2018 Volume 24 Issue 3 Pages 455-463
We investigated the effect of carbonation treatment with heating (CH) in the presence of glycerin fatty acid esters on Bacillus subtilis spores. The inactivation effect of CH was the highest in the presence of monoglycerol monocaprate (MC10). Monoglycerol monolaurate showed the highest bacteriostatic effect. The resistance of spores to CH with MC10 did not change with pre-heat activation. Freshly prepared spores from the surviving spores following CH with MC10 showed resistance similar to that of normal spores. The resistance of spores might be affected by the composition of the spore-forming medium. CH with MC10 enhanced adhesion of MC10 to spores and dipicolinic acid (DPA) release from spores, but did not induce DNA fragmentation. Inactivation of B. subtilis spores by CH with MC10 may be attributable to the impairment of physiological germination and/or decrease in heat resistance, which were induced by enhanced MC10 adhesion and the resultant DPA release.
Bacterial spores are highly resistant to various stresses such as heat, pressure, drugs, and ultraviolet rays. The spores that survive the stresses in the production of food products can germinate and proliferate, causing food spoilage. Currently, retort treatment is used as a sterilization method for food. However, the excessive heating of this method causes deterioration of food quality and loss of nutritional components. Carbonation treatment, performed by dissolving CO2 into a liquid food under pressure, has been proposed as an alternative to heat treatment. It has been reported that carbonation treatment has the potential to sterilize vegetative cells at a lower temperature than heat treatment (Rao et al., 2016). However, carbonation treatment has a poor inactivation effect on bacterial spores; treatment at 80°C, 5 MPa for 30 min yielded only a 1 log-order inactivation effect on Bacillus subtilis spores (Noma et al., 2011).
Glycerin fatty acid ester (FAE) is a compound in which a fatty acid molecule is ester-linked to a glycerin molecule. FAEs are widely used as food additives and are known to have a bacteriostatic effect (Shibasaki, 1979). In our previous studies, we observed a > 3 log-order inactivation effect on Bacillus and Geobacillus spores when carbonation under heating (CH) was performed in the presence of 2 mM monoglycerol caprate (MC10), an FAE (Klangpetch et al., 2013; Nakai et al., 2014). However, the bactericidal mechanism of the combination treatment of carbonation with MC10 against bacterial spores remains unknown. To increase the bactericidal effect of this combination treatment, elucidation of the underlying inactivation mechanism is required.
In this study, we aimed to investigate the bactericidal effect of CH in the presence of various FAEs, understand factors involved in the resistance of B. subtilis spores to this combined treatment, and elucidate the inactivation mechanism of B. subtilis spores with respect to the adhesion of FAE and spore injury.
Preparation of spore suspension Bacillus subtilis MGNA-A001 (original number, 168) was used in this study. For spore formation, the stocked spore suspension was plated on nutrient agar (NA; Difco, BD, Sparks, USA) plates after heating at 85°C for 30 min, and then incubated at 30°Cfor approximately 3 d. Spore formation (> 90%) was confirmed using a phase contrast microscope (ECLIPSE E600, Nikon, Tokyo, Japan). To determine the effects of spore forming media on spore resistance, spores were formed on Schaefer's medium and in modified G medium by the methods reported previously (Schaefer et al., 1965; Stewart and Halvorson, 1953). The spores formed were washed three times in sterilized water by centrifugation at 10 000 × g at 4°C for 3 min. The spore suspension was purified using Percoll PLUS (GE Healthcare, Uppsala, Sweden) with the pH adjusted to 7.0. The spores were washed three times as before. The spores were suspended in nutrient broth (NB; Difco, BD) to give a spore concentration of approximately 106 CFU/mL. Each glycerin fatty acid, summarized in Table 1, was added to the spore suspension at a final concentration of 0.1, 0.2, 1.0, or 2.0 mM.
