Bacillus strains tested in this study, including B. licheniformis (KCTC 1918), B. cereus (KCTC 3624), B. coagulans (KCTC 3625), and B. subtilis (KCTC 3135), are type strains and were obtained from the Korean Collection for Type Culture (KCTC; Daejeon, South Korea). The strains were grown in tryptic soy broth (TSB; Becton Dickinson, Sparks, MD, United States) for 24 h at the temperatures recommended by the KCTC (B. licheniformis and B. coagulans at 37°C, for B. subtilis and B. cereus at 30°C), and then kept as glycerol stock cultures (60%, v/v) in a freezer at −20°C.

To compare the heat resistance (D_100°C-values) of spores of the four Bacillus species presented in the current and previous studies, literature was searched using search engines, including Web of Science and Google Scholar. Literature data compiled in Figure 2 and Supplementary Tables 1–4 were selected based on the following criteria: (1) minerals should not be added into the sporulation media in spore crop preparation and (2) D_100°C-values should be obtained through heat treatment in a solution with a pH of approximately 7.0.

Nutrient agar (NA; MB cell, Seoul, South Korea) served as the basal sporulation medium. Calcium chloride dihydrate (CaCl₂⋅2H₂O; Duchefa Biochemie, Harleem, Netherlands) and manganese sulfate monohydrate (MnSO₄⋅H₂O; Biosesang, Seoul, South Korea) acted as the cations added to NA. Diverse concentrations of Ca²⁺ (0.00, 0.25, 0.50, 1.00, and 2.00 mM) and Mn²⁺ (0.00, 0.10, 0.25, and 0.50 mM) were used to supplement the basal sporulation medium, similar to a previous report by Sinnelä et al. (2019) as based on earlier studies (Amaha and Ordal, 1957; Levinson and Hyatt, 1964). All sporulation media had a pH of 6.95–7.00, and prepared in Petri dishes with a diameter of 90 mm (SPL Life Sciences, Pocheon, South Korea).

The glycerol stocks of Bacillus strains were pre-activated before use by three successive subculturings in TSB for 24 h at the temperatures recommended for vegetative growth of respective strains described above. To form Bacillus spores, the sporulation media, described in the previous section, was inoculated with 0.1 mL of vegetative cell suspension by spreading, and the cells were sporulated for 2 days (Bai, 2013) at the temperatures for vegetative growth of respective Bacillus strains. Spores were then harvested by flooding the agar surface with M/15 Sörensen’s phosphate buffer (5.675 g of sodium phosphate dibasic and 3.63 g of potassium phosphate monobasic in 1 L of distilled water, pH 7.0; chemicals from Sigma-Aldrich Co., St. Louis, MO, United States) and scraping with a cell scraper (SPL Life Sciences). The spores from multiple plates were pooled, washed three times in a centrifuge (1736R; Gyrozen Co., Ltd., Daejeon, South Korea) at 19,000 × g for 10 min at 4°C, resuspended in the same phosphate buffer (to a final concentration of 9 log CFU/mL), and refrigerated at 4°C prior to being tested for heat resistance. The procedure for spore formation is illustrated in Figure 1 (step 1–5).

The D-values (time required for a 10-fold reduction in viable spores) of Bacillus spores were determined by using the multiple-point method. Fifty microliters of spore suspension (prepared in section “Spore Formation”) were injected into a glass capillary tube with an inner diameter of 1.8 mm and an outer diameter of 3.0 mm (Corning Inc., Corning, NY, United States) using a pipette, and then both ends of the tubes were heat sealed. The tubes were fully immersed in a 100°C oil bath (Thermo Fisher Scientific Inc., Newington, NH, United States) filled with ethylene glycol (Samchun Pure Chemical Co., Ltd., Pyeongtaek, South Korea) set at 100 ± 0.015°C and heated for five different time intervals (B. licheniformis, 20 s intervals; B. cereus, 10 s intervals; B. coagulans and B. subtilis, 50 s intervals). After heating, the tubes were removed from the oil bath, cooled immediately in a crushed ice water bath, and washed with 70% ethyl alcohol. Both ends of the tubes were cut aseptically, and the content was flushed out with 3 mL of the phosphate buffer (Sinnelä et al., 2019). To enumerate spore survival, the treated suspension was 10-fold serially diluted in sterile 0.1% peptone water, and 0.1 mL of each dilution was spread onto plate count agar (PCA; Becton Dickinson) plates and incubated for 24 h at temperatures for vegetative growth of respective Bacillus strains described above. Following, the colonies of spore survivors were counted. The procedure for heat treatment of spores is illustrated in Figure 1 (step 6–10).

