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. 2024 May 14;33(14):3357–3366. doi: 10.1007/s10068-024-01577-1

Inactivation effects of combined thermosonication and potassium sorbate treatments on Bacillus subtilis spores

Jiajia Li 1, Zhong Zhang 1,✉, Qinghuan Li 1, Yongxia Liu 1, Yichang Liu 1
PMCID: PMC11422313  PMID: 39328230

Abstract

This study aimed to investigate the inactivation effect of combined TS (thermosonication) and PS (potassium sorbate) treatments on Bacillus subtilis spores. The inactivation effect and potential mechanisms were examined using plate counts, OD600 values, nucleic acid leakage, DPA (dipicolinic acid) leakage, flow cytometry, and FTIR (Fourier transform infrared spectroscopy). The results showed that, after TS + PS treatments, the integrity of the inner membrane was lost, the permeability of the inner membrane to water molecules was increased, and the intraspore substances leaked. Furthermore, the OD600 value was reduced, indicating that the spore core hydration was enhanced. Spores proportion with damaged inner membrane was significantly increased to 66%, the ordered secondary structure of the protein was changed into a disordered structure and nucleic acid was fragmented after TS + PS treatment. The results indicated that the combined TS and PS treatments may be a useful method for inactivating bacterial spores in food processing and sterilization.

Keywords: Bacillus subtilis, Thermosonication, Potassium sorbate, Spore, Inactivation

Introduction

The gram-positive Bacillus subtilis bacteria are common spoilage bacteria in foods (for example, milk, soy milk, pear, garlic and onion, etc.) and have the ability to change into metabolically inactive spores, allowing it to endure harsh environmental conditions like intense heat, high-power ultrasound, chemicals, and ultra-high pressure (Cho and Chung, 2020; Fan et al., 2019a; Lyu et al., 2023; Rao et al., 2020). Due to their great resistance to sterilization, these dormant spores can survive in food and swiftly germinate into vegetative cells in favorable conditions, leading to food poisoning or deterioration (Fan et al., 2019a). Different from vegetative bacteria, Bacillus subtilis spores have multi-layer resistant structures with complex chemical properties (Sun et al., 2022). The cortex consists of a cross-linked peptidoglycan matrix that maintains the lower water activity in the spore core and provides heat tolerance to spores (Lyu et al., 2023). Additionally, the inner membrane makes spores resistant to chemicals because of its limited permeability to small molecule substances even for water molecules (Rao et al., 2020). The integrity of the spores’ inner membrane was one aspect that helped them resist sterilization (Hauck-Tiburski et al., 2019). The spore core is dehydrated and contains dipicolinic acid (DPA), nucleic acid substances, and small acid-soluble proteins (Cho and Chung, 2020). The dehydration of the spore core is a significant factor in the spore’s resistance to various sterilization treatments.

Thermal sterilization is a conventional and effective method for inactivating bacterial spores. However, it may adversely affect the nutritional quality and flavor of food. Therefore, the focus has shifted to the combination of heat and other treatments to preserve the nutritional and sensory qualities of food while inactivating spores (Cho and Chung, 2020; Lyu et al., 2023). As a promising technology, US (ultrasonic) has been widely used in food processing and microbial inactivation. The frequency of an oscillating sound pressure wave known as an ultrasound is higher than the range of human hearing. Ultrasonic waves in a frequency range of 20 to 40 kHz were categorized as high-energy or high-power (Mason, 1999). The alternating compression and expansion cycles are generated when high-power ultrasonic propagates in a liquid medium. Acoustic cavitation caused by these cycles is thought to be the primary factor in the inactivation of microorganisms. Violent molecular collisions and microflow caused by acoustic cavitation result in significant shear forces, shock waves, and damage to the cell membrane (José et al., 2014). Additionally, acoustic cavitation produced free radicals and hydrogen peroxide, which led to sonochemical reactions and spore oxidative damage (Alzate et al., 2020). However, it was demonstrated that while vegetative bacteria could be efficiently inactivated by US treatment, bacterial spores could not. Therefore, several researchers have attempted to improve the effect of spore sterilization by combining treatments such as US and heat (Evelyn et al. 2023; Fan et al. 2019a; 2019b).

