Original papers
Control of Bacillus subtilis Spores by Intermittent Treatment Using Heating after Carbonation in the Presence of Germinants and Bacteriostatic Agents
2018 Volume 24 Issue 3 Pages 403-411
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2018 Volume 24 Issue 3 Pages 403-411
We investigated the inactivation effect of intermittent treatments using carbonation under heating (CH) and second heating (HT), in the presence of germinants or monoglycerol caprate (MC10) on Bacillus subtilis spores. Intermittent treatments by CH, followed by incubation and a second HT, in the presence of germinants showed up to 2.6 log-order inactivation effects on spores. Compared with the effects of glycine, sodium benzoate, and potassium sorbate, MC10 increased the inactivation effect of CH. MC10 decreased the D value and activation energy for inactivation of the spores. Intermittent treatments with MC10, conducted by a CH, followed by HT, inactivated spores by 3.6 log-orders. CH with MC10 induced the release of dipicolinic acid (DPA) and increased the stainability of the spores by DAPI. This suggests that germination-like phenomena, during CH, were included in the inactivation effects. Spores, which survived the intermittent treatment combined with MC10, did not proliferate in a liquid medium.
Bacterial spores are difficult to inactivate because they show high resistance to physical and chemical treatments such as heat, pressure, ultraviolet light, and chemical agents. Spores, which survive after inactivation treatments, germinate and proliferate, causing food spoilages. Retort treatments, of approximately 120°C for 30–60 min, are used in spore sterilization, but these treatments deteriorate food quality because of excessive heating.
Carbonation treatments are performed by dissolving carbon dioxide (CO2) into liquid foods under pressure. Although liquid food is temporarily acidified by the dissolved CO2, pH levels can be restored by removal of CO2 (Nakai et al., 2013). Furthermore, carbonation treatments have minor influence on food quality (Zhou et al., 2015). Carbonation treatments, under conditions yielding a supercritical state of CO2, can effectively inactivate bacterial spores (Watanabe et al., 2003); treatments under milder conditions show a high inactivation effect on bacterial vegetative cells (Klangpetch et al., 2011). Therefore, carbonation treatments can be used to inactivate microorganisms in neutral pH foods as an alternative to retort treatment. Although carbonation using gaseous CO2 exerted only weak inactivation effect on bacterial spores, treatment at 80°C and 5 MPa for 30 min induced an entire log-order inactivation effect on Bacillus subtilis spores (Noma et al., 2011).
Intermittent treatment is usually carried out in three steps. The first step is a sub-lethal heating of the bacterial spores, followed by incubation for several hours, and then a second heating. Spores, activated by the first heating step, germinate during the incubation period, and then these germinated spores are easily inactivated by second heating. Therefore, germinating the spores, before the second heating, is important for obtaining high inactivation effects by intermittent treatment.
Germination of bacterial spores is caused by sequential reactions of spore-lytic germination enzymes (Physiological germination; Foster and Johnstone, 1990). Germinants, including l-alanine and l-valine, activate spore-lytic germination enzymes. AGFK (composed of l-asparagine, d-glucose, d-fructose, and KCl) can efficiently germinate Bacillus subtilis 168 spores. Decrease in heat resistance, release of dipicolinic acid, increase in stainability, and decrease in optical density occur during spore germination. In addition, carbonation treatment increases the stainability of the spores with DAPI (Noma et al., 2011). This indicates that carbonation treatment enhances the permeation of substances with small molecular weights, such as germinants, into the spores. Carbonation treatment may increase the ratio of spores more likely to germinate physiologically after incubation, which decreases their heat resistance.
Nisin and fatty acid esters are widely used food additives with bacteriostatic activity against Bacillus spores (de Arauz et al., 2009; Nakayama et al., 2003). The addition of nisin enhances the inactivation effects of carbonation under heating on B. subtilis spores (Rao et al., 2016). The inactivation effect of carbonation under heating, on Bacillus and Geobacillus spores, is increased by the addition of fatty acid esters (Klangpetch et al., 2013; Nakai et al., 2014). These motivated us to investigate the inactivation effect of carbonation in the presence of various bacteriostatic agents.