Survivor curves were plotted on a semi-log chart (log survivors versus time) to determine D-values. The D-values were obtained by taking the negative reciprocals of the slopes from linear regression of the survivor curves in Supplementary Figures 1–4.

The measurement of D_100°C-values was conducted in triplicate, and the data were expressed as mean and standard deviation of the values obtained from a single spore batch. All values in Figure 2 were calculated from D_100°C-values of different spore batches. Statistical analysis was performed by one-way analysis of variance (ANOVA) with Fisher’s pairwise comparison test. Differences were considered statistically significant at p-values of <0.05.

Heat resistance profiles of spores of B. licheniformis, B. cereus, B. coagulans, and B. subtilis formed on basal media without mineral supplementation were compiled in Figure 2 (also refer to Supplementary Tables 1–4). In the present study, the D_100°C-values of the spores of the type strains were determined to be 3.91 ± 1.65 min for B. licheniformis, 0.49 ± 0.28 min for B. cereus, 1.70 ± 1.63 min for B. coagulans, and 2.73 ± 1.71 min for B. subtilis, which were similar to those of type (representing each species) and reference (serving as definitive control) strains examined in previous studies, respectively (p > 0.05). The reported D_100°C-values were 1.94 ± 1.30 min for B. licheniformis (Nakayama et al., 1996; Esteban et al., 2015), 1.05 ± 0.17 min for B. cereus (Beaman et al., 1982; Rajkowski and Mikolajcik, 1987; Penna and Moraes, 2002; De Vries et al., 2004), 4.15 ± 0.91 min for B. coagulans (Nakayama et al., 1996; Majeed et al., 2021), and 1.64 ± 0.86 min for B. subtilis (Nakayama et al., 1996; Esteban et al., 2015). Note that type strains represent each species in taxonomic studies, and reference strains are used as definitive control strains for various microbiological testing procedures (National Collection of Type Cultures, 2015), regardless of the isolation sources. In some cases, type strains can be used as reference strains depending on the research purpose. It is also noteworthy that, for the above comparison, all literature data as much as possible were collected based on the following criteria: (1) minerals should not be added into the sporulation media in spore crop preparation and (2) D_100°C-values should be obtained through heat treatment in a solution with a pH of approximately 7.0. The reasons for setting these criteria were to precisely compare the heat resistance profiles of spores of four Bacillus spp. to those of previous studies conducted under the same test conditions as the current study, and avoid influencing factors, such as strains tested, sporulation methods and media, suspension media, and heating methods (Den Besten et al., 2018). Based on the observation in the present study, mean D_100°C-value of the spores of B. licheniformis calculated from the heat resistance is significantly higher than those of B. subtilis and B. coagulans (although the mean value for B. subtilis spores is higher than that of B. coagulans spores), while that of B. cereus is the lowest (p < 0.05). In literature, mean D_100°C-value of the spores of B. coagulans is the highest, followed by B. licheniformis, B. subtilis, and B. cereus (although there were no statistical differences among the latter three Bacillus species). Such observation is expected to be important information that can be utilized to selectively reduce the spores of undesirable pathogenic B. cereus.

Meanwhile, in previous studies, the D_100°C-values of the spores of the isolated Bacillus strains have been determined to be 3.11 ± 2.57 min for B. licheniformis (Janštová and Lukášová, 2001; André et al., 2013), 1.73 ± 2.00 min for B. cereus (Rajkowski and Mikolajcik, 1987; Janštová and Lukášová, 2001), 5.28 ± 5.69 min for B. coagulans (Janštová and Lukášová, 2001; André et al., 2013), and 1.70 ± 1.58 min for B. subtilis (Janštová and Lukášová, 2001; Movahedi and Waites, 2002; Conesa et al., 2003). The D_100°C-values of the type and reference strains in the present and previous studies fall within the ranges of D_100°C-values for the isolated strains (p > 0.05). Such wide ranges of D_100°C-values for the isolated strains might be attributed to strain diversity, growth conditions, and the sources from which the strains were isolated. As type and reference strains have been cultured for a long time in the laboratory conditions preferable for growth, phenotypic characteristics of the strains might be stable (Fux et al., 2005), whereas isolated strains may be more adapted to the environment in which they are naturally found. Furthermore, not only are natural environments often more diverse and extreme than laboratory conditions (Fux et al., 2005), but strain diversity can also influence heat resistance properties (Carlin et al., 2006; Luu-Thi et al., 2014; Berendsen et al., 2015). Therefore, it seems inevitable that characteristics and resistances of spores of isolated strains differ from laboratory strains. Indeed, several studies reported that Bacillus strains isolated from some processed foods (particularly food products processed using heat treatment) had more heat resistance than type and reference strains (André et al., 2013; Den Besten et al., 2018). In addition, regarding the isolated strains, there was no statistically significant difference between the heat resistances of the spores of four Bacillus species, unlike the aforementioned type strains.