Some studies have proved that US and heat have a synergistic effect on the inactivation of vegetative bacteria or spores, and this effect seems to be stronger at high temperatures. For example, Khanal et al. (2014) found that the morphological surface of three Bacillus species spores was completely broken after TS (thermosonication) treatment at 500 W-63 °C-30 min, indicating that TS has the ability to directly inactivate Bacillus spores. In another study, Evelyn et al. (2023) found that the mold spores in orange juice were decreased by 1.70 log after TS (250 W-90 °C-30 min) treatment. The PS (potassium sorbate) was acknowledged to be safe (Code of Federal Regulations. Title 21, § 582.3640) when used in accordance with manufacturing specifications, and the PS has been used for food preservation for a long time (Mohammadzadeh-Aghdash et al., 2018; Trevisan et al., 2022). It was reported that the antiseptic activity of PS is linked with the number of carboxyl groups and carbon atoms (Mohammadzadeh-Aghdash et al., 2018). A study by den Besten et al. (2012) showed that the germination and growth of Bacillus spores were stressed by sorbic acid, which was verified by flow cytometry analysis. Qian et al. (2023) found that the Pichia membranaefaciens in soy sauce was synergistically inactivated by TS (300 W-60 °C) combined with PS (0.1 mg/mL) treatment. However, to the best of our knowledge, no studies have investigated the sporicidal effects of TS + PS treatments on bacterial spores.

Therefore, this study aimed to explore the inactivation effects of combined TS and PS treatments on Bacillus subtilis spores and discuss underlying mechanisms. FTIR (Fourier transform infrared spectroscopy) was used to analyze the changes in the molecular structure of spore proteins and nucleic acids. Flow cytometry analysis was performed to evaluate the damage in the inner membrane of spores after various treatments. Additionally, the count of surviving spores, OD600 values, and DPA and nucleic acid leakage were measured.

Materials and methods

Spore preparation

The Bacillus subtilis spore culture and spore suspension were prepared as described by Rao et al. (2020) with some modifications. The strain of Bacillus subtilis As 1.433 used in this study was obtained from the China General Microorganism Culture Collection and Management Center. Under the aseptic conditions, the Bacillus subtilis spores of the third generation were inoculated on a nutrient agar medium and incubated at 37 °C for 7 days and were then washed with distilled water and sifted through a screen with the pore size of 38 μm into a sterilized conical flask. The collected spore suspension was centrifuged at 4 °C and 9000 × g for 15 min and the supernatant was discarded, then the spores were resuspended and washed by centrifugation again. The washing of spores by centrifugation was repeated three times. The concentration of spores was adjusted to 1.5 × 109 CFU/mL by PBS buffer and stored at 4 °C.

Sterilization treatment

The spore suspention was centrifuged at 4 °C and 9000 × g for 15 min, and the supernatant was discarded. The spores were then resuspended in distilled water or UHT (Ultra-high temperature) sterilized milk, and the concentration of spore suspensions was adjusted to 1.5 × 108 CFU/mL. For PS treatment, the PS was added to spore suspentions at concentration of 0.2% (PS was obtained form Shanghai Macklin Biochemical Co., Ltd., SH, CHN). An ultrasonic homogenizer (JY92-IIDN, Ningbo Scientz Biotechnology Co., Ltd., ZJ, CHN) consisted of three parts: main engine, transducer, and amplitude transformer was used, and the ultrasonic frequency was 20–25 kHZ. The ultrasonic temperature was 25/90 °C, ultrasonic power was 300 W/600 W, the duty cycle was 1:1 (ultrasonic time 2 s, intermittent time 2 s), and ultrasonic treatment time was 30 min, and each treatment was performed in triplicate. The temperature sensor was used to continuously monitor the sample temperature throughout the experiment. The ultrasonic probe had to be submerged in the sample and was placed 15 mm from the conical flask’s bottom. After the experiment, the ultrasonic probe was thoroughly sterilized and cleaned using 75% ethanol.

Counting of spore survivors

The spore counting was performed as described by Meng et al. (2016) with some modifications. The spore untreated and treated spore suspensions were diluted, and 15–20 mL of trypticase soy-yeast extract agar medium were mixed with 1 mL spore suspension. The whole process was carried out under sterilized conditions. The solidified mediums were incubated at 37 °C for 24 to 48 h, and the number of spore survivors was counted. The number of surviving spores was expressed in logarithmic values.