In this study, we examined the inactivation effect of intermittent treatment by carbonation, subsequent incubation, and second heating in the presence of germinants, against B. subtilis spores. We also assessed the inactivation effect of carbonation, in the presence of several bacteriostatic agents, against B. subtilis spores. The bacteriostatic agent, with the highest inactivation effect, was used for intermittent treatment with no incubation.
Preparation of spore suspension B. subtilis MGNA-A001 (original name, B. subtilis 168) was obtained from the National Bioresource Project, Japan. Spores were formed by plating 100 µL of the stock spore suspension on nutrient agar (NA; Becton, Dickinson and Company, Sparks, USA) and incubating at 30°C for approximately 3 days. Spores were then harvested and washed three times by centrifuging at 10 000 × g at 4°C for 3 min in sterile deionized water. Then, residual vegetative cells were removed by a density-gradient centrifugation of 7 000 × g at 4°C for 15 min using Percoll Plus (GE Healthcare, Uppsala, Sweden), with pH adjusted to 7.0 using 0.1 N HCl. Purified spores were then washed three times by centrifuging at 10 000 × g at 4°C for 3 min in sterile deionized water. Phase contrast microscopy (ECLIPSE E600, Nikon, Tokyo, Japan) was used to ensure that the number of refractive spores, in the suspension, exceeded 90% of the bacterial population. After that, the spore suspension was stored at 4°C until use. B. subtilis spores were suspended in nutrient broth (NB; Difco, BD), and each individual germinant, l-asparagine, d-glucose, d-fructose, KCl, l-alanine, or l-valine, was added at a final concentration of 10 or 100 mM to the suspension. A mixed germinant, AGFK, was used at a final concentration of 0.4- or 4-fold more than the concentrations used in a study by Yamazaki et al. (1997). In another set of experiments, bacteriostatic agents, glycine (Wako Pure Chemical Industries, Ltd, Osaka, Japan), sodium benzoate (Nacalai Tesque, Kyoto, Japan), potassium sorbate (Nacalai Tesque), and monoglycerol caprate (MC10; Taiyo Kagaku Co., Mie, Japan), were added to the suspension at a final concentration of 0.7, 0.2, 0.2, and 0.05%, respectively, instead of the germinants.
Induction of changes in the optical density of spores by germinants The initial OD630 value, of spore suspensions with germinants, was set at approximately 0.8. Spore suspensions (150 µL) were placed in 96-well plates and incubated at 30°C for 0–3 h while shaking at 650 rpm (96-well plate shaker J849050, Greiner Bio One International GmbH, Kremsmünster, Austria). The OD630 of spore suspensions was measured using a microplate reader (CHROMATE4300, Awareness Technology, Inc., Palm City, USA). Relative OD630 was calculated using the following equation: relative OD630 (%) = N/N0 × 100, where N0 and N are the OD630 values before and after incubation, respectively.
Inactivation treatments Intermittent treatment (IT), in the presence of germinants, was performed by subjecting the spore suspension to carbonation with heating (CH), followed by incubation, and then a second heating (HT). Spore concentrations were adjusted to approximately 106 CFU/mL. CH was conducted at 40, 60, and 80°C and 5 MPa for 15 min, as described previously (Klangpetch et al., 2013). Briefly, 1.5 mL of the spore suspension in a test tube (φ10 mm×100 mm) was placed in a pressurization vessel (φ95 mm×135 mm), and heated at the desired temperature using a thermal bath (Kato Kagaku Co., Ltd., Tokyo, Japan). CO2 gas was introduced into the vessel until a pressure of 5 MPa was achieved via the CO2 gas cylinder. After CH, the spore suspensions were incubated at 30°C, for 0–24 h, while shaking at 250 strokes per minute (spm) using a shaker (MMS, EYELA, Tokyo, Japan). Immediately after incubation, HT was performed at 90°C for 5 min using a thermal block (MG-2000, EYELA).
CH, in the presence of each bacteriostatic agent, was performed at 80°C and 5 MPa for 30 min. Time-dependent inactivation behaviors were determined in the presence of MC10. HT and CH were performed at 70–95°C, for 0–30 min, and at 70–90°C and 5 MPa for 0–30 min, respectively. Intermittent treatment with no incubation (ITWNI), in the presence of MC10, was carried out by subjecting the spore suspension to HT2 immediately after CH, where CH was performed at 80°C and 5 MPa for 10 min, and HT2 was carried out at 90°C for 5 min. HT1 at 80°C for 10 min followed by HT2 was also performed for a control experiment of CH.