Heat resistance profiles of B. licheniformis spores formed under different supplementation conditions were compiled in Table 1 (also refer to Supplementary Figure 5). B. licheniformis spores displayed the greatest heat resistance (D_100°C-value of 5.07 ± 0.20 min) when sporulated on the non-supplemented media, whereas the smallest heat resistance (2.17 ± 0.04 min) of the spores was observed when sporulated on media supplemented with both a moderate level of calcium (1.00 mM) and the highest concentration of manganese (0.50 mM). Janštová and Lukášová (2001) reported that for spores of 21 B. licheniformis strains formed on NA media without mineral supplementation, the highest D_100°C-value was found to be 5.06 min; conversely, Nakayama et al. (1996) reported a lower D_100°C-value of 1.02 min when the spores were formed under the same non-supplementation condition. The former result was almost identical to the highest D_100°C-value obtained in the present study from B. licheniformis spores formed under the same non-supplementation condition. Conversely, the latter result was somewhat lower than the lowest D_100°C-value obtained in the current study from spores prepared with mineral supplementation. This difference may be due to the diversity of strains tested in the current and the previous research (Carlin et al., 2006; Luu-Thi et al., 2014; Berendsen et al., 2015).

Similar to B. licheniformis spores, the greatest heat resistance of B. cereus spores (0.29 ± 0.01 min) was achieved when sporulated on the non-supplemented media, as shown in Table 1 (also refer to Supplementary Figure 6). In contrast, B. cereus spores exhibited the smallest (0.21 ± 0.01 min) and the second smallest heat resistance (0.22 ± 0.01 min) when sporulated on media supplemented solely with calcium at a concentration of 2.00 and 1.00 mM, respectively. The media supplemented with lower levels of both calcium (0.50 mM) and manganese (0.25 mM) also resulted in a D_100°C-value of 0.22 ± 0.00 min (another second smallest heat resistance). Although limited information is available about the effects of calcium and manganese on the heat resistance of B. cereus spores, D_100°C-values, ranging from approximately 0.2–0.6 min, similar to the present study, have been obtained by multiple researchers (Gonzáles et al., 1997; Mazas et al., 1997a,b, 2009; González et al., 1999). On the other hand, Janštová and Lukášová (2001) reported much higher D_100°C-values for B. cereus spores ranging from 0.71 to 1.48 min. The previous studies have shown that research methods, such as mineral (and other additives) supplementation, sporulation temperature, heating medium (and its pH and a_w), and strain tested, could have a significant impact on the D-values of B. cereus spores.

As for both B. licheniformis and B. cereus spores formed on media supplemented only with a single mineral in this study, 0.50 mM calcium supplementation significantly enhanced the heat resistance of the spores compared to the other concentrations of calcium. Nevertheless, the D_100°C-values of all the spores obtained on media supplemented only with calcium were lower than that of the spores sporulated on media without mineral supplementation. Unlike calcium, variation in the concentration of manganese as the sole mineral in the sporulation media insignificantly affected the heat resistance of spores of both Bacillus species. Thus, the heat resistance profiles of B. licheniformis spores and B. cereus spores sporulated on media supplemented with a single mineral were almost similar to each other, showing that a calcium concentration of 0.50 mM is the most effective in enhancing heat resistance. Moreover, both B. licheniformis spores and B. cereus spores exhibited the greatest heat resistance when formed under non-supplementation conditions, as described above.

Interestingly, when manganese was supplemented along with 0.50 mM calcium, the concentration of manganese seemed to adversely affect the heat resistance of spores of both Bacillus species. However, manganese supplementation alongside 1.00 mM calcium, affected the heat resistance of spores of the two species in opposite ways: the heat resistance of B. licheniformis spores was weakened as the manganese concentration increased, while that of B. cereus spores was enhanced. It is also noteworthy that the lowest D_100°C-values of B. cereus spores for each concentration of manganese (except for non-supplementation of manganese) were observed when the calcium concentration was 0.50 mM, but B. licheniformis spores did not exhibit such a calcium concentration-specific decrease in heat resistance. Incidentally, note that the mineral supplementation at all levels resulted in a decrease in the heat resistance of both B. licheniformis spores and B. cereus spores as compared to non-supplementation conditions.