Measurement of OD600 value

The OD600 value of samples was measured as described by Tehri et al. (2017) with some modifications. Three mL of untreated and treated Bacillus subtilis spore suspension was taken and shaken. The absorbance values at 600 nm were recorded using an ultraviolet-visible spectrophotometer (754 PC, Jinghua Science and Technology Instrument Co., Ltd., SH, CHN). All measurements were performed in triplicate and the instrument was calibrated with distilled water before determination.

Measurement of DPA and nucleic acid leakage

The DPA leakage of samples was measured as described by Lv et al. (2019) with some modifications. The supernatant of spore suspension was used for the DPA measurement. Five mL spore samples of different treatments were centrifuged (4 °C-9000 × g) for 15 min and then 4 mL supernatant and 1 mL DPA chromogenic agent (Composed of 1 M sodium acetate buffer, 1% ascorbic acid, and 1% ammonium ferrous sulfate) were vortex mixed. After 90 min, the absorbance value was measured at 440 nm using an ultraviolet-visible spectrophotometer. All measurements were carried out in triplicate and distilled water was used to calibrate the instrument before determination. The treatment of 121 °C-30 min was used as a positive control. The content of DPA was calculated according to a standard curve.

For the DPA standard curve: a gradient standard DPA solution (60-1200 µM) of 4 mL was prepared and mixed with 1 mL DPA chromogenic agent. After 90 min, the absorbance value was measured at 440 nm using an ultraviolet-visible spectrophotometer. All measurements were carried out in triplicate and distilled water was used to calibrate the instrument before determination.

The leakage of nucleic acid was measured as described by Majeed et al. (2023) with some modifications. Five mL of untreated and treated Bacillus subtilis spore suspension was centrifuged at 4 °C-9000 × g for 10 min and the supernatant was collected. The absorbance value was measured at 260 nm using an ultraviolet-visible spectrophotometer. All measurements were performed in triplicate and the instrument was calibrated with distilled water before determination.

Flow cytometry analysis

Flow cytometry analysis was conducted as described by Majeed et al. (2023) with some modifications. The concentration of spore suspension was diluted to 1 × 106 CFU/mL. Two mL spore suspension was mixed with propidium iodide (PI) at a concentration of 13 µg/mL. The mixture was incubated at room temperature in the dark for 15 min. After incubation, the spore suspension was centrifuged and washed twice with sterilized water at 9000 ×g for 10 min to remove the free PI. Cube 8 flow cytometry (Sysmex Corporation, TKY, JPN) was used to collect the FSC light (forward scattered), SSC light (side scattered), and fluorescence channel FL2 of spore suspension at 488 nm excitation light. Approximately 30,000 spores were collected from each sample. The FSC Express software Version 7.0 was used to analyze exported data, and the results were represented in a histogram.

FTIR analysis

The FTIR analysis was conducted as described by Shen et al. (2023) with some modifications. The untreated and treated spore suspension was centrifuged and freeze-dried. The freeze-dried spore powder and dried potassium bromide powder were ground and mixed in an agate mortar at a mass ratio of 1:100 mg and were then pressed into tablets at 14 MPa. A Two Fourier infrared spectrometer (Perkin Elmer Co., Ltd., MA, US) was used and a potassium bromide tablet was used as a background. The scanning range was set from 1800 to 800 cm−1 with a scanning resolution of 4 cm−1 and 64 scans per sample. The Origin Software, Version 2021, was used for plotting and data analysis and the Peakfit software, Version 4.12, was used for peak fitting analysis of the secondary structure proportions of spore proteins. Multiple fittings were conducted until the R2 value was ≥ 0.999.

Statistical analysis

Data obtained from triplicate measurements were statistically analyzed and expressed as means ± standard deviation. The results were plotted using the Origin Software, Version 2021 (OriginLab Corp., MA, USA) and Peakfit software, Version 4.12 (Systat Software Inc., CA, USA). Analysis of variance and Duncan test at p < 0.05 was carried out using the SPSS software, Version 23 (IBM, NY, USA). Data from the flow cytometry analysis were analyzed and plotted using the FSC Express software, Version 7.0 (De Novo Software Corp., CA, USA).