Determination of viable spores Viable spore counts were determined using the plate count method. Spore suspensions were diluted with sterile water, plated onto NA plates, and incubated at 30°C for 24 h. Then, the colonies were counted. The effects of food additives and CO2 solubilized into spore suspension on viable spore count are limited, because agitation and dilution decreased concentrations of CO2 and food additives.
D values and Arrhenius plot The decimal reduction time (D value) was defined as the treatment period required for reducing 90% of the viable count. D value was calculated from the reciprocal of the initial slope of inactivation curve. The death rate constant was calculated from the D value using equation (1), and energy of activation (Ea) for inactivation was calculated from the Arrhenius law (2):
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where A is the frequency factor, R is gas constant (8.31 J/mol·K), and T is temperature (K).
Determination of concentration of dipicolinic acid (DPA) The concentration of DPA, released from the spores, was measured using terbium chloride as previously described (Shibata et al., 1993). Spore concentration was adjusted to approximately 108 CFU/mL, and the spore suspension was centrifuged at 10 000 × g for 3 min. Then, 100 µL of the resultant supernatant were mixed with 100 µL of 1 mM terbium chloride solution (Nacalai Tesque) and 800 µL of 25 mM Tris/HCl buffer (pH 7.5). Then, fluorescence intensity was measured by a fluorescence spectrophotometer (RF-5300PC, Shimadzu, Kyoto, Japan) at an excitation wavelength of 282 nm and an emission wavelength of 544 nm.
4′,6-diamidino-2-phenylindole (DAPI) staining Spore concentration was adjusted to approximately 108 CFU/mL. Spores, subjected to CH, were stained with DAPI (Nacalai Tesque) as described previously (Noma et al., 2011). Stained spores were then imaged by a phase contrast microscope (BX50, Olympus Co., Tokyo, Japan) equipped with accessories for observing fluorescence (BH2-RFL-T3, U-ULS100HG, and BX-KLA; Olympus).
Estimation of bacteriostatic effect Spore suspension (1.5 mL), subjected to ITWNI in the presence of MC10, was incubated in a test tube (φ10 mm×100 mm) at 30°C for 5 d, with shaking at 200 spm, using the shaker. OD630 was measured with a microplate reader and used to estimate the degree of proliferation.
Statistical analysis Data are presented as mean ± standard deviation of experiments performed in triplicate. Significant difference was determined by t-test after performing an f-test (p < 0.05).
Germination behaviors of spores in the presence of germinants Achieving a higher inactivation effect by second heating, when using IT, requires an increase in the germination ratio of B. subtilis spores. Spores lose their refractive properties because of water incursion into the spores during germination (Hachisuka, 1988). This loss of refractility induces decreases in the turbidity of spore suspensions. Therefore, to determine incubation time and concentration of germinants required for spore germination, we used decreases in the turbidity of spore suspensions. Figure 1 shows the changes in relative OD630 after incubation for 0–3 h in the presence of each germinant. Addition of l-asparagine, d-glucose, d-fructose, or KCl, at 10 and 100 mM, did not affect the turbidity of spore suspensions. l-valine, at 100 mM, decreased turbidity; l-alanine, at 10 mM and 100 mM, decreased turbidity within 1 h to a maximum degree of 88 and 83%, respectively. AGFK, at 0.4 and 4 times the original concentration, reduced turbidity within 1 h to 91 and 85%, respectively. These results indicate that when using 10 mM of l-alanine or 0.4 times the concentration of AGFK, incubation times needed to exceed 1 h in order to induce germination in most spores. The decrease in turbidity was reversely increased after approximately 1 h of incubation in the presence or absence of germinants. This may be due to the proliferation of vegetative cells, obtained from outgrown spores, at an earlier time of incubation.
Change in relative OD630 of spore suspension during incubation in the presence of each germinant. Incubation was performed at 30°C for 0–3 h while shaking. Graph. (A) and (B) show 100 mM and 10 mM of each individual germinant, or 4 and 0.4 times of AGFK, respectively. No germinant (black diamonds), l-asparagine (white diamonds), d-glucose (black squares), d-fructose (white squares), KCl (black triangles), l-alanine (white triangles), l-valine (black circles), and AGFK (white circles).