| FAE | Abbreviation | Glycerin*1 | Fatty acid*2 | Molecular weight | HLB*3 |
|---|---|---|---|---|---|
| Monoglycerol monocaprate | MC10 | 1 | 10 | 246 | 6.5 |
| Monoglycerol monolaurate | MC12 | 1 | 12 | 274 | 5.3 |
| Monoglycerol monostearate | MC18 | 1 | 18 | 358 | 4.1 |
| Diglycerol monolaurate | DC12 | 2 | 12 | 348 | 8.5 |
| Decaglycerol monocaprylate | Dec8 | -*4 | 8 | 876*5 | 16.1 |
| Decaglycerol monolaurate | Dec12 | -*4 | 12 | 932*5 | 14.7 |
Heat treatment and carbonation treatment under heating For heat treatment (HT), the spore suspension (1.5 mL) in a test tube (φ13 × 100 mm) was immersed in a thermal bath (MG-2000, TOKYO RIKAKIKAI Co., Ltd., Tokyo, Japan) at 80°C. For carbonation treatment under heating (CH), the spore suspension (1.5 mL) in the tube was immersed in water at 80°C in a CO2-dissolving vessel. CO2 gas was introduced at 5 MPa and dissolved in the spore suspension with stirring. After standing for 30 min, the CO2 gas in the vessel was released via a pressure-control valve. The equipment for carbonation is described in a previous paper (Noma et al., 2011).
Heat activation of spores The spore suspension was incubated at 80°C for 10 min in the thermal bath as with the HT method.
Surviving spore count Each treated spore suspension was serially diluted with sterile water, and 100 µL of this was plated onto NA. After incubation at 30°C for 24 h, the number of viable spores was enumerated as CFU by colony counting.
Estimation of FAE bacteriostatic activity A spore suspension treated with each FAE at 0.1 mM was incubated at 30°C in a microtiter plate with shaking at 700 rpm using a microplate shaker (96-well plate shaker J849050, Greiner Bio One International GmbH, Kremsmünster, Austria). The change in OD630 was used as a criterion for proliferation and was measured after incubation at 30°C for 18 h by a microplate reader (CHROMATE4300, Awareness Technology, Inc., Palm City, USA).
Analysis of MC10 adhesion to spores Spores subjected to either HT or CH in the presence of 2 mM MC10 were washed three times in sterile water by centrifugation at 10 000 × g at 4°C for 3 min to remove un-adhered MC10. To recover MC10 molecules adhered to spores, a 50% ethanol solution and 30 mg of glass beads (0.038–0.053 µm) were added to the resultant spore pellet and the mixture was vortexed for 8 min (VORTEX-GENIE 2, Scientific Industry Inc., Bohemia, USA). The suspension was then heated at 95°C for 5 min and centrifuged at 10 000 × g at 4°C for 3 min. Derivatization of MC10 by 3,5-dinitrobenzoyl was performed according to a previous report (Murakami et al., 1988), with slight modifications. The supernatant was collected in a glass test tube, and 1.5 mL of dichloroethane (DCE) was added. The mixture was vortexed at the maximum level for 5 s and allowed to stand until the mixture separated into two layers; 1200 µL of the DCE layer was transferred to a sample tube and dehydrated with anhydrous sodium sulfate, and 700 µL of the solution was dried using a centrifugal evaporator (VC-15SP, Taitec, Saitama, Japan). After the residue was dissolved in 600 µL of DCE, 8 mg of 3,5-dinitrobenzoyl chloride and 625 µL of triethylamine-dichloroethane solution (4% triethylamine) were added and heated at 55°C for 15 min. After the solution was dried using a centrifugal evaporator, the residue was dissolved in tetrahydrofuran-acetonitrile solution (1:1). The solution was filtered and used for RP-HPLC analysis. RP-HPLC consisted of an L-6200 intelligent pump, L-5090 degasser, and L-2400 UV detector (Hitachi, Tokyo, Japan). The mobile phase consisted of pure water (solvent A) and acetonitrile (solvent B), and the elution was performed with a programmed gradient from 65% (0 min) to 100% (5 min) of solvent B. Separation was achieved on an Atlantis T3 analytical column (3 µm, 2.1 mm × 100 mm, Waters, Milford, USA) at a flow rate of 0.5 mL/min and 25°C, with detection performed at 230 nm. The MC10 concentration was determined by calculating the peak area of the derivative.
