spores during heat treatment would only take place at low heating temperatures, when the relatively lower rate of heat inactivation of the spores would allow this process to occur before spores are killed. Demineralized spores would show no additional demineralization in neutral buffer at any heating temperature, and so their heat resistance would be relatively higher at low heating temperatures. As a result their z values would be lower than those of native spores.

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).

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.

Our results indicate that sporulation temperature increases heat resistance by increasing the mineralization of the spores. They also seem to indicate that the loss of heat resistance of B. subtilis spores in acidic media is not due to demineralization taking place during heating.