The inactivation of Bacillus subtilis spores using a combination of potassium sorbate and thermal treatments and exploring the underlying mechanisms

doi:10.21203/rs.3.rs-7700416/v1

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The inactivation of Bacillus subtilis spores using a combination of potassium sorbate and thermal treatments and exploring the underlying mechanisms

https://doi.org/10.21203/rs.3.rs-7700416/v1

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This study evaluated potassium sorbate (1 and 2 g/L) combined with heat (80, 90, and 100°C for 30, 60 and 90 min) for inactivating Bacillus subtilis spores. Plate counts, Fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM) assessed lethality and structural responses. The combination yielded synergistic inactivation; the strongest effect (2.12 log CFU/mL reduction) occurred at 2 g/L sorbate and 90°C for 60 min. Flow cytometry showed a rightward shift of FL2 histograms and higher propidium-iodide fluorescence, evidencing increased inner-membrane permeability. FTIR indicated perturbation of membrane lipids, peptidoglycan disruption, and denaturation of nucleic acids and proteins, aligning with SEM-observed surface damage. Collectively, sorbate plus moderate heat compromises multiple protective barriers of spores and enhances killing, offering mechanistic insight and a practical route to lower-intensity sterilization in food processing.

Potassium sorbate

thermal inactivation

spore structure

Bacillus subtilis

FTIR

flow cytometry

food sterilization

Bacterial endospores, particularly those belonging to the genus Bacillus, are key targets for food sterilization (Cho and Chung, 2020). Bacillus cereus and Bacillus anthracis stand as representative pathogenic spore-forming bacteria. Bacillus subtilis and Bacillus amyloliquefaciens are among the primary bacteria responsible for the degradation of processed foods (Higgins and Dworkin, 2012). Therefore, it is important for food preservation to explore methods for effectively inactivating these spores. Although not categorized as a human pathogen, Bacillus subtilis has been reported to be associated with cases of food poisoning. For instance, in February 2000, an outbreak of foodborne intoxication occurred in a small kindergarten in the town of Split (Croatia), which was linked to Bacillus subtilis contamination (Pavic et al., 2005; Logan, 2012). Previously, attention has been directed towards non-thermal inactivation methods such as high-pressure processing, ultrasound treatment, pulsed electric fields, and cold plasma technology to target the inactivation of Bacillus spores (Cho and Chung, 2020). However, it's noteworthy that these sterilization methods alone may not be sufficient to completely inactivate Bacillus spores; they often require combination with heat treatment for effective sterilization.

Heat treatment stands as a simple and effective method of sterilization, protecting food from pathogenic microorganisms. However, this processing method can have adverse effects on the nutritional and organoleptic qualities of food (Rozali et al., 2017). Combining heat treatment with preservatives may lead to a synergistic bactericidal effect, potentially reducing the intensity of heat treatment required to inactivate the spores. Sorbates rank as the third most significant antimicrobial preservative in both the food and pharmaceutical industries (Nemes et al., 2020). Some studies have found that potassium sorbate inhibits Listeria monocytogenes, Saccharomyces cerevisiae, Escherichia coli, and fungi (García et al., 2015; González and Dominguez, 2007; Pylypiw and Grether, 2000). Mira et al. (2010) reported that the antimicrobial mechanism of potassium sorbate may involve obstructing microbial catabolic pathways. Potassium sorbate is effective in inhibiting the formation of bacterial cell membranes and it is commonly utilized as a bacteriostatic agent in various food products such as milk, fruit juices, and meat products (Subils et al., 2012). Mansour et al. (1998) found that potassium sorbate did not directly inactivate spores, however, it interfered with spore germination, particularly at high concentrations and low pH levels. This study aimed to explore the efficacy of combining potassium sorbate with heat treatment to inactivate Bacillus subtilis spores, offering a novel approach to food sterilization. Flow cytometry was employed to investigate the damage to the spore's inner membrane caused by the combined treatments. Additionally, Fourier-transform infrared spectroscopy (FTIR) was utilized to analyze alterations in the spore's inner membrane fatty acids, peptidoglycan layer, nucleic acids, and proteins. Furthermore, scanning electron microscopy (SEM) was employed to observe changes in the morphology of Bacillus subtilis spores following treatments. Moreover, the inactivation mechanism of Bacillus subtilis spores by combining potassium sorbate with heat treatment was thoroughly explored.