Dipicolinic acid (DPA) release The DPA concentration was determined according to a previously reported method (Shibata et al., 1993). Briefly, the spore suspension (about 108 CFU/mL) subjected to CH with MC10 was centrifuged at 10 000 × g for 3 min, and the resulting supernatant (100 µL) was mixed with both 100 µL of 1 mM terbium chloride (Nacalai Tesque, Kyoto, Japan) solution and 800 µL of 25 mM Tris/HCl buffer (pH 7.5). Fluorescence intensity of this solution was measured using a fluorescence spectrophotometer (F-7000, Hitachi High-Technologies Co., Tokyo, Japan) at an excitation wavelength of 282 nm and an emission wavelength of 544 nm.
Analysis of spore DNA fragmentation Spore DNA was prepared using the NucleoSpin Microbial DNA kit (MACHEREY-NAGEL GmbH & Co. KG., Düren, Germany). The prepared DNA was subjected to agarose gel electrophoresis. The agarose (for ≥ 1 kbp fragments, Nacalai Tesque) gel was prepared in TAE buffer at 0.5%. The marker used was the 1 kbp DNA Ladder One (Nacalai Tesque). Electrophoresis was performed using a Mupid-2plus (Mupid Co., Ltd., Tokyo, Japan) at room temperature and 100 V for 40 min.
Statistical analysis The data presented are the mean ± standard deviation of three replicated experiments. A student's t test after the f test and Tukey's test were employed to determine statistically significant differences (p < 0.01 and 0.05).
Effect of each FAE on the inactivation of B. subtilis spores by CH The sporicidal effect of CH (80°C and 5 MPa for 15 min) on B. subtilis spores in the presence of various FAEs at 2 mM was investigated (Fig. 1A). The highest sporicidal effect was observed in the presence of MC10, a monoglycerin molecule ester-linked to one molecule of capric acid with 10 carbon atoms. The inactivation effect decreased with elongation of the fatty acid carbon chain within the monoglycerol fatty acid esters. An increase in the glycerin chain length appeared to decrease the inactivation effect. The effects of the hydrophile-lipophile balance (HLB) and molecular weight of the FAE on inactivation are presented in Fig. 1B and 1C, respectively. These data were calculated from the results shown in Fig. 1A. It is likely that the optimal HLB value, 6.5, enhanced the inactivation effect of CH (Fig. 1B). An increase in the molecular weight tended to decrease the inactivation effect of CH (Fig. 1C).
Surviving spores after treatment with CH with 2 mM of each FAE (A). Effects of HLB (B) and molecular weight (C) of the FAE on the inactivation effect of CH on B. subtilis spores. Change in OD630 values of untreated spore suspensions in the presence of 0.1 mM of each FAE after incubation at 30°C for 18 h (D). Columns in Fig. A denote spores untreated (white) and treated by CH with FAE (gray). CH was performed at 80°C, 5 MPa for 30 min. N0 and N denote surviving spore number before and after CH, respectively (Figs. B and C). Symbols “*” and “**” represent significant difference vs. the “not added” control of p < 0.05 and p < 0.01, respectively
The bacteriostatic effect of treatment with each FAE at 0.1 mM on previously untreated B. subtilis spores is presented in Fig. 1D. The strength of the bacteriostatic effect of most FAEs appeared to be correlated with their inactivation effect (Fig. 1A). However, MC12 showed a dramatically higher bacteriostatic effect than the other FAEs, completely inhibiting the increase in OD630.
Effect of heat activation on spore resistance to CH with MC10 The influence of heat activation on spore survival following CH with MC10 is shown in Fig. 2. After heat activation of the spores at 80°C for 10 min, the combination of CH with MC10 was performed at 80°C and 5 MPa for 30 min. The number of surviving spores following heat activation was similar to that without heat activation. This result suggests that heat activation does not affect the resistance of spores to CH with MC10.
Effect of heat activation on survival of B. subtilis spores after CH with or without MC10. Non-activated (white) and activated (gray) spores were used. Heat activation was carried out at 80°C, 0.1 MPa for 10 min. HT was carried out at 80°C, 0.1 MPa for 30 min. CH was performed at 80°C, 5 MPa for 30 min. MC10 was added at 1 mM.
