Highlights
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This study focused on the molecular regulatory mechanisms of spores under non-thermal processing.
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Data-independent-acquisition method was used to quantify the protein responses to stress.
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Spores down-regulated key proteins in energy metabolic and transportation pathways under stress.
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Spores obtained stress resistance under the external stress.
Keywords: Ultrasound, Proteomic response, Electrolyzed water, Cross-protection, Stress resistance
Abstract
Ultrasound is a green nonthermal technology with promising applications in microbial inactivation. Electrolyzed water has been investigated and found to have a synergistic inactivation effect of ultrasound on spores. This study used a data-independent-acquisition method to analyze the stress response of Bacillus cereus spores following ultrasound combined with electrolyzed water treatment. We identified 197 differentially expressed proteins under ultrasound combined with an electrolyzed water treatment for which the ratio in the metabolic pathway was the highest. Spores downregulated key proteins in energy metabolic and transportation pathways, in particular in phosphotransferase systems and ATP synthase under ultrasound, electrolyzed water, and combined stress. The results of this study revealed that the key proteins in intracellular metabolism decreased after ultrasound treatment, and the expression of small acid-soluble spore protein and cell wall biosynthesis protein increased. Meanwhile, DNA integration, recombination, and inversion protein and small acid-soluble spore protein were upregulated after electrolyzed water treatment. In general, the spores exhibited stress resistance under external stress. The inactivation of spores by further stress was reduced, which we called “cross-protection.”
1. Introduction
Spores provide an adaptation strategy under environmental stress. They are not metabolically active, and this enables the microorganism to survive over long periods of time under extreme environmental conditions [1]. Spores are considered to be a major threat in heat-treated food plants [2]. Bacillus are major agents of food spoilage and food-borne diseases and are extremely resistant to most killing agents [3]. Bacillus cereus is a conditionally pathogenic bacterium that most commonly causes food poisoning through the production of toxins that cause diarrhea and vomiting. Diarrheal toxins and vomiting toxins leading to food poisoning, which lead to food poisoning. Occasionally, infections through the bacterium can cause diseases, such as ocular disease, endocarditis, meningitis, and bacteraemia, in humans [4]. Therefore, an analysis of the stress response of spores holds strong significance.
Nonthermal microbial inactivation technology offers the advantages of microbial inactivation conditions, being easy to control, and being less influenced by external conditions. In the process of nonthermal microbial inactivation technology, foods can be maintained at a relatively low temperature. This treatment has little effect on the color, flavor, or nutritional composition of the food. These technologies help to maintain the physiological activity of the various functional components in food, which can meet the requirements of consumers for high-quality food [5]. The main nonthermal microbial inactivation techniques used to inactivate spores in the food industry include ultrasound, electrolyzed water, high hydrostatic pressure (HHP) technology, nonthermal plasma (NTP) technology, ultraviolet radiation (UV) technology, and pulsed electric field (PEF) technology.
Ultrasound treatment is a mechanical vibration propagation process in a medium that can be applied to microbial inactivation, extraction, and drying [6]. Ultrasound treatment can reduce the intensity of microbial inactivation, maintain food quality, and reduce the loss of functional components [7]. Ultrasound cavitation is generated by transmitting sound waves in a nonflowing liquid system caused by a dissimilarity in the pressure amplitude [8]. This “cavitation” phenomenon was surmised by Euler in his hypothesis on water turbines. It can ensure a noninvasive, nonionizing, and nonpolluting form of processing technique [9], [10]. Sesal et al. found that the particular sonication specifications used were anticipated to be efficient on Gram-negative bacterial inactivation, especially in the case of the ultrasound treatment for a long time [11].
The combined microbial inactivation technology of ultrasound also has a wide range of application prospects [12]. The synergistic effect of ultrasound and acidic oxidation potential water can significantly improve the microbial inactivation rate, and the mechanism of action may be related to the action of air and ultrasound to promote a chemical reaction; in addition, under the action of ultrasound accelerates the microbial cell membrane potential change and membrane permeability increases thus promoting the microbial inactivation rate [13]. Linma Jiang et al. found that ultrasound treatment in combination with slightly acidic electrolytic water resulted in a significant reduction in the number of surviving but nonculturable bacteria; the appearance and ultrastructure of Staphylococcus aureus were more damaged by the two treatments than by the treatment alone [14]. Forghani et al. applied slightly acidic electrolyzed water in combination with ultrasound for decontamination of kashk, found that combined treatment induced 1.87 log CFU/mL reductions of Staphylococcus aureus [15]. But currently, no research has focused on the molecular mechanisms of inactivation induced by ultrasound combined electrolyzed water treatment.