Inactivation effect of IT using CH in the presence of germinants IT was performed by subjecting the spore suspension to CH at 40–80°C and 5 MPa for 15 min, followed by incubation at 30°C for 0–24 h, and then a subsequent HT at 90°C for 5 min (Fig. 2). To increase the ratio of germinated spores, incubation time was set to 3 and 24 h. l-alanine at 10 mM, and AGFK at 0.4 times the concentration, were used for this experiment. HT alone, expressed as “no CH”, represents the inactivation effect of HT in the presence or absence of germinants. Additionally, the difference in the viable count before and after germination corresponds to the number of germinated spores, since their heat resistance decreases after germination. Incubation of spores in the presence of the germinant for 3 and 24 h induced slightly higher inactivation effects, suggesting that incubation with the germinant did not confer a notable effect in inducing spore germination.
Viable spores after IT, in the presence of 10 mM l-alanine and AGFK at 0.4 times concentration. CH was performed at 40–80°C and 5 MPa for 15 min. Incubation was conducted at 30°C for 0–24 h. HT was carried out at 90°C for 5 min. Incubation periods and CH temperatures are shown in the graph. White, gray, and dark gray bars indicate no addition, l-alanine, and AGFK, respectively.
Spores subjected to CH, in the presence of l-alanine or AGFK, followed by incubation for 3 or 24 h and HT, were slightly more inactivated than spores without germinants. Increasing the temperature of CH tended to enhance the inactivation effects of the IT in the presence of germinants, suggesting that an increase in the CH temperature enhanced the number of germinated spores. However, the inactivation effects of the IT, using CH and germinants, were less than 2.6 log-orders.
Inactivation effects of CH in the presence of bacteriostatic agents To determine which bacteriostatic agent most enhanced the inactivation effect of CH, we performed CH at 80°C and 5 MPa for 30 min in the presence of different bacteriostatic agents (Fig. 3). The agents were added at the maximum concentration, which is usually reserved to achieve bacteriostasis in food manufacturing processes. CH, in the presence of glycine, sodium benzoate, or potassium sorbate, yielded approximately 2.4–2.8 log-order inactivation effects. CH, in the presence of MC10, had a 3.4 log-order inactivation effect. These results indicate that the addition of MC10 most efficiently augmented the inactivation effect of CH against the spores of B. subtilis. We also reported that HT alone (80°C and 0.1 MPa for 30 min) or pressurization alone (80°C and 5 MPa for 30 min, pressurized by N2 gas) in the presence or absence of MC10 induced no inactivation effects (Noma et al., 2015). These results showed that the inactivation effect in the presence of MC10 was demonstrated not only by heating or pressurization but by CH.
Effects of bacteriostatic agents on inactivation effect of CH. CH was performed at 80°C and 5 MPa for 30 min. Concentration of glycine, sodium benzoate, potassium sorbate, and MC10 was 0.7, 0.2, 0.2, and 0.05%, respectively. White and gray bars indicate the levels before and after CH, respectively.
Figure 4 shows the inactivation curve for B. subtilis spores, subjected to HT at 70–95°C for 0–30 min, or to CH at 70–90°C and 5 MPa for 0–30 min, in the presence of MC10. D value was calculated from the initial slope of the inactivation curve because it reflected the resistance of most of the spores in the culture (Table 1). At 70 and 80°C, the D values for CH were lower than those for HT; D values for CH, in the presence of MC10, were approximately 4 times lower than those for CH in the absence of MC10. These results indicate that CH, in the presence of MC10, induced the largest death rate in B. subtilis spores. Arrhenius plots of the logarithm of the rate constants, for inactivation of B. subtilis spores by HT or CH in the presence of MC10, are shown in Fig. 5. Ea, for inactivation of B. subtilis spores, was calculated from the slopes of the regression lines between lnk and 1/T (Table 2). The values of Ea for CH were lower than those for HT; Ea for CH, in the presence of MC10, was lower than that for CH in the absence of MC10. Therefore, the energy, required for inactivation of B. subtilis spores with CH, was decreased by the addition of MC10.