2.1 Preparation of spores and bacterial suspension

The Bacillus subtilis spores utilized in this study were prepared following the method described by Fan et al. (2018), with some modifications. Bacillus subtilis was first activated by subculturing for at least three generations in nutrient broth at 37°C for 24 h under shaking conditions (150 rpm) to ensure active growth. After activation, the culture was inoculated into a sterilized spore-promoting manganese salt nutrient agar. The medium was composed of 33 g of nutrient agar, 0.1538 g of manganese sulfate, and 1000 mL of distilled water, adjusted to pH 7.0. The use of manganese was based on its reported role in enhancing sporulation efficiency in Bacillus subtilis (Fan et al., 2018), serving as an effective alternative to calcium in some sporulation systems. The medium was boiled three times, dispensed into test tubes, and sterilized at 121°C for 15 min. The inoculated tubes were then incubated at 37°C for 7 d to allow for sporulation.

The resulting spore cultures were washed three times with sterile distilled water. After each wash, the suspensions were centrifuged at 6,600 ×g at 4°C for 15 min. The supernatant and the upper portion of the pellet, which contained residual cell debris, were discarded, and the bottom layer enriched in spores was retained and resuspended.

Spore purification was conducted following the method of Tavares et al. (2012) with slight modifications. The spore suspensions were again centrifuged at 6,600 ×g at 4°C for 10 min and resuspended in 50 mM Tris-HCl buffer (pH 7.2) containing 50 µg/mL of lysozyme. The mixture was incubated at 37°C for 1 h to remove remaining vegetative cell debris. After washing once with distilled water, the pellets were vortexed in 0.05% SDS solution to further purify the spores. The samples were washed three times with distilled water and resuspended. The spore integrity was confirmed by phase contrast microscopy to ensure no germination or structural damage occurred during the lysozyme and SDS treatments. The final spore suspension was adjusted to a concentration of 1.5 × 10⁹ CFU/mL, stored at 4°C, and used within one month. Before use, the suspension was diluted to approximately 1 × 10⁸ CFU/mL.

2.2 Treatment of Bacillus subtilis spores

Aliquot of the Bacillus subtilis spore’s suspension (30 mL) was put into a 50 mL sterile PTFE centrifuge tube, which had been autoclaved at 121 ℃ for 15 min before use. The tube was then centrifuged at 6,600 ×g for 15 min, after which the supernatant was discarded. Then, 30 mL of 1 g/L and 2 g/L potassium sorbate solutions were added into separate tubes, respectively, and thoroughly mixed by vortexing. The contents were subsequently transferred to sterile polyethylene plastic bags that had been sterilized under UV irradiation light for 30 min prior to use. These bags were heat-sealed using a vacuum packaging machine and then placed in a water bath for treatment. The treatment temperatures were 80 ℃, 90 ℃ and 100 ℃, and the treatment durations were 30 min, 60 min, and 90 min respectively, and the samples were cooled in ice water immediately after treatment.

2. 3 Determination of dipicolinic acid (DPA) leakage

The DPA leakage was determined as described by Zhang et al. (2012) with some modifications. The treated Bacillus subtilis spore’s suspension was centrifuged at 4 ℃ and 6,600 × g for 15 min. Following centrifugation, 4 mL of the supernatant was mixed with 1 mL of DPA color test reagent (0.5 mol/L sodium acetate buffer 100 mL, 1 g each of ascorbic acid and ferrous ammonium sulphate). The absorbance value was then measured at 440 nm within a time of 1 to 2 h.

2.4 Flow cytometry analysis

Flow cytometry analysis was carried out as reported by Antolinos et al. (2014) with some modifications. Aliquot of untreated and treated Bacillus subtilis spore suspension (1 mL) was taken, washed by centrifugation, and resuspended in distilled water. Then, the samples were stained by adding 0.75 µL of 20 mmol propidium iodide (PI), followed by incubation in darkness at room temperature for 15 min. Prior to flow cytometric analysis, the stained spore suspensions were examined under an epifluorescence microscope (excitation at 488 nm, emission at 615 nm) to verify effective PI staining and confirm the absence of dye aggregates or visible cellular debris. Forward scattered light (FSC), side scattered light (SSC), and fluorescence channel FL2 were detected by flow cytometry. Approximately 30,000 cells were detected for each sample. Under 488 nm excitation light, the PI-labelled cells emitted red fluorescence at 615 nm in the FL2 channel. Subsequently, the collected data were analyzed using FSC Express Version 3.0 software.