With the development of proteomics technology, the protein-regulated mechanisms during spore formation can be studied in greater depth. Differentially expressed proteins (DEPs) are proteins with significantly different expression levels obtained under different conditions based on the analysis performed using proteomics techniques (e.g., two-dimensional gel electrophoresis, mass spectrometry). These DEPs often can be used to identify potential biomarkers that can help us better understand the physiological and pathological mechanisms of complex biological systems and diagnose and treat diseases. For example, in cancer research, by comparing DEPs in cancer cells and normal cells, potential therapeutic targets can be identified or new diagnostic tools can be developed. Therefore, DEPs are of great importance in proteomics and biomedical research. Proteomics is a technique to study the composition, structure and function of proteins, and it can detect and identify a large number of proteins simultaneously. Proteomics analysis can provide insight into which proteins are expressed and regulated during spore formation and their role in spore formation. In addition, proteomics can provide greater insight into protein interactions and signaling mechanisms. Wang et al. investigated the mechanism of high pressure processing and slightly acidic electrolyzed water on Bacillus cereus spore inactivation using a label-free quantitative proteomics approach [16]. They found that the metabolic, degradation, signaling, and biosynthesis pathways were involved in high pressure processing combined with slightly acidic electrolysed water mediated spore inactivation. Analysis of ultrasound induced spore inactivation mechanism by using proteomics technology also can provide a more reliable theoretical basis for application and industrial development of ultrasound treatment.
2. Materials and methods
2.1. Spore suspension preparation
We obtained Bacillus cereus ATCC 14579 from Haibo Bio-Technology Co.Ltd (Qingdao, China). We cultured the lyophilized strain in a pure form by inoculation in nutrient broth (NB; 10% peptone, 3% beef extract, 5% sodium chloride [NaCl], and 15% agar; Haibo Bio-Technology Co., Ltd.). Glycerol tubes were stored to −80 °C in the refrigerator for backup. After overnight incubation in a shaker until the stabilization phase, the supernatant was discarded after centrifugation. The spore suspension was taken and spread on nutrient agar plates that were supplemented with manganese sulfate tetrahydrate, which were then incubated at 37 °C for 2–7 days to induce spore formation. We stored the samples at −20 °C in the refrigerator for backup.
2.2. Ultrasound and electrolyzed water treatment
We performed the ultrasound treatment with a probe ultrasound processor with a 900 W, 20 kHz, duty cycle of 1:1, with a 10-mm-diameter titanium solid probe. The ultrasound probe was immersed 1.5 cm into a cylindrical ultrasound glass tube (85 mL) containing 25 mL of diluted budding cell suspension (approximately 8 log CFU/mL) of spore suspension. A constant temperature of 20 ± 1 °C was maintained with a water bath during this period.
We performed electrolyzed water treatment in the same 85-mL sonication tube. We took 2.5 mL of spore suspension and mixed it thoroughly with 22.5 mL of the electrolyzed water sample (available chlorine concentration of 30 mg/L, pH of 3.2, oxidation–reduction potential of 956 mV). After a specific treatment time at 20 ± 1 °C, the samples were mixed with the neutralization solution (0.85% NaCl + 0.5% Na2S2O3) to neutralize the residual electrolyzed water in the samples and end the electrolyzed water treatment.
For ultrasound combined electrolyzed water treatment, we took 2.5 mL of spore suspension and mixed it with 22.5 mL of electrolyzed water. We simultaneously turned on the ultrasound treatment and placed the sample in a water bath to maintain a constant temperature of 20 ± 1 °C. To test the effect of ultrasound on the properties of electrolyzed water, we mixed 2.5 mL of sterile water with 22.5 mL of electrolyzed water.
2.3. Spore protein extraction and quantification
We added 10 mL of lysate to 1 mL of spore suspension and suspended it and added a small amount of glass beads. We crushed it for 5 min using a crusher, centrifuged the spore suspension at 30/s, 10,000 r/min for 10 min, and stored the supernatant at − 20 °C. We performed protein quantification and quality control and continued to the next step of detection. We determined soluble whole-cell proteins using a modified Bradford method protein concentration assay kit. The protein content was determined by the enzyme labeling method using Thomas Brilliant Blue. The equation of the standard curve for the determination of protein content by the Kaumas Brilliant Blue method is A = 0.0022C + 0.3136 (R2 = 0.9912) [17]. Protein extraction can also be performed using a bidirectional polyacrylamide gel electrophoresis technique [18].