As opposed to B. licheniformis spores and B. cereus spores, both B. coagulans spores and B. subtilis spores exhibited the greatest heat resistance when sporulated on media supplemented solely with the highest concentration of calcium (2.00 mM), as shown in Table 2 (also refer to Supplementary Figures 7, 8). Another greatest heat resistance of spores of both Bacillus species was also achieved when sporulated on media supplemented with moderate levels of both calcium (1.00 mM) and manganese (0.25 mM). The highest D_100°C-values of B. coagulans spores formed under each supplementation condition were 5.76 ± 0.33 and 5.75 ± 0.31 min, respectively, and those of B. subtilis spores were 6.54 ± 0.26 and 6.54 ± 0.66 min, respectively. On the contrary, the smallest heat resistance of B. coagulans spores and B. subtilis spores was observed when sporulated on media supplemented solely with manganese at a concentration of 0.25 and 0.50 mM, respectively. The lowest D_100°C-values of spores of the two species were 3.01 ± 0.23 and 2.83 ± 0.35 min, respectively. Nakayama et al. (1996) reported two D_100°C-values of 4.6 and 4.79 min obtained from B. coagulans spores formed on NA media without mineral supplementation, which was almost identical to the D_100°C-value of 4.51 ± 0.12 obtained from B. coagulans spores formed under the same supplementation condition in this study (Table 2). However, it is worth mentioning that a large difference in the D_100°C-value of B. coagulans spores appeared when the spores were heat treated in different heating menstrua (Mallidis et al., 1990). In the previous study, the reported D_100°C-values of B. coagulans spores were as follows: 4.15 min in tomato serum (pH 4.24), 4.9 min in citrate buffer (pH 4.5), and 21.0 min in phosphate buffer (pH 7). Meanwhile, Conesa et al. (2003) reported a D_100°C-value of 6.5 min obtained from B. subtilis spores formed on PCA media without mineral supplementation, which was identical to the highest D_100°C-value of B. subtilis spores in this study. In addition, it seems most likely that basal sporulation media have a significant impact on the D-values of B. subtilis spores as spores formed on NA media without mineral supplementation have been reported to range from 0.85 to 2.13 min (Nakayama et al., 1996; Janštová and Lukášová, 2001), which is much lower than the former study using non-supplemented PCA media. While in the present study, B. subtilis spores also displayed a lower D_100°C-value of 3.95 ± 0.29 min when sporulated on NA media with no supplementation (Table 2).

As for both B. coagulans spores and B. subtilis spores formed on media supplemented with only a single mineral, the concentrations of calcium and manganese significantly affected the heat resistance of the spores in opposite ways. In detail, the heat resistance of spores of both Bacillus species was enhanced as the calcium concentration increased. On the contrary, with the exception of supplementation only with 0.50 mM manganese for B. coagulans spores, the heat resistance of the spores of the two species was weakened as the manganese concentration increased. Amaha and Ordal (1957) empirically revealed that both calcium and manganese significantly enhanced the heat resistance of B. coagulans spores. Olivier et al. (2012) also reported that mineral supplementation, consisting of calcium, magnesium, and manganese, resulted in an increase in the heat resistance of B. coagulans spores compared to that of the spores formed under non-supplementation conditions. The previous studies are in agreement with the results obtained from supplementation with calcium, but contrary to the results obtained from supplementation with manganese in the current study. In B. subtilis, calcium has been well known to increase the heat resistance of spores (Bender and Marquis, 1985; Marquis and Bender, 1985; Oomes et al., 2007). However, the literature provides little information on the effect of manganese as a single mineral on the heat resistance of B. subtilis spores.

Interestingly, the lowest D_100°C-values of both B. coagulans spores and B. subtilis spores formed at each concentration of manganese were observed when calcium was not supplemented. With a few exceptions, the D_100°C-values generally rose as the calcium concentration increased. The results indicated that calcium is necessarily required to enhance the heat resistance of spores of both B. coagulans and B. subtilis, which is in agreement with the results obtained from the supplementation with calcium as a single mineral. In contrast, spores of both B. licheniformis and B. cereus did not exhibit such calcium concentration-dependent heat resistance. It is also worth mentioning that, unlike calcium, increasing the concentration of manganese often adversely affected the heat resistance of both B. coagulans spores and B. subtilis spores formed at each concentration of calcium, with a few exceptions. This is in accordance with the results obtained from the supplementation with manganese as a single mineral.

Taken together, depending on the response of spores to supplementation, the Bacillus spp. were split into two distinguished heat resistance groups: B. licheniformis and B. cereus; and B. coagulans and B. subtilis. In both B. licheniformis and B. cereus, the greatest heat resistance of spores was achieved on media without mineral sporulation, which did not significantly affect the heat resistance of B. coagulans spores and B. subtilis spores. Alternatively, the heat resistance of spores of both B. coagulans and B. subtilis was enhanced when sporulated on media with high levels (1.00–2.00 mM) of calcium supplementation, but that of B. cereus was significantly lowered under supplementation solely with 2.00 mM calcium.