2.5 Fourier transform infrared spectroscopy (FTIR) analysis

The FTIR analysis was performed as described by Liu et al. (2020) with some modifications. The samples were prepared using the KBr pressing method. The KBr, which was purified to chromatographic grade, underwent baking in an oven at 121°C for over 24 h. Subsequently, it was dried and sealed for storage. The FTIR spectrometer and other instruments were sterilized using alcohol-soaked cotton balls. Then, 200 mg of KBr and 2 mg of sample were ground in an agate mortar and pressed into tablets. The pressed tablets were scanned using an FTIR spectrometer within a wavelength range of 500–4000 cm^− 1. The spectrometer was set to conduct 64 scans at a resolution of 4 cm^− 1. The spectral data were analyzed using Omnic 8.0 software, where baseline correction and smoothing were applied as part of the analysis process.

The bands within the range of 1700 − 1600 cm^− 1 mainly represent the amide I band, which provides insight into the secondary structure of the protein. The Omnic 8.0 software was used and the data of the amide I band were collected. The data were then subjected to FTIR deconvolution curve fitting analysis. Peak Fit 4.12 software was used to perform curve fitting on the spectra peaks, and multiple fittings were conducted to minimize the residual value (R² ≥ 0.999) until the R² value remained unchanged. After the correspondence between each sub-peak and each secondary structural unit was determined, the relative content of each secondary structure was calculated based on the peak area.

2.6 Scanning electron microscopy (SEM) analysis

The SEM analysis was performed as reported by Liu et al. (2017) with slight modifications. Aliquot of untreated and treated bacterial suspension (8 mL) was transferred to a sterilized Eppendorf tube. The sample tubes were then centrifuged for 15 min at 6,600 × g to remove the supernatant and collect the bacterial precipitate. The spore samples were washed for 3 times with distilled water, and then freeze-dried for 5 h. The samples were affixed to a metal sample stage using conductive adhesive. They were then subjected to vacuum coating and observed using a scanning electron microscope.

2.7 Statistical analysis

All experiments were carried out in triplicates and means and standard deviations were calculated from the results obtained in independent trials. The data were subjected to analysis of variance (ANOVA), followed by Duncan tests at a significance level of p < 0.05. The statistical analysis was performed using SPSS software (Version 26.0, IBM Corporation) for Windows. Omnic 8.0 and Origin 2018 were used to perform two-point baseline correction on the FTIR data. The 5-point Savizky-Golay function was employed to smooth and deconvolve the second-order derivative processing of the data. Peak Fit 4.12 was used for peak fitting analysis. Statistical analysis and graphical representation of the experimental results were performed using Origin 2018 software.

3.1 The spore inactivation effect of combined treatments

The Bacillus subtilis spores’ inactivation effect resulting from various mass concentrations of potassium sorbate (1 g/L, 2 g/L) combined with heat at temperatures of 80 ℃, 90 ℃, 100 ℃ treatments for 60 min is shown in Fig. 1A. The initial count of Bacillus subtilis spores before treatment was about 1×10⁸ CFU/mL, and no significant change in the total number of colonies was observed after treating the spores at 80 ℃ alone. However, the addition of potassium sorbate at 80°C resulted in noticeable inactivation of Bacillus subtilis spores. This suggests that the combination of potassium sorbate with heat treatment was more effective in inactivating the spores. This is consistent with the results reported by Tominaga et al (2018). Additionally, it can be seen that the concentration of surviving Bacillus subtilis spores decreased significantly (p < 0.05) with increasing temperature and potassium sorbate concentration.