2.4. Enzymatic digestion of spore proteins
For each sample, we added 100 μg of protein solution to Trypsin and digested it at 37 °C for 4 h. To complete the digestion, the same weight of Trypsin was added and digestion was continued at 37 °C for 8 h. The digested peptides were desalted using a Strata X column and then were vacuum dried to obtain the digested peptides.
2.5. High pH isolated peptide
We mixed all of the extracted peptide samples with 10 μg each and added 2 mL of mobile phase A (4% acetonitrile) to the sample. The samples were separated in liquid phase using a Shimadzu LC-20AB liquid phase system with a Gemini C18 column (5 μm inner diameter, 25 cm column length, and 4.6 mm inner diameter of the column material) as the separation column. The elution rate was 1 mL/min. The elution peaks at 214 nm were monitored, and one fraction was collected every minute, and the samples were finally combined with the chromatographic elution peak spectra to obtain 10 fractions, which were finally freeze-dried.
2.6. High-performance liquid chromatography
The drained peptide sample was redissolved in mobile phase A (2% acetonitrile, 0.1% acetic acid), and the supernatant was taken into the sample. Then, we separated the peptide using UltiMate 3000 ultra-performance liquid chromatography.
2.7. DDA and DIA mass spectrometry detection
We performed both data-dependent acquisition (DDA) and data-independent acquisition (DIA) analysis on a Q-Exactive HF mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA). For DDA analysis, the parameters were as follows: ion source voltage was 1.6 kV, mass spectrometry (MS) scan range was from 350 to 1500 m/s, MS resolution was 60,000; MS/MSHCD scan range was from 15,000, m/z 100, dynamic exclusion duration was 30 s, and AGC settings were as follows: primary 3E6, secondary 1E5. Intensity threshold was 10,000. The charge exclusion was as follows: exclude greater than +7 or less than 2+. DIA analysis was conducted according to the following parameters: ion source voltage of 1.6 kV, full scan range of 350–1500 m/z at a resolution of 120,000, and loop count of 40. Fragment ions were detected with an Orbitrap column, at a dynamic exclusion duration of 30 s, and AGC settings were as follows: primary 3E6, secondary 1E5.
2.8. Bioinformatics analysis
We divided the process of bioinformatic analysis into DDA analysis, DIA analysis, and MSstats variance analysis. Among these analyses, DDA analysis was based mainly on the sample data generated by the high-resolution MS. The spectral library was constructed using Spectronaut after the identification was completed using the Andromeda engine integrated with MaxQuant. When the number of samples was large, we used DIA to analyze data. Then, the constructed spectral library information was used to deconvolute the data for extraction, and the mProphet algorithm was used to complete the quality control analysis of the data, which made the analysis results more reliable. In addition, we included gene ontology (GO) analysis, COG analysis, and pathway function annotation analysis.
3. Results and discussions
3.1. Ultrasound combined electrolyzed water treatment of DEPs and their functional annotation
3.1.1. Identification and quantification of DEPs
Ultrasound combined with electrolyzed water treatment of spores had a synergistic inactivation. Using protein DIA quantification to identify differential proteins after treatment, the results showed that a total of 197 proteins had significant changes in expression after treatment (fold change ≥ 2, p < 0.05). Wang et al [16] used electrolyzed water treatment on Bacillus cereus and obtained less than 100 DEPs using iTRAQ technique. Although previous studies have shown that ultrasound had little inactivation effect on Bacillus cereus spores, a large number of proteins were regulated to respond to the ultrasound treatment. This was a possible reason for the decrease in resistance after ultrasound treatment [19]. This decrease indicated that there was no correlation between the inactivation effect and the number of DEPs. Fig. 1 shows the volcano plots of DEPs after ultrasound combined with electrolyzed water treatment, which allowed for a visual screening of DEPs. Proteins that had significant changes in expression represented a smaller percentage of all of the proteins and a greater number of proteins with downregulated expression.
Fig. 1.
Volcano plot of changes in the levels of identified Bacillus cereus spore proteins after simultaneous ultrasound and electrolyzed water treatment.