Characterization of surviving spores Figure 3A shows the effects of single and double treatments of CH with MC10 on surviving spores. An approximately 3 log-order of spore inactivation was achieved after the first CT with MC10; however, the inactivation effect was not notably enhanced by a second treatment performed immediately after the first. These results suggest the existence of highly resistant spores that can survive even after repeated treatments of the spore suspension. The spores that survived after the first treatment were plated on NA for re-sporulation, and the freshly prepared spores were subjected to CH with MC10 under the same conditions (Fig. 3B). The re-sporulated spores did not show higher resistance than the original spores, suggesting that the spores surviving after CH with MC10 do not have a genetically higher resistance to CH with MC10.
Effect of repeated CH with MC10 treatments on the survival of B. subtilis spores (A). Survival of normal and re-sporulated spores after CH with MC10 (B). Spores were either left untreated (white) or treated (gray) by CH with FAE. CH was performed at 80°C, 5 MPa for 30 min. MC10 was added at 2 mM.
Effect of sporulation medium on spore resistance The spores formed on NA, in modified-G medium, and in Schaefer's spore-forming medium were subjected to CH (80°C, 5 MPa, 30 min) with MC10 (1 mM), and the numbers of surviving spores were compared (Fig. 4). After HT with and without MC10, spores formed in the modified-G medium showed the highest resistance, followed by the spores formed in Schaefer's medium. Similar resistance patterns were observed after CH without MC10. However, the degree of difference between the spore formation conditions became more notable after CH with MC10, with the spores formed in the modified-G medium showing prominently higher resistance. Therefore, the sporulation medium can affect spore resistance to CH with MC10.
Survival of spores sporulated using NA (white), modified-G medium (light gray), or Schaefer's medium (dark gray) after CH with or without MC10. CH was performed at 80°C, 5 MPa for 30 min. MC10 was added at 1 mM.
MC10 spore adhesion After spores were subjected to HT or CH in the presence of 2 mM MC10, adhered and/or penetrated MC10 molecules were recovered in ethanol, and their concentrations were determined (Fig. 5). Adhesion of MC10 was not significantly enhanced by HT, but tended to be increased by HT (p = 0.063). The degree of MC10 sorption after CH with MC10 was notably higher than that after HT with MC10.
Adhesion of MC10 to spores after HT and CH in the presence of 2 mM MC10. HT was carried out at 80°C, 0.1 MPa for 30 min. CH was performed at 80°C, 5 MPa for 30 min. Different characters (a vs. b) show the significant difference determined by Tukey's test (p < 0.05).
DPA release The effects of HT and CH on the DPA release from B. subtilis spores are shown in Fig. 6. In untreated spores, the addition of MC10 did not affect the DPA release from B. subtilis spores. HT increased the DPA release, and the release was significantly higher with MC10 treatment than without it. The DPA release after CH without MC10 was similar to the release after HT with MC10. The DPA release after CH was significantly increased by the addition of MC10.
DPA release from spores subjected to HT and CH with or without MC10. HT was carried out at 80°C, 0.1 MPa for 30 min. CH was performed at 80°C, 5 MPa for 30 min. MC10 was added at 2 mM. Significant differences were determined by the Student's t test (*p < 0.05, **p < 0.01) after the f test.
DNA fragmentation DNA was extracted from spores after CH with MC10 and subjected to agarose gel electrophoresis (Fig. 7A). The CH conditions and the accompanying MC10 concentrations were selected to obtain approximately a 1 log-order inactivation effect (Fig. 7B). No DNA fragmentation appeared to be induced by the treatments.
Agarose gel electrophoresis of DNA extracted from B. subtilis spores subjected to CH (A). Survival counts of B. subtilis untreated spores (white) or spores after CH (gray) (B). CH was performed at 80°C, 5 MPa. Lanes in panel A: 1, marker; 2, untreated spores without MC10; 3, untreated spores with 2 mM MC10; 4, spores treated with CH for 30 min without MC10; 5, spores treated with CH for 15 min with 1 mM MC10; 6, spores treated with CH for 5 min with 2 mM MC10.