Time-dependent changes in viable spores after HT (A) and CH (B) in the presence or absence of MC10 at 0.05%. Closed and opened symbols indicate “in the absence of MC10” and “in the presence of MC10”, respectively. Symbols denote: diamond, 70°C; square, 80°C; circle, 85°C; triangle, 90°C; reverse triangle, 95°C.
| MC10 | Temperature (°C) | |||||
|---|---|---|---|---|---|---|
| (0.05%) | 70 | 80 | 85 | 90 | 95 | |
| HT | − | 169 | 45.7 | 19.6 | 2.51 | 0.857 |
| + | 96.2 | 41.7 | 16.8 | 1.80 | 0.889 | |
| CH | − | 35.7 | 18.1 | * | 2.63 | * |
| + | 8.40 | 4.38 | * | 2.32 | * | |
Arrhenius plots of the logarithm of the rate constant for inactivation of B. subtilis spores by HT (A) and CH (B). Closed and opened symbols indicate “in the absence of MC10” and “in the presence of MC10”, respectively.
| MC10 | Ea | |
|---|---|---|
| (0.05%) | (kJ/mol) | |
| HT | − | 227 |
| + | 208 | |
| CH | − | 134 |
| + | 66.7 |
Inactivation effect of ITWNI in the presence of MC10 The inactivation rate was decreased after a certain treatment period. This phenomenon is called tailing. Because of tailing, we did not expect the addition of MC10 to further increase the inactivation effect of CH (Fig. 4). In addition, we previously reported that CH in the presence of MC10 inhibited germination of B. subtilis spores (Noma et al., 2015). Therefore, we investigated the inactivation effect of ITWNI, CH at 80°C and 5 MPa for 10 min, without incubation, followed by HT2 at 90°C for 5 min, in the presence of MC10 (Fig. 6). Regardless of the presence or absence of MC10, inactivation effects of CH alone, and HT2 alone were ≤ 1 log-order. In the presence and absence of MC10, HT1 followed by HT2 yielded an inactivation effect of approximately 2 log-orders. In the absence of MC10, ITWNI yielded 1.5 log-order inactivation effects. In the presence of MC10, ITWNI had a 3.6 log-order inactivation effect. These results indicate that ITWNI, in the presence of MC10, can induce higher inactivation effect than HT2 or CH alone. The inactivation effect of the ITWNI, obtained in 15 min, was equivalent to CH applied for 30 min, in the presence of MC10.
Viable spores after ITWNI, in the presence of MC10 at 0.05%. CH was performed at 80°C and 5 MPa for 10 min, and HT2 was carried out at 90°C for 5 min. HT1 was carried out at 80°C for 10 min. White and gray bars indicate “in the absence of MC10” and “in the presence of MC10”, respectively. * and ** indicate significant difference at p < 0.05 and 0.01, respectively in Welch's t-test.
DPA release and DAPI staining of B. subtilis spores after CH in the presence of MC10 To investigate the mechanisms driving the high inactivation effects of ITWNI, we examined the influence of MC10 on spore germination via CH. This was conducted by examining DPA release and DAPI staining. Figure 7 shows the effects of CH and HT on DPA release in the presence of MC10. Untreated spores, and those after treatment with HT, released 0.4–0.6 µg/mL and 1.1 µg/mL of DPA, respectively. MC10 did not affect the levels of DPA release. In addition, CH with MC10 induced 3.1 µg/mL of DPA release, whereas CH without MC10 led to a release of 0.9 µg/mL DPA in the spores. Consequently, CH, in the presence of MC10, induced an approximately 3.4-fold greater DPA release from the spores than it did in the absence of MC10 (p < 0.01). This indicates that a combination of CH and MC10 physically triggered the release of DPA.
DPA concentration of spore suspension after HT and CH in the presence of MC10 at 0.05%. CH was performed at 80°C and 5 MPa for 10 min and HT was carried out at 80°C for 10 min. White and gray bars indicate “in the absence of MC10” and “in the presence of MC10”, respectively. ** significant difference of p < 0.01 in Welch's t-test.