3.2 Effect of combined treatments on the release of DPA

The effect of potassium sorbate combined with heat treatment on the release of DPA from Bacillus subtilis spores is shown in Fig. 1B. The release of DPA increased with the increasing temperature and potassium sorbate concentration. Bacterial spores utilize an unusually high concentration (approximately 10% dry weight of spores) of DPA to protect themselves, and its release is a key step in inactivating them under mild pressure and temperature conditions (Akasaka et al., 2017). In the control group, no DPA release was observed. However, DPA release increased significantly after treatment (p < 0.05), and the amount of release was positively related to the potassium sorbate concentration. The core of the spores undergoes a hydration process after the release of DPA, which makes them susceptible to inactivation. The release of DPA indicates a change in the inner membrane permeability of the spores after treatment. The combination of potassium sorbate and heat treatment on the spores could intensify the damage to the spores and enhance the release of DPA.

3.3 Effect of combined treatments on the inner membrane of spores

The flow cytometry results for untreated and treated Bacillus subtilis spores are shown in Fig. 2. Propidium iodide (PI), a membrane-impermeable dye, selectively stains spores with compromised inner membranes, emitting red fluorescence upon binding to nucleic acids. Untreated spores displayed minimal fluorescence intensity, indicating intact inner membranes and low PI uptake. In contrast, treated spore populations exhibited a clear rightward shift in fluorescence histograms, reflecting increased PI uptake and thus enhanced membrane permeability.

As the treatment temperature and potassium sorbate concentration increased, the overall fluorescence intensity of the spore populations also increased, suggesting a greater extent of inner membrane damage. For example, spores treated at 90°C for 60 min in the presence of 2 g/L potassium sorbate showed a pronounced fluorescence enhancement compared to those treated at 1 g/L or under milder thermal conditions. Similarly, spores treated at 100°C combined with 2 g/L potassium sorbate exhibited the highest fluorescence intensity among all groups. This trend supports the hypothesis that both temperature elevation and potassium sorbate synergistically compromise membrane integrity.

These observations align with previous studies, such as Bi et al. (2023), which reported that ε-polylysine combined with heat treatment disrupts the inner membrane of Bacillus subtilis spores. In this study, the observed surface damage might be a direct result of sorbate interaction or an indirect consequence of enhanced thermal sensitivity.

3.4 FTIR spectra

As shown in Fig. 3A, the spores subjected to potassium sorbate combined with heat treatment exhibited changes in peak patterns within specific spectral bands. To improve the intuitive differentiation of characteristic band changes in the spectra, it is necessary to apply second derivative processing to the data. Second derivative processing is a commonly used method for analyzing infrared data (Al-Qadiri et al., 2008). The bands within the range of 3000 − 2800 cm^− 1 contain signals arising from C-H stretching vibrations related to fatty acids and cell membrane phase transitions (Girardeau et al., 2021). Protein bands are observed with the 1700 − 1600 cm^− 1 range, including signals from the C-O stretching vibrations and N-H bending vibrations of proteins (Dong et al., 2022; Sun et al., 2021). The spectra bands in the range from 1300 to 900 cm^− 1 includes signals associated with nucleic acids and cell wall components (Movasaghi et al., 2008).

3.5 FTIR spectra within the 3000 − 2800 cm^− 1 range

The band at 2872 cm^− 1 mainly represents the CH₃ symmetric telescopic vibrations of membrane lipids (Yu and Joseph, 2005). From Fig. 3B, compared with the untreated sample, the FTIR absorption bands at 2872 cm^− 1 shifted to 2877 cm^− 1 after treatment with 2 g/L potassium sorbate combined with heat. This shift is consistent with an alteration in the structure of the methyl chains in the membrane lipids of the spores (Bi etal., 2023). The vibrational intensity of the FTIR spectra showed significant changes with the increase in potassium sorbate concentration. This phenomenon could be attributed to several factors, including the weakening of hydrogen bonding between molecules of the inner membrane phospholipids in the spores, the transition of inner membrane phospholipids from a gel state to a liquid crystal state, and an increase in the fluidity of the inner membrane. These changes likely enhance the membrane's permeability to water molecules and consequently reduce the spore’s resistance (Ulmer et al., 2002; Rupert et al., 1988).