We previously showed that the inactivation effect of CH on B. subtilis spores was increased by the addition of FAEs, including MC10 and MC12 (Klangpetch et al., 2013). In the present study, we investigated the inactivation effect of CH with a greater variety of FAEs on B. subtilis spores and explored factors related to spore resistance. In addition, we addressed the mechanism of inactivation of B. subtilis spores by CH with MC10. Our results can be summarized as follows. First, the FAE most effective at increasing the sporicidal effect of CH was MC10, while MC12 showed the strongest bacteriostatic effect. Second, spore resistance to CH with MC10 was potentially impacted by the spore-forming conditions used, including the composition of the spore-forming medium. Third, three inactivation models for CH with MC10 can be proposed based on our data: 1) CH increased the surface hydrophobicity of spores, resulting in enhanced sorption of MC10 to the spores; 2) The inner membrane of the spores was injured, and the DPA within the core was released; and 3) the DPA release induced impairment of physiological germination and/or decrease in the heat resistance, resulting in a loss of proliferative ability.
Inactivation effect of CH with FAE The highest inactivation effect was observed when MC10 was used with CH; however, the FAE with the highest bacteriostatic effect was MC12. The inactivation effect of CH with monoglycerol esters tended to decrease with the elongation of the fatty acid chain, whereas a similar tendency was not observed for the strength of the bacteriostatic effect. In addition, the inactivation effect of CH with MC12 was similar to that of CH with DeC12; however, the bacteriostatic effect of MC12 was greater than that of DeC12. Therefore, it is suggested that the increase in inactivation effect with an FAE is not always correlated with its bacteriostatic effect. An HLB of 6.5 (MC10) appeared to be optimal for enhancing the inactivation effect of CH, and increases in the molecular weight of FAE appeared to weaken the inactivation effect. Therefore, we concluded that the ideal FAE is characterized by an optimal HLB for accessing the spores and a small molecular weight for penetrating the spores.
Factors influencing spore resistance to CH with MC10 Heat treatment under sub-lethal conditions reversibly activates bacterial spores, making them more likely to induce physiological germination. In addition, heat activation induces the rearrangement of spore coat proteins by the protease YabG and the transglutaminase Tgl, also located on the spore coat (Kuwana, 2009). Germination triggered by a germinant such as L-alanine occurs rapidly and completely following activation. The results in the present study indicate that heat activation before CH with MC10 did not alter the resistance of B. subtilis spores to CH with MC10 (Fig. 2), indicating that the substantial changes mediated by heat activation did not contribute to spore resistance.
The spores surviving after the first CH with MC10 were not completely inactivated by a second CH with MC10 performed immediately after the first (Fig. 3A). We also noted that the spore inactivation rate decreased after a certain time when determining the time-dependent changes in spore survival following CH (80°C, 5 MPa) with MC10 (data not shown). These results indicate that the surviving spores had adequate resistance to CH with MC10 under these conditions. During inactivation treatment under milder conditions, a decrease in the inactivation rate is sometimes observed after a certain period. This phenomenon is called tailing. The mechanism of tailing has been well-studied using vegetative bacteria. Humpheson et al. (1998) reported that tailing in Salmonella enteritidis PT4 during the thermal inactivation curve may be induced by the de novo synthesis of proteins, such as heat shock proteins, during heat treatment. Welch et al. (1993) noted that the synthesis of heat shock- or cold shock-inducible proteins is accelerated during a pressure up-shift from atmospheric pressure to about 55 MPa. However, bacterial spores are in a dormant state, and such vigorous protein synthesis is unlikely to be induced. On the other hand, Hauben et al. (1997) obtained pressure-resistant E. coli MG1655 cells by subjecting the cells to repeated high-pressure treatments. We have also confirmed that E. coli O157:H7 cells surviving in the tail portion have markedly high resistance to hydrostatic pressure treatment (Noma et al., 2006). These reports motivated us to investigate whether the spores surviving after CH with MC10 indicate the low probability occurrence of genetically resistant spores present in the spore suspension. However, freshly prepared spores from the population of spores surviving after CH with MC10 were inactivated to a similar degree by CH with MC10 as the normal spores (Fig. 3B). A possible explanation for the high resistance of the surviving spores is that the limited heterogeneity of conditions for spore formation, such as temperature, oxygen, and available nutrients, yielded only a small number of spores with high resistance.
It is considered that the conditions for spore formation, such as temperature, humidity, oxygen, and available nutrients, might not be completely uniform among spores on NA. The results of Fig. 4 showed that differences in the composition of the spore forming medium affected the resistance of B. subtilis spores to CH with MC10. This result may provide evidence that the conditions during spore formation significantly affect spore resistance. We also observed that spores formed in modified-G medium contained protein(s) at higher expression levels. Analyses of the effect of the composition of the spore-forming medium and the protein(s) may contribute to clarification of spore resistance to CH with MC10 in the future.
The mechanism of B. subtilis spore inactivation by CH with MC10 HT is known to increase the surface hydrophobicity of spores and cause the formation of spore clumps (Furukawa et al., 2005; Sakaguchi and Amaha, 1951; Toda and Aiba, 1966). We found that CH (80°C, 5 MPa for 30 min) enhanced the hydrophobicity of the spore surface more so than did treatment with heat and hydrostatic pressure. This increase in surface hydrophobicity promoted the formation of spore clumps as well as the attachment of spores to hydrophobic surfaces (Noma et al., 2018). Faille et al. (2001) showed that Bacillus spores with high hydrophobicity adhere to hydrophobic surfaces in custard. MC10, a hydrophobic FAE with an HLB of 6.5, showed greater spore adhesion after CH than after HT (Fig. 5). In addition, we demonstrated that CH induced a germination-like phenomena, such as an increase in DAPI staining and a decrease in the heat resistance of the spores (Noma et al., 2011); this indicates that CH increased the permeability of spores to substances, including MC10.
DPA exists within the spore core with chelating Ca2+ ions, and contributes to decreases in the core water content and resistance to hydrogen peroxide, formaldehyde, and iodine-based disinfectants (Paidhungat et al., 2000). In addition, DPA promotes the protection of spore DNA from dry heat or desiccation (Espitia et al., 2002). Significantly higher DPA release was observed after CH with MC10 than after HT with MC10 (Fig. 6). DPA is released through the physiological germination process. There is an optimal pH for nutrient germination, and a pH downshift may inhibit the germination (Sale et al., 1970; Bender and Marquis, 1982). Since CH decreases the pH of a spore suspension to pH 3.2 during treatment (Spilimbergo et al., 2005), the DPA release mediated by physiological germination may be inhibited by CH. We suggest that the enhanced DPA release by CH with MC10 was due to damage of the spore inner membrane. The release of DPA may result in a loss of the ability for physiological germination and subsequent proliferation. DPA release also decreases the heat resistance of spores. We have shown that the heat resistance of B. subtilis spores was decreased to a greater extent by CH with MC10 than by CH alone (Noma et al., 2015). In addition, the inactivation effect of CH with MC10 was more notable at temperatures above 70°C (Klangpetch et al., 2013). DPA release may reduce the resistance of DNA to CH with MC10. However, CH with MC10 did not appear to induce DNA fragmentation (Fig. 7).
The inactivation mechanism of spores can be summarized in the model proposed in Fig. 8. CH increases the surface hydrophobicity of spores, inducing enhanced sorption of the hydrophobic FAE, MC10. When combined with CH, MC10 induces injury in the inner membrane of spores to release DPA. The DPA release impairs the physiological germination ability of the spores, resulting in a loss of proliferative capacity. DPA release also decreases heat resistance, and the spores were inactivated by the heating component of CH. We have shown that CH with 0.1 mM MC10 exhibits a bacteriostatic effect, while 0.1 mM MC10 treatment alone did not (Noma et al., 2015). This result is consistent with the proposed inactivation mechanism. The apparent increase in the bacteriostatic activity of MC10 treatment may be due to the enhanced sorption of MC10 to spores during the combined use of CH and MC10.
Proposed mechanism for B. subtilis spore inactivation by CH with MC10.
We investigated factors contributing to spore resistance to CH with MC10 and presented a mechanism for B. subtilis spore inactivation by CH with MC10. It has been reported that the inner membrane is an important target for the inactivation of bacterial spores (Setlow et al., 2015; Rao et al., 2016). The combined use of any treatment that attacks the inner membrane of spores and CH with MC10 may bring about further increases in the inactivation effect on spores.
Acknowledgements This work was supported by JSPS KAKENHI Grant Number 26350094. FAEs were kindly provided by Taiyo Kagaku Co. (Mie, Japan).