B. subtilis spores, subjected to CH and MC10, were stained with DAPI and imaged under a fluorescence microscope (Fig. 8). For untreated spores, a small number of spores was DAPI positive; however, most spores after HT were stained with DAPI. MC10 did not affect the DAPI stainability of the spores in untreated and HT spore groups. CH, combined with MC10, induced strong stainability in most spores, whereas spores treated with CH without MC10 were poorly stained with DAPI. These results demonstrate that CH, in the presence of MC10, accelerated germination more efficiently than did HT.
DAPI staining of spores subjected to no treatment, HT, and CH, in the presence of MC10 at 0.05%. CH was performed at 80°Cand 5 MPa for 10 min, and HT was carried out at 80°C for 10 min.
Bacteriostatic effect after ITWNI in the presence of MC10 Figure 9 shows the change in OD630 during a 5 days incubation of a spore suspension subjected to the ITWNI in the presence of MC10. In the absence of MC10, increases in OD630 were observed. However, in the presence of MC10, OD630 did not change, indicating the inhibition of spore growth.
Change in OD630 values of spore suspensions in the presence of 0.05% MC10 after incubation at 30°C for 5 d. ITWNI was performed by CH at 80°C and 5 MPa for 10 min followed by HT2 at 90°C for 5 min. White and gray bars indicate “in the absence of MC10” and “in the presence of MC10”, respectively.
This study investigated the inactivation effects of intermittent treatment, using CH and HT, in the presence of germinants or the bacteriostatic agent MC10, on the spores of B. subtilis. The results indicate that the presence of germinants during CH, combined with an incubation period (IT), did not increase the inactivation effects of a second HT. Conversely, intermittent treatments with no incubation (ITWNIs) significantly increased the inactivation effects of either CH or HT in the presence of MC10. The bacteriostatic effect of MC10 was observed on spores subjected to the ITWNI.
The optimal pH for physiological germination is between 7 and 9, while the optimal pH for spore-lytic germination enzymes is between 5 and 8 (Adbelmadjid and Foster, 2001). The pH of the spore suspension decreased to approximately 3.2 during CH (Spilimbergo et al., 2005), recovered to approximately 5.6 just after CH, and reached a neutral pH during incubation with shaking (Nakai et al., 2013). This indicates that a CH-mediated pH change of the spore suspension did not inhibit physiological germination. We, therefore, expected that the germinants would trigger physiological germination, and CH-mediated permeation of nutrient components would accelerate spore germination during post-CH incubation. However, IT using CH, in the presence of germinants, yielded up to only 2.6 log-order inactivation effects (Fig. 2). There are two possible explanations for the disappointing inactivation effects. It is possible that the pH of the spore suspension was recovered to a neutral pH, while the intraspore pH remained unrecovered. Another likely reason is that germination receptors were impaired by CH and could not contribute to initiating a germinant-mediated physiological germination. Furthermore, B. subtilis spore germination is usually enhanced by the addition of germinants, whereas B. coagulans spores, frequently detected from various kinds of foods, are not easily germinated. Therefore, IT is not a promising method for inactivation of spores of various Bacillus species in food.
The inactivation effect of high-pressure treatment against B. subtilis spores is increased by the addition of sodium benzoate (Balasubramanian and Balasubramaniam, 2003). Neosartorya fischeri is inactivated to a greater degree by heating in the presence of sodium benzoate or potassium sorbate (Rajashekhara et al., 2000). Glycine is usually used for bacteriostasis of Bacillus spores. Moreover, MC10 enhances the inactivation effect of heating at ultrahigh temperatures on the spores of G. stearothermophilus (Fujimoto et al., 2006). The glycerin fatty acid ester yielding the strongest inactivation effect of CH was MC10 (Hirokado et al, 2018). We observed that the increases in the inactivation effect of CH by the addition of 0.05% sucrose fatty acid esters (fatty acid molecule(s) with 12–18 carbon chain length is ester-bonded with one sucrose molecule) were ≤ 0.5 log-order (data not shown). These results motivated us to choose MC10 as a representative emulsifying agent in this experiment. We assessed the effects of four kinds of bacteriostatic agents on inactivation of B. subtilis spores by CH. We found that the addition of these bacteriostatic agents increased the inactivation effect of CH. MC10 most efficiently enhanced the inactivation effect of CH (Fig. 3) and contributed to decreasing the activation energy of B. subtilis by CH (Table 2). Therefore, the intermittent treatment performed via CH followed by HT (ITWNI) was performed in the presence of MC10.