Results and discussion

Sporicidal effect of different treatments

Since Bacillus subtilis frequently causes spoiling in a variety of food types, it was chosen as the target microorganism, and the sporicidal effect of US, PS, TS, and combined TS and PS treatments on Bacillus subtilis spores were evaluated. As shown in Fig. 1, in distilled water and UHT sterilized milk, the count of spores in the untreated samples was 8.11 ± 0.04 log and 8.13 ± 0.06 log respectively, and the sporicidal effect after the treatment with 0.2% PS alone (25 °C) was not significant (p > 0.05) for the two samples. As can be seen from Fig. 1A, the counts of Bacillus subtilis spores in distilled water, treated at 300 W/600 W-25 °C, were 8.01 ± 0.19 log and 7.56 ± 0.10 log, respectively. However, the count of Bacillus subtilis spores in distilled water was 5.89 ± 0.11 log and 5.58 ± 0.22 log when the temperature was increased to 90 °C, respectively. This indicated that the Bacillus subtilis spores in distilled water were significantly (p < 0.05) inactivated with the increase in temperature at the same ultrasonic power. Similar sporicidal effect was observed for the spores in UHT sterilized milk. Evelyn and Silva (2016) found that the D value of the Alicyclobacillus acidoterrestris spores treated by TS (202 W-78 °C) was 6 times lower than that treated at 78 °C without TS treatment. Other studies also found that TS treatment of bacterial spores had a synergistic sporicidal impact by damaging the inner membrane, hydrating the spore core, and reducing resistance (Evelyn and Silva, 2020; Fan et al., 2019b; Kakagianni et al., 2020). As shown in Fig. 1A, compared with the untreated samples, Bacillus subtilis spores in distilled water, treated at 300 W/600 W-90 °C-0.2% PS, were inactivated by 2.88 ± 0.13 log and 3.26 ± 0.11 log respectively. As shown in Fig. 1B, compared with the untreated samples, Bacillus subtilis spores in UHT sterilized milk, treated at 300 W/600 W-90 °C-0.2% PS, were inactivated by 2.45 ± 0.10 log and 2.96 ± 0.14 log respectively. These results demonstrated that the combination of TS and PS treatments significantly promoted the Bacillus subtilis spore inactivation in both distilled water and UHT sterilized milk. It is worthy noting that the sporicidal effect in UHT sterilized milk was slightly lower than that in distilled water, which can be attributed to the protective effect of milk components. Qian et al. (2023) found that the inactivation effect of Pichia membranaefaciens in soy sauce after TS + PS (300 W-60 °C-0.1 mg/mL PS) treatment was greater than that of US, US + PS, and TS treatments, which is consistent with our experimental results. Tiburski et al. (2014) reported that the inactivation effect was positively correlated with the degree of spore hydration. Therefore, our next experiment determined the spore core hydration degree under various treatments. However, because milk will hinder the determination of subsequent experimental results, only distilled water was used as spore-suspending medium in subsequent experiments.

Fig. 1.

Fig. 1

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Sporicidal effects of different thermosonication treatments and combined treatments of thermosonication and potassium sorbate (PS) for 20 min on Bacillus subtilis spores. Values with different lowercase letters indicate significant differences atp < 0.05. A distilled water; B UHT sterilized milk

The changes in the OD600 value after treatments

The OD600 value was used to characterize the spore core hydration of Bacillus subtilis (Tehri et al., 2017), and a sporicidal effect was verified. As shown in Fig. 2, after treatment at 90 °C alone, the OD600 value of spore suspension was decreased to 0.794 ± 0.006. However, after the treatment at ultrasonic power of 300 W, the OD600 value decreased to 0.728 ± 0.007, and the OD600 value significantly (p < 0.05) decreased to 0.700 ± 0.002 when the ultrasonic power was increased to 600 W. These results indicate that the core hydration degree of spores treated with TS was greater than that of thermally treated ones. Fan et al. (2019a) found that the reduction in OD600 value of Bacillus subtilis spores treated at 330 W-25 °C-0.02% nisin was greater than that of spores treated with US (330 W-25 °C). This was attributed to that ultrasonication enhanced the effect of bioactive substances (Fan et al. 2019a). On the other hand, when PS of 0.2% concentration was added, the OD600 value of spores suspension was reduced significantly (p < 0.05) to 0.643 ± 0.002 after treatment at 300 W-90 °C , and the OD600 value significantly reduced to 0.602 ± 0.004 as the power increased to 600 W at the same temperature. This indicates that the addition of 0.2% PS enhanced the spore’s hydration under the TS treatment, which is supported by the spore count results.