3.6 FTIR spectra in the 1300 − 900 cm^− 1 range

Figure 3C shows the second-order derivative spectra in the 1300 − 900 cm^− 1 range for the untreated and treated spores. This region mainly reflects the vibrational characteristics of cell nucleic acids and cell wall components (Goodacre and Kell, 1996). The bands located at 1080 cm^− 1 and 1220 cm^− 1 represent symmetric and antisymmetric telescopic vibrations of the nucleic acid phosphodiester backbone P = O (Al-Qadiri et al., 2008).

The spore cortex consists of a thick and loosely cross-linked peptidoglycan layer, which prevents the hydration of the germinal core. Significant changes in the amplitude of the C-O-C stretching vibration bands were observed in the 1200 − 900 cm^− 1 spectra. High concentrations of potassium sorbate caused the functional groups to vibrate more vigorously. This may be due to the disruption of the peptidoglycan layer structure by the combined potassium sorbate and heat treatments. The heat resistance of the spores is related to the peptidoglycan in the spore cortex (Gould et al., 1991). When the peptidoglycan layer is disrupted, the resistance of the spores to heat is significantly reduced. It can be seen from Fig. 3C that under the same heat treatment conditions, as the concentration of potassium sorbate increased, the vibration intensity of the FTIR spectra at 1220 com^− 1 and 1080 cm^− 1 also increased. It may be because potassium sorbate can interact with nucleic acids. Denaturation of nucleic acids is consistent with loss of spore viability, as they are unable to continue growing. Although spore DNA is tightly bound by small acid-soluble proteins (SASPs), structural damage observed in FTIR spectra may still suggest partial conformational changes. Furthermore, repair mechanisms post-germination may mitigate some of the damage, which warrants further investigation.

3.7 Effect of combined treatments on the secondary structure spore proteins

The fitting curves of the spore protein’s amide I band spectra for both the untreated and treated spores are shown in Fig. 4. The sub-absorption peaks were obtained by deconvolution fitting using the second-order derivative and Gaussian method. The corresponding relationship between each sub-peak and the secondary structure was identified as follows: 1650–1660 cm^− 1 represents α-helical structure, 1610–1640 cm^− 1,1690–1700 cm^− 1 represents a β-sheet structure, 1660–1690 cm^− 1 represents a β-turn structure, and 1640–1650 cm^− 1 represents a random coil structure (Baltacioglu et al., 2017; Wang et al., 2012). Each sub-peak was confirmed, and the content of each structure was calculated.

Changes in the secondary structure of Bacillus subtilis spore proteins are shown in Fig. 5. According to the curve fitting analysis, the untreated Bacillus subtilis amide I contained 30.28% α-helix, 27.95% β-folding, 25.29% β-turning, and 16.48% random curling structures. With the increase of temperature and potassium sorbate concentration, the content of α-helix decreased, while the content of random coil increased. It can be concluded that the secondary structure underwent a transition from an ordered to a disordered state, leading to a decrease in protein stability and a reduction in spore’s resistance. This is in agreement with the findings of Meng et al. (2016). Also, Sofos, Busta, and Allen (1986) reported that sorbic acid may interfere with the function of lipids, proteins, and DNA.

It was reported that once DPA was fully released from the spores, the proteins of the spores were readily denatured at mild temperatures, such as 80°C, which was much lower than the commonly used temperature of 121°C for inactivating spores (Akasaka et al., 2017). In our system, DPA leakage increased with both treatment temperature and potassium sorbate concentration, a trend consistent with diminished spore resistance. These observations admit two non-mutually exclusive interpretations: (i) progressive DPA efflux accompanies core rehydration and lowers protein thermal robustness, facilitating inactivation; (ii) potassium sorbate perturbs the spore envelope and increases inner-membrane fluidity/permeability, rendering membrane-associated functions required for germination and metabolism more susceptible to thermal damage.

3.8 Changes in morphology of Bacillus subtilis spores

Changes in the morphological structure of spores after combined treatments are shown in Fig. 6 (A), as captured using a scanning electron microscope. The untreated Bacillus subtilis spores had a full and rounded surface with a uniform size distribution. The structure of Bacillus subtilis spores after heat treatment at 90°C for 60 min exhibited damage, with the surface showing severe depressions. This is consistent with the findings of Rozali et al. (2017). The spore surface after heat treatment with 2 g/L potassium sorbate combined with heat at 90 ℃ for 60 min showed more severe crumpling. Peptidoglycan plays a crucial role in maintaining the structural integrity of bacterial spores. The FTIR analysis revealed the destruction of the peptidoglycan structure, which is consistent with the results of SEM analysis.