The inactivation effect of ITWNIs, in the presence of MC10, was 3.6 log-orders (Fig. 6). Physiological germination was not included for this inactivation effect because incubation was not performed between CH and the second HT. However, in the experiments shown in Figs. 7 and 8, CH combined with MC10 induced DPA release and increased the DAPI stainability of the spores; CH without MC10 did not produce the same effect. These results suggest that CH, in the presence of MC10, physically caused the phenomena observed in the spores, which was similar to physiological germination. Therefore, the addition of MC10 may have accelerated the physical germination, induced by CH, producing high levels of spore inactivation. Previously, we showed that the DAPI stainability of B. subtilis spores suspended in water was enhanced after CH (Noma et al., 2011). This was possibly due to the easier physical germination in water (when compared to NB) mediated by the pH downshift. The induction of the germination-like phenomena during CH may not be affected by the physiological properties of spores associated with ease of germination, because we found that CH in the presence of MC10 inactivated spores of B. coagulans, B. cereus, B. licheniformis, and Geobacillus stearothermophilus (Klangpetch et al., 2013). We intend to investigate the inactivation effect of ITWNI in the presence of MC10 against the spores of various Bacillus species in the future.
Monoglycerol fatty acid esters possessing medium chain lengths, such as MC10, exert a bacteriostatic effect, inhibiting the growth of bacterial spores (Kimsey et al., 1981). The viable number of B. subtilis spores does not increase during a 30-day storage, after treatment with CH combined with fatty acid esters (Klangpetch et al., 2013). In this study, MC10 also inhibited the growth of B. subtilis spores before and after the ITWNI. Nakayama et al. (2015) described that MC10 treatment of B. subtilis spores at pH 8.0 resulted in half of the minimum bactericidal concentration of MC10 at pH 6.0. The pH value before and after ITWNI in the presence of MC10 was 6.9 and 6.4, respectively. The difference in pH values before and after ITWNI in the presence of MC10 was smaller in this study than in the 2015 report, and ITWNI did not decrease MC10 concentration (data not shown). Therefore, we feel that the bacteriostatic effect of MC10 was not inhibited by pH change and MC10 degradation through ITWNI.
Release of DPA was promoted by CH (80°C, 5 MPa, 10 min) in the presence of MC10 (Fig. 7). This accelerated release may have resulted from damage to the inner membrane of spores (Hirokado et al., in press). HT (80°C, 10 min) also tended to enhance the DPA release (Fig. 7). DPA release is physiologically controlled in the germination process of intact Bacillus spores. Therefore, we inferred that CH in the presence of 0.05% MC10 impaired the physiologically controlled release of DPA, resulting in bacteriostasis. Nakayama et al. (2015) described that MC12 at minimum bactericidal concentration did not affect the germination of Bacillus spores, whereas it inactivated vegetative cells generated after outgrowth. Therefore, it is inferred that the mechanism of action of MC10 is different from that of MC12.
In conclusion, our results indicate that ITWNI via CH followed by HT effectively increased the inactivation effect of CH against B. subtilis spores when combined with the bacteriostatic agent MC10. The inactivation effects of ITWNIs, combined with MC10, did not reach 6 log-orders, which is a criterion for sterilization; however, within the tested conditions, the treated spores did not proliferate during the incubation that followed. This enables the distribution of food under normal temperature. Additionally, the second HT caused an increase in the inactivation effects and can remove the remaining CO2 gas from the food. Therefore, ITWNIs combined with MC10 can be potentially used as an alternative method to extend the preservation period of retort food with high water content, such as meat extract and chicken soup. However, the effects of fatty acid esters on the inactivation effects of ITWNI may vary among Bacillus species. Determination of the inactivation effect of ITWNI in the presence of MC10 on the other Bacillus species is required before this method may be used in practical applications.
Acknowledgments Part of this work was supported by the Japan Society for the Promotion of Science (grant numbers 26350094 and 17K00818). The authors are thankful to Taiyo Kagaku Co. for providing MC10.