Fig. 2.

Fig. 2

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The OD600 values of Bacillus subtilis spores following different thermosonication treatments and combined treatments of thermosonication and potassium sorbate (PS) for 20 min. Values with different lowercase letters indicate significant differences at p < 0.05

The leakage of DPA and DNA under different treatments

The reduction in the OD600 value is related to the leakage of intraspore substances such as DPA and DNA (Lv et al., 2019). The DPA is a unique substance to bacterial spores, accounting for 5 to 15% of the dry matter content of spores (Aldrete-Tapia and Torres, 2020). Lv et al. (2019) found that TS treatment caused destruction to resistant structures such as the spore inner membrane and enhanced the leakage of spore DPA. According to the DPA standard curve (y = 0.0016015x + 0.0272157, R2 = 0.9992), the leakage amount of DPA from the positive control was 68.651 ± 0.308 µg/mL (data not shown). However, the DPA leakage was 12.683 ± 0.781 µg/mL after the treatment at 90 °C for the same time. On the other hand, the DPA leakage of spores increased to 23.798 ± 1.320 µg/mL after sonication at a power of 300 W, and the DPA leakage increased significantly (p < 0.05) to 32.352 ± 2.073 µg/mL as the sonication power increased to 600 W. These results indicate that the increase in ultrasonic power and temperature enhanced the leakage of DPA. It is worth noting that the DPA leakage may be caused by many reasons, such as spore germination, the change of membrane permeability, and spore inactivation (Lv et al., 2019). Treatment with potassium sorbate alone caused small DPA leakage by unknown mechanism which need further study in the future. Lv et al. (2019) found that the DPA leakage of Bacillus cereus spore after TS treatment (800 W-80 °C) was significantly greater than that after US (800 W) treatment alone, which is consistent with our results. Pandey et al. (2015) reported that PS and heat had synergistic effects on the DPA leakage and spore core hydration. As shown in Fig. 3A, after the 600 W-90 °C-0.2% PS treatment, 43.53 µg/mL of spore DPA was leaked, and compared with the positive control, the relative DPA leakage rate was 63.41% (relative DPA leakage rate = DPA leakage of treatment/DPA leakage of positive control × 100%). This indicates that the addition of 0.2% PS enhanced the DPA leakage significantly (p < 0.05) under the TS treatment conditions.

Fig. 3.

Fig. 3

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The DPA and nucleic acid release of Bacillus subtilis spores following different thermosonication treatments and combined treatments of thermosonication and potassium sorbate (PS) for 20 min. Values with different lowercase letters indicate significant differences at p < 0.05. A DPA release; B nucleic acid release

The leakage of nucleic acid and permeability of the inner membrane could be characterized by OD260 values of spore samples (Hauck-Tiburski et al., 2019; Majeed et al., 2023). For example, Cao et al. (2018) found that the OD260 values of bacterial suspension increased as the nucleic acid leakage increased. The changes in OD260 value after different treatments are shown in Fig. 3B. The OD260 value for the untreated spore suspension was 0.051 ± 0.006, and there was no significant difference of OD260 values between the untreated sample and the sample treated with potassium sorbate alone, which indicated treatment with potassium sorbate alone didn’t cause nucleic acid leakage. However, after the treatment at 90 °C alone, the OD260 value was 0.151 ± 0.008. Moreover, the OD260 value increased to 0.260 ± 0.007 and 0.307 ± 0.006 after TS treatment at the power of 300 and 600 W, respectively. These results reveal that the leakage of nucleic acid was significantly enhanced as the ultrasonic power of TS treatment increased at the same temperature, indicating an increase in the inner membrane permeability. On the other hand, after treatment with TS at 300 W/600 W-90 °C with the addition of 0.2% PS solution, the OD260 value of spore suspension increased to 0.337 ± 0.012 and 0.354 ± 0.007, respectively. After the 600 W-90 °C-0.2% PS treatment, the leakage of spore nucleic acid and inner membrane permeability were the most significant. Compared with the TS treatment alone, the leakage of the spore’s nucleic acids was increased significantly after the addition of 0.2% PS (p < 0.05). These results indicate that inner membrane permeability to intraspore substances was enhanced by combining TS and PS treatments, which is supported by the results of OD600 value.