Combining potassium sorbate and thermal treatments resulted in significant inactivation effect on Bacillus subtilis spores. The inactivation effect enhanced as the concentration of potassium sorbate and temperature increased. Under combined potassium sorbate–heat treatments, spores exhibited cortical peptidoglycan damage, alterations in inner-membrane phospholipid phase state, inner-membrane injury, increased core hydration, and denaturation of nucleic acids and proteins; overall, the spores were substantially inactivated under the tested conditions. This study elucidated the inactivation mechanism of Bacillus subtilis spores through the combined use of potassium sorbate and heat treatment, providing a theoretical basis for the application of this technology in food sterilization. Further research is needed to investigate the effectiveness of these combined treatments in inactivating microorganisms within complex food matrices.

DPA, Dipicolinic Acid

PI, Propidium Iodide

FTIR, Fourier Transform Infrared.

SEM, Scanning Electron Microscope.

Conflicts of Interest

The authors declare that there is no conflict of interest.

Author Contribution

K.B. performed the experiments, collected and analyzed the data, and drafted the manuscript.Y.L. assisted with experimental design, data validation, and interpretation.Z.Z. conceived and supervised the study, acquired funding, and revised the manuscript critically for important intellectual content.All authors have read and approved the final version of the manuscript.

Acknowledgements

This study was supported by the Natural Science Foundation of China (No. 32260633) and the Natural Science Foundation of Ningxia (No. 2023AAC03136).

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You are reading this latest preprint version

The inactivation of Bacillus subtilis spores using a combination of potassium sorbate and thermal treatments and exploring the underlying mechanisms

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials and methods

2.1 Preparation of spores and bacterial suspension

2.2 Treatment of Bacillus subtilis spores

2. 3 Determination of dipicolinic acid (DPA) leakage

2.4 Flow cytometry analysis

2.5 Fourier transform infrared spectroscopy (FTIR) analysis

2.6 Scanning electron microscopy (SEM) analysis

2.7 Statistical analysis

3. Results and discussion

3.1 The spore inactivation effect of combined treatments

3.2 Effect of combined treatments on the release of DPA

3.3 Effect of combined treatments on the inner membrane of spores

3.4 FTIR spectra

3.5 FTIR spectra within the 3000 − 2800 cm^− 1 range

3.6 FTIR spectra in the 1300 − 900 cm^− 1 range

3.7 Effect of combined treatments on the secondary structure spore proteins

3.8 Changes in morphology of Bacillus subtilis spores

4. Conclusions

Abbreviations

Declarations

Conflicts of Interest

Author Contribution

Acknowledgements

References

Additional Declarations

Status:

Version 1

The inactivation of Bacillus subtilis spores using a combination of potassium sorbate and thermal treatments and exploring the underlying mechanisms

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials and methods

2.1 Preparation of spores and bacterial suspension

2.2 Treatment of Bacillus subtilis spores

2. 3 Determination of dipicolinic acid (DPA) leakage

2.4 Flow cytometry analysis

2.5 Fourier transform infrared spectroscopy (FTIR) analysis

2.6 Scanning electron microscopy (SEM) analysis

2.7 Statistical analysis

3. Results and discussion

3.1 The spore inactivation effect of combined treatments

3.2 Effect of combined treatments on the release of DPA

3.3 Effect of combined treatments on the inner membrane of spores

3.4 FTIR spectra

3.5 FTIR spectra within the 3000 − 2800 cm− 1 range

3.6 FTIR spectra in the 1300 − 900 cm− 1 range

3.7 Effect of combined treatments on the secondary structure spore proteins

3.8 Changes in morphology of Bacillus subtilis spores

4. Conclusions

Abbreviations

Declarations

Conflicts of Interest

Author Contribution

Acknowledgements

References

Additional Declarations

Status:

Version 1

3.5 FTIR spectra within the 3000 − 2800 cm^− 1 range

3.6 FTIR spectra in the 1300 − 900 cm^− 1 range