3.1.2. Differential protein GO enrichment analysis
The categorical functional annotation of all 197 DEPs using the Hierarchical Structured Vocabulary GO [20] enrichment analysis method is shown in Fig. 2. According to biological processes, DEPs were distributed mainly in the cellular processes, metabolic processes, and localization. According to molecular functions, DEPs were distributed mainly in the annotated entries of catalytic activity, binding, and translocation activities. According to protein localization, differential proteins were enriched mainly in the membranes, membrane fractions, and cells, and the distribution of differential proteins after co-treatment was closer to that after ultrasound treatment. We also identified some distributions that had highly analyzed complexes, organelles, organelle fractions, and nuclei, and majority of proteins in these parts were downregulated expression.
Fig. 2.
Functional categorization based on gene ontology (GO) analysis of significantly expressed proteins of Bacillus cereus spores after simultaneous ultrasound and electrolyzed water treatment. Red columns represent upregulated DEP numbers, and blue columns represent downregulated DEP numbers. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
3.1.3. Differential protein pathway enrichment analysis
Pathway databases, especially Kyoto Encyclopedia of Genes and Genomes (KEGG), have been widely used as a reference knowledge base for biomedical scientists to interpret their experimental findings [21]. The KEGG pathway classification statistics of the DEPs after ultrasound combined with electrolyzed water treatment are shown in Fig. 3. The differential proteins were distributed in four main categories: cellular process, environmental information processing, genetic information processing, and metabolism. Among these proteins, one differential protein was annotated to a cellular process pathway and was involved in the transport and catabolic pathway, two proteins were annotated to an environmental information processing pathway and were involved in membrane transport, and seven proteins were annotated to a genetic information processing pathway and were involved in translation and a folding, sorting, and degradation pathway. Another 88 proteins were annotated to a metabolic pathway and were involved in nine pathways, which included a global overview map, amino acid metabolism, carbohydrate metabolism, and cofactor and vitamin metabolism. A higher percentage of differential proteins were annotated to metabolic pathways after ultrasound combined with electrolyzed water treatment compared with ultrasound alone and compared with electrolyzed water treatment alone. Significantly downregulated protein short-chain dehydrogenases are key regulatory proteins, which are classified into initial short-chain dehydrogenases and extended short-chain dehydrogenases, depending on their length and cofactor binding sequences [22], [23]. Jörnvall et al. [24], [25], [26] focused on short-chain dehydrogenases and isolated short-chain fatty acids of different structures in bacterial and yeast organisms. Most of the short-chain fatty acids played key roles in cell differentiation and signaling, including carbohydrate metabolism. In the metabolic pathways of fatty acid and ketone body synthesis in Bacillus cereus spores, short-chain dehydrogenases are key regulatory proteins [27], and the downregulation of their expression leads to the blockage of several pathways.
Fig. 3.
Distribution of DEPs in Bacillus cereus spores after simultaneous ultrasound and electrolyzed water treatment in KEGG pathways.
Fig. 4 shows the pathway statistics of the significant enrichment of DEPs after ultrasound combined with electrolyzed water treatment. Only photosynthesis and oxidative phosphorylation pathways were enriched with differential proteins. In the photosynthesis pathway, three DEPs had significantly decreased expression. The expression of 12 DEPs in the oxidative phosphorylation pathway also showed a significant decrease. This result indicated that the level of oxidative phosphorylation in the spore was significantly decreased after ultrasound combined with electrolyzed water treatment, and the ability to generate ATP was reduced. The number of significantly enriched pathways was less than that with ultrasound treatment alone (Fig. 5).
Fig. 4.
Pathway with significant enrichment after simultaneous ultrasound and electrolyzed water treatment.
Fig. 5.
Pathway network diagram with enrichment after simultaneous ultrasound and electrolyzed water treatment.
Compared with ultrasound treatment alone, the number of enriched pathways was smaller and relatively less closely associated. The enriched pathways, however, were mainly centered on oxidative phosphorylation and amino acid metabolism after ultrasound combined with electrolyzed water treatment. This result indicated that ultrasound combined with electrolyzed water treatment primarily affected intracellular ATP synthesis and amino acid metabolic pathways in the spores.