The inner membrane damage after different treatments

The increase in inner membrane permeability probably resulted from inner membrane damage which was measured by flow cytometry. The broken membrane could be penetrated by PI, which stained the DNA and stimulated the red fluorescence (Majeed et al., 2023). The flow cytometry histogram (Fig. 4) was divided into two regions, M1 (negative region) and M2 (positive region). The M1 region indicated that the spores population was not stained by PI and the inner membrane was intact, whereas the M2 region indicated that the spores population was stained and the inner membrane was damaged (Kim et al., 2021). The M2 region for the untreated spore sample was 2.18%, however, the M2 region increased significantly (p < 0.05) after TS treatment (300 W/600 W-90 °C) to 50.94% and 52.51%; respectively, demonstrating damage to the spore’s inner membrane, which is consistent with the findings of Fan et al. (2019b). The inner membrane damage might be caused by acoustic cavitation effect (José et al., 2014). The M2 region was further increased with the addition of 0.2% PS, reaching a maximum value of 66.35% at 600 W-90 ℃-0.2% PS. These results verified that the combination of TS + PS treatments had a synergistic damage effect on the spore inner membrane, which was enhanced with the increase of ultrasonic power and the addition of PS.

Fig. 4.

Fig. 4

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Flow cytometry histogram of Bacillus subtilis spore suspension stained with PI. A Untreated; Btreated at 90 °C; C treated at 300 W-90 °C; D treated at 600 W-90 °C; E treated at 300 W-90 °C-0.2% PS; F treated at 600 W-90 °C-0.2% PS. (the PS stands for potassium sorbate)

Effects of different treatments on the DNA structure of spores

The Fourier transform infrared spectroscopy measures changes in chemical structure and relative content, and the results are unaffected by the physical state of samples (Perna et al., 2020). The FTIR was used to scan the samples in the wavenumber range of 1800–800 cm−1, as shown in Fig. 5. The characteristic absorption peak at 1076 cm−1 is attributed to the C=O stretching vibration of the nucleic acid skeleton. After treatment with a combination of TS + PS, the absorption peak at 1076 cm−1 changed to two peaks at 1054 cm−1 and 1093 cm−1, which were assigned to C=O deoxyribose stretching vibration and phosphate symmetric vibration (Ovissipour et al., 2019), respectively. The peak intensity ratio at 1054 cm−1/1093 cm−1 indicates the fragmentation of nucleic acid structure (Ovissipour et al., 2019). This peak intensity ratio was increased with the addition of 0.2% PS and the increase of ultrasonic power, indicating nucleic acid denaturation. These results are consistent with the finding of Bermudez-Aguirre et al. (2011) in which OH−, H+, and hydrogen peroxide free radicals were produced under ultrasonic treatments, which targeted bacterial DNA and resulted in nucleic acid denaturation.

Fig. 5.

Fig. 5

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Effects of different thermosonication treatments and combined treatments of thermosonication and potassium sorbate (PS) for 20 min on infrared spectra of Bacillus subtilis spores in the range of 1800–800 cm−1. (the CK stands for control)

The wavenumber range of 900–700 cm−1 was classified as a “fingerprint region”, which is unique to certain bacteria (Dong et al., 2022). The characteristic absorption peak at 889 cm−1, shown in Fig. 5, represents the C=C, C=N, and C–H out-of-plane bending vibrations of DNA nucleotide rings (Aboelkheir et al., 2019). The intensity of the absorption peak also increased by combining TS + PS treatments, reaching a maximum value after 600 W-90 °C-0.2% PS treatment. Our experimental results indicated that combining TS and PS treatments had a synergistic effect on DNA denaturation, and this effect was enhanced with the addition of PS and the increase of ultrasonic power. These results are consistent with the changes in OD600, OD260, and DPA leakage, as well as the flow cytometry results.

Effects of different treatments on the protein structure of spores

In addition, the absorption peaks at 1661 cm−1 and 1554 cm−1 in Fig. 5 represent the C=O stretching vibrations of protein amide I band, and the N-H in-plane bending vibration and C–N stretching vibration of the protein amide II band, respectively (Dong et al., 2022; Sun et al., 2021). With the addition of PS and the increase of ultrasonic power, the absorption peak intensity at 1661 and 1554 cm−1 were gradually increased. It was reported that ultrasonic triggered the breaking of hydrogen bonds and van der Waals interactions in the peptide chain, changing the secondary structure of the protein (José et al., 2014). The free radicals excited by TS treatment were bound to spore protein, in which the oxygen of carbonyl (C=O) was used as a hydrogen bond receptor, increasing the absorption peak intensity (Bene and Jordan, 1999; Yeargers, 2018). These results indicated that combined TS + PS treatments lowered spore protein stability and damaged the spore coat, which mainly consisted of proteins. In addition, PS is a linear structure containing a carbonyl group, and strong covalent binding was formed between the amino groups of amino acids and PS after nucleophilic attack (Hauck-Tiburski et al., 2019). The amide I band (1700–1600 cm−1) was fitted and the sub-peaks of the secondary structures were identified and merged, which was used to calculate the proportion of the protein’s secondary structures (Sun et al., 2021). As shown in Fig. 6, the α-helix and β-fold proportions of the untreated spores were 28.65% and 39.47%, while β-rotation and random coil proportions were 21.05% and 10.83%, respectively. After TS treatments, the proportion of α-helix and β-fold were decreased, and the proportion of β-rotation and the random coil was increased. These results indicated that the ordered structure of spore proteins was changed into a disordered structure under TS treatments (Çınar and Czeslik, 2015). With the addition of 0.2% PS, these changes were enhanced, especially after the 600 W-90 °C-0.2% PS treatment, the proportions of α-helix, β-fold, β-rotation, and random coil were changed to 14.13%, 27.06%, 32.95%, and 25.86%, respectively. These results showed that the protein’s stability was significantly reduced by combining TS and PS treatments.

Fig. 6.

Fig. 6

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Changes in the secondary structure proportion of Bacillus subtilis spore protein following different thermosonication treatments and combined treatments of thermosonication and potassium sorbate (PS) for 20 min. (the CK stands for control)

Effects of combined thermosonication (TS) and Potassium Sorbate (PS) treatments on Bacillus subtilis spores were evaluated using plate counts, OD600 values, nucleic acid leakage, DPA leakage, flow cytometry, and FTIR. Comparing the effects of TS with PS addition treatments to a TS treatment, the TS with PS addition treatments synergistically inactivated spores. The main reasons for the loss in spore resistance were damage to the spores’ inner membrane and the release of significant chemical compounds. The inner membrane’s integrity was destroyed, the membrane’s permeability to water molecules increased, and the intraspore chemicals leaked after TS and TS + PS treatments. Following TS + PS treatment (600 W-90 °C-0.2% PS), about 3.26 ± 0.11 log Bacillus subtilis spores were inactivated and 63.41% of DPA was released. Additionally, after the TS + PS treatment (600 W-90 °C-0.2% PS), nucleic acids were extensively leaked (p < 0.05), and the structure of spore proteins, DNA, and the inner membrane was significantly altered. Furthermore, the OD600 value was reduced by 0.398 ± 0.004 after TS + PS treatment, indicating that the spore core hydration was enhanced. Spores proportion with damaged inner membrane was significantly (p < 0.05) increased to 66%, and spore protein was denatured after TS + PS treatment. The ordered secondary structure of proteins changed into a disordered structure, and nucleic acids were fragmented following TS + PS treatment. The findings suggest that combining TS and PS treatments may be a useful technique for sterilization and bacterial spore inactivation. PS could enhance the sterilization effect of TS, which would be beneficial to the sensory and nutritional qualities of foods.

Acknowledgements

This study was supported by the Natural Science Foundation of Ningxia [2023AAC03136] and the National Natural Science Foundation of China [Grant No. 32260633 and 31760474].

Declarations

Conflict of interest

The authors declare that there are no conflict of interest.

Footnotes

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