3.1.4. Analysis of differential protein interactions
Fig. 6 shows the network diagram of the differential protein interactions before and after the ultrasound combined with electrolyzed water treatment. According to this figure, the correlation between the differential proteins after ultrasound combined with electrolyzed water treatment was not as strong as that after treatment with electrolyzed water alone. The combined ultrasound treatment may have had a disruptive effect on the interactions between the differential proteins. The spores contained many acid-soluble small molecules of spore proteins (SASP), which accounted for approximately 20% of the total spore core proteins. These double-stranded DNA-binding proteins changed the DNA into an a-like conformation [28]. Thus, the presence of SASP could protect DNA within the spore core from damage to a greater extent [29]. The significant upward trend in SASP expression after electrolyzed water treatment suggested that spores express SASP in large amounts to protect the core material of the spore in response to the external oxidizing environment. Raju et al. [30] found that the α/β type of SASP played an important role in the thermal resistance of Clostridium perfringens spores. Table 1 summarizes information about some of the key protein clusters after ultrasound combined with electrolyzed water treatment. The interactions between differential proteins consisted of two main parts: the first and largest protein cluster involved the metabolism-related proteins, including carbohydrate metabolism, oxidative phosphorylation, and the tricarboxylic acid (TCA) cycle. Among them, proteins such as ATP synthase, NADH dehydrogenase, cytochrome c oxidase, and succinate dehydrogenase were central to the interactions among this protein cluster. The second protein cluster involved relatively few proteins, which were all spore stress-related proteins, including the two-component signaling system, chemotactic proteins, and flagella. The two-component signaling system was a key signaling system for microorganisms to sense changes in the external environment and to respond further by converting changes in the external environment into signals to alter the intracellular physiological state. This system consisted of two main components, in which histidine protein kinase could sense signals in the environment, and homologous responses regulated proteins, output signals, and conducted them to the intracellular state [31]. In contrast, the expression of the two-component signaling system was significantly downregulated after the action of ultrasound combined with potentiated water, indicating a reduced ability of the spore to sense external environmental changes to conduct signals in response.
Fig. 6.
Protein–protein interaction networks of DEPs of Bacillus cereus spores after simultaneous ultrasound and electrolyzed water treatment pathways.
Table 1.
Part of the DEPs in protein–protein interaction networks after simultaneous ultrasound and electrolyzed water treatment.
| Protein ID | Ratio | Changes | Protein Name |
|---|---|---|---|
| NP_834227.1 | −1.57 | down | succinate dehydrogenase cytochrome b558 subunit |
| NP_833662.1 | −1.69 | down | cytochrome c oxidase polypeptide I |
| NP_833661.1 | −1.34 | down | cytochrome c oxidase polypeptide III |
| NP_830510.1 | −1.54 | down | cytochrome aa3 quinol oxidase polypeptide I |
| NP_830511.1 | −1.17 | down | cytochrome aa3 quinol oxidase polypeptide II |
| NP_834494.1 | −1.96 | down | cytochrome d ubiquinol oxidase subunit I |
| NP_834956.1 | −4.71 | down | NADH dehydrogenase subunit L |
| NP_834964.1 | −1.42 | down | NADH dehydrogenase subunit A |
| NP_834955.1 | −2.41 | down | NADH dehydrogenase subunit M |
| NP_834595.1 | −2.33 | down | hypothetical protein BC4923 |
| NP_834974.1 | −1.41 | down | ATP synthase F0F1 subunit A |
| NP_832492.1 | −2.44 | down | preprotein translocase subunit SecY |
| NP_835141.1 | −3.81 | down | OxaA-like protein precursor |
| NP_834972.1 | −1.30 | down | ATP synthase F0F1 subunit B |
| NP_833572.1 | 1.38 | up | glycerol-phosphate acyltransferase PlsX |
| NP_830007.1 | −1.14 | down | 30S ribosomal protein S7 |
| NP_833911.1 | 2.47 | up | elongation factor P |
| NP_833443.1 | 2.01 | up | PTS system-fructose-specific subunit IIABC |
| NP_832528.1 | 2.94 | up | dihydrolipoamide dehydrogenase |
| NP_833769.1 | 1.08 | up | BigG family transcription antiterminator |
| NP_831083.1 | −1.40 | down | two component system histidine kinase |
| NP_830628.1 | −2.81 | down | PTS system-sucrose-specific subunit IIBC |
3.2. Key proteins for spore stress response
Microorganisms can respond rapidly to changing environments by regulating protein expression. Many responses to stress are specific, with regulatory mechanisms tailored to external signals [32]. The general stress response, however, was activated once microorganisms entered dormancy or were subjected to nutrient depletion. This unique feature is the common and most extensive output in response to different external signals. In many different microorganisms, the key regulator of the general stress response is a specific sigma factor—the promoter-specific subunit of RNA polymerase [33].
Ultrasound treatment is a physical processing technique that causes mechanical damage to spores structure such as promotion the detachment of the exosporium and damage the permeability of the inner membrane [34]. Electrolyzed water, in contrast, has a high redox potential as well as strong oxidizing properties (active chlorine) to chemically damage the structure of proteins as well as nucleic acid in the Bacilli [35]. To investigate the mechanisms underlying the general stress response of Bacillus cereus spores in response to external stress stimuli, we analyzed and screened DEPs in the spores under different treatment conditions. The results yielded 15 proteins whose expression was significantly altered after ultrasound, electrolyzed water, and ultrasound combined with electrolyzed water treatments (Table 2). These proteins played an important role in the response of spores to different external stress stimuli.
Table 2.
DEPs after different treatments.
| Protein ID | Changes |
Ratio |
Protein Name | ||
|---|---|---|---|---|---|
| US | AEW | US-AEW | |||
| NP_829918.1 | down | −1.11 | −1.07 | −1.69 | D-alanyl-D-alanine carboxypeptidase |
| NP_830628.1 | down | −2.02 | −1.50 | −2.81 | PTS system-sucrose-specific subunit IIBC |
| NP_831015.1 | down | −1.83 | −1.43 | −2.67 | sodium/proline symporter |
| NP_831145.1 | down | −1.36 | −1.84 | −1.68 | hypothetical protein BC1364 |
| NP_832024.1 | down | −1.35 | −1.59 | −3.37 | cobalt-zinc-cadmium resistance protein czcD |
| NP_832245.1 | up | 1.63 | 1.78 | 3.24 | hydroxymethylglutaryl-CoA lyase |
| NP_833130.1 | down | −1.21 | −1.01 | −1.19 | hypothetical protein BC3395 |
| NP_833437.1 | up | 2.60 | 2.87 | 3.22 | DNA integration/recombination/invertion protein |
| NP_833511.1 | down | −2.34 | −2.32 | −3.06 | nucleoside ABC transporter permease |
| NP_834013.1 | down | −1.11 | −1.61 | −2.24 | metal-dependent phosphohydrolase |
| NP_834102.1 | up | 1.05 | 1.04 | 1.06 | TPR repeat-containing protein |
| NP_834350.1 | down | −1.47 | −1.66 | −2.72 | PhnB protein |
| NP_834891.1 | down | −1.01 | −1.29 | −1.78 | L-lactate permease |
| NP_834956.1 | down | −1.99 | −1.92 | −4.71 | NADH dehydrogenase subunit L |
| NP_834967.1 | down | −1.03 | −1.16 | −1.53 | ATP synthase F0F1 subunit epsilon |
The upregulated and downregulated expression of DEPs was consistent after different treatments (Table 2). Fig. 7 shows the main protein regulations of Bacillus cereus spores under stress. Among them, the proteins whose expression was significantly downregulated after treatment were related mainly to energy metabolism and material transport, specifically the PTS system sucrose-specific subunit, sodium/proline transporter, ATP synthase, nucleoside ABC transporter permease, and lactate permease. The three proteins with significantly upregulated expression were hydroxymethylglutaryl-coAlyase; DNA integration, recombination, and transformation protein; and TPR repeat-sequence protein. The PTS system was responsible for bacterial intracellular carbohydrate translocation and utilization, and more than 20 intracellular carbohydrates were translocated using the PTS system [36]. In addition, the PTS system could transmit signals that inhibited further intracellular catabolism. The PTS system ensured maximum carbohydrate utilization by microorganisms under external stress [37]. After ultrasound combined with electrolyzed water stress, the expression of sucrose-specific subunits of the PTS system followed a significant downward trend, and the ability of the spore to translocate and utilize carbohydrates decreased. The expression of ATP synthase also followed a significant downward trend. Meanwhile, the intracellular ability to synthesize ATP decreased, and the core processes of energy metabolism were affected.
Fig. 7.
Schematic diagram of main protein regulations of Bacillus cereus spores under stress.
3.3. Synergistic inactivation mechanism of spores by ultrasound and electrolyzed water
The combined action of ultrasound and electrolyzed water had a synergistic effect on the inactivation of Bacillus cereus spores [38]. The cavitation effect of ultrasound made microorganisms more susceptible to electrolyzed water action and also may be related to the free radicals generated in the liquid phase [39]. The cavitation effect of ultrasound had a significant disruptive effect on the dense structure of the spores from the phenotypic properties, which facilitated the penetration of electrolyzed water into the interior of the spores. The high redox potential of electrolyzed water as well as the effective chlorine disrupted the intracellular protein and nucleic acid structures. To elucidate the synergistic inactivation mechanism of ultrasound combined with electrolyzed water on spores from a molecular perspective, we analyzed the changes in the expression of key proteins of spore stress after ultrasound combined with electrolyzed water treatment and screened the proteins associated with this synergistic effect (Table 3).
Table 3.
The key proteins in the synergistic effect of ultrasound and electrolyzed water.
| Protein ID |
Ratio |
Protein Name | ||
|---|---|---|---|---|
| US | AEW | US-AEW | ||
| NP_831083.1 | −1.61 | −0.70 | −1.40 | two component system histidine kinase |
| NP_831431.1 | 0.07 | −1.49 | −2.36 | chemotaxis protein CheV |
| NP_831422.1 | −0.98 | −0.02 | −1.75 | flagellar MS-ring protein |
| NP_831417.1 | 0.71 | −1.92 | −2.20 | flagellar protein fliS |
| NP_834960.1 | −1.74 | 0.33 | −1.19 | NADH dehydrogenase subunit H |
| NP_834964.1 | −1.62 | 0.27 | −1.42 | NADH dehydrogenase subunit A |
| NP_834955.1 | −2.47 | 0.09 | −2.41 | NADH dehydrogenase subunit M |
| NP_834956.1 | −1.99 | −1.92 | −4.71 | NADH dehydrogenase subunit L |
| NP_834972.1 | −2.54 | 0.28 | −1.3 | ATP synthase F0F1 subunit B |
| NP_834974.1 | −2.34 | 0.24 | −1.41 | ATP synthase F0F1 subunit A |
| NP_834967.1 | −1.03 | −1.16 | −1.53 | ATP synthase F0F1 subunit epsilon |
In general, after ultrasound combined with electrolyzed water treatment, the expression of key proteins to stress response showed a significant increase compared with the treatment alone. After ultrasound treatment, the expression of the ATP synthesis-related and oxidative phosphorylation pathway and TCA cycle proteins showed a significant downregulation trend. The expression of a large number of key proteins in the key pathways of spore metabolism was downregulated, and the energy metabolism of spores was hindered. As a result, the energy required for various life activities could not be guaranteed. The expression of proteins related to energy metabolism did not change significantly after electrolyzed water treatment, whereas the expression of chemotactic proteins and flagellin showed a significant decrease. The expression of proteins related to energy metabolism in the spore was significantly downregulated after ultrasound combined with electrolyzed water treatment, and the expression of histidine kinase, a two-component signaling system, was also significantly downregulated. Additionally, the expression of chemotactic proteins and flagellin was significantly downregulated. The key proteins of intracellular stress revealed a superimposed effect after the combined treatment, which was presumed to be the molecular mechanism of the synergistic inactivation effect.
3.4. Spore’s cross-protection mechanism
The inactivation of spores treated with different sequences of ultrasound and electrolyzed water did not increase significantly compared with a single treatment. This result was probably due to the stress resistance and cross-protection phenomenon [40]. The cross-protection meant bacterial under mild inactivation stress obtained adaptation to the next inactivation treatment. The cross-protection phenomenon of spores seriously jeopardizes food safety and induces different stress environments, and it has different triggering mechanisms. In addition to electrolyzed water combined with ultrasound treatment, high osmotic pressure and cold stress also could cause cross-protection [41]. Table 4, Table 5 show protein expression after ultrasound combined with electrolyzed water treatment, indicating this possible mechanism of cross-protection.
Table 4.
DEPs after ultrasound treatment.
| Protein ID | Ratio | Changes | Protein Name |
|---|---|---|---|
| NP_833661.1 | −1.67 | down | cytochrome c oxidase polypeptide III |
| NP_833662.1 | −1.61 | down | cytochrome c oxidase polypeptide I |
| NP_833663.1 | −1.37 | down | cytochrome c oxidase polypeptide II |
| NP_830510.1 | −1.53 | down | cytochrome aa3 quinol oxidase polypeptide I |
| NP_830511.1 | −1.37 | down | cytochrome aa3 quinol oxidase polypeptide II |
| NP_834494.1 | −2.31 | down | cytochrome d ubiquinol oxidase subunit I |
| NP_834974.1 | −2.34 | down | ATP synthase F0F1 subunit A |
| NP_834971.1 | −1.20 | down | ATP synthase F0F1 subunit delta |
| NP_834972.1 | −2.54 | down | ATP synthase F0F1 subunit B |
| NP_831301.1 | −1.17 | down | menaquinol-cytochrome c reductase iron-sulfur subunit |
| NP_831303.1 | −1.43 | down | menaquinol-cytochrome c reductase cytochrome c subunit |
| NP_834227.1 | −1.35 | down | succinate dehydrogenase cytochrome b558 subunit |
| NP_834955.1 | −2.47 | down | NADH dehydrogenase subunit M |
| NP_834956.1 | −1.99 | down | NADH dehydrogenase subunit L |
| NP_834964.1 | −1.62 | down | NADH dehydrogenase subunit A |
| NP_834480.1 | −3.24 | down | ABC transporter substrate-binding protein |
| NP_834916.1 | −1.42 | down | ABC transporter permease |
| NP_832880.1 | 1.52 | up | oxidoreductase |
| NP_830238.1 | 2.09 | up | oxidoreductase |
| NP_830323.1 | 1.22 | up | cell wall biosynthesis glycosyltransferase |
| NP_830661.1 | 1.52 | up | small acid-soluble spore protein |
| NP_830616.1 | 1.73 | up | DNA-binding protein |
| NP_830300.1 | 1.14 | up | general stress protein 26 |
| NP_834573.1 | 1.14 | up | general stress protein 13 |
Table 5.
DEPs after electrolyzed water treatment.
| Protein ID | Ratio | Changes | Protein Name |
|---|---|---|---|
| NP_832528.1 | 2.36 | up | dihydrolipoamide dehydrogenase |
| NP_833443.1 | 2.74 | up | PTS system-fructose-specific subunit IIABC |
| NP_833560.1 | −1.79 | down | tRNA (guanine-N(1)-)-methyltransferase |
| NP_831431.1 | −1.49 | down | chemotaxis protein CheV |
| NP_833607.1 | −1.44 | down | carbamoyl phosphate synthase small subunit |
| NP_829979.1 | −1.20 | down | lysyl-tRNA synthetase |
| NP_831391.1 | −1.13 | down | Zn-dependent hydrolase |
| NP_834890.1 | −1.02 | down | thiamine biosynthesis protein ThiC |
| NP_833517.1 | −1.29 | down | translocation-enhancing protein tepA |
| NP_833437.1 | 2.87 | up | DNA integration/recombination/invertion protein |
| NP_832834.1 | 2.14 | up | small acid-soluble spore protein |
| NP_834926.1 | −1.16 | down | UDP-glucose 4-epimerase |
| NP_834940.1 | −1.18 | down | tyrosine-protein kinase |
| NP_832835.1 | −1.24 | down | glutathione-dependent formaldehyde dehydrogenase |
After ultrasound treatment, the expression of proteins related to energy metabolism in the spores showed a significant decrease, including ATP synthase and oxidative phosphorylation key proteins. The expression of key proteins in the main metabolic pathways decreased, and intracellular metabolism was affected. The spores had relatively weak metabolic and vital activities, which was one of the reasons for the high resistance of the spores. After ultrasound treatment, the metabolism of the spores was at a lower level, and resistance was then increased. The ultrasound treatment caused some structural damage to the spores. However, the expression of cell wall biosynthetic glycosyltransferase was significantly upregulated, which indicated that ultrasound treatment induced the spores to accelerate cell wall synthesis and repair. The acid-soluble small molecule spore protein SASP played a key role in spore resistance, and the expression of SASP was significantly increased after ultrasound treatment. In addition, the expression of general stress proteins was significantly elevated after ultrasound treatment. Overall, after ultrasound treatment, the intracellular stress level was elevated significantly. The surviving spores were more resistant to the subsequent stress. Therefore, the total inactivation of spores was not significantly increased by the ultrasound combined with electrolyzed water treatment, which resulted in the phenomenon of cross-protection, which is shown in Fig. 8.
Fig. 8.
Schematic diagram of the synergistic mechanism of Bacillus cereus spores under simultaneous ultrasonic and acidic electrolyzed water treatment.
As shown in Table 5, the expression of DNA integration, recombination, and transformation proteins was significantly increased after electrolyzed water treatment, and the repair capacity of intracellular DNA was strongly enhanced. In addition, the expression of SASP increased and spore resistance was elevated. After the electrolyzed water treatment, the less resistant part of the spores had been inactivated, and the surviving spores had a significant increase in resistance because of regulation at the molecular level. The subsequent ultrasound treatment had no significant inactivation effect on the treated spores.
4. Conclusions
In this study, we identified how spores responded to stress using ultrasound combined with electrolyzed water treatment. We achieved these results by regulating intracellular metabolism and material transportation. In addition, key proteins in energy metabolism and two component systems showed superimposed effects after combined treatment, which caused a synergistic inactivation effect of the spores. The resistance of spores was significantly increased under the external stress, and the inactivation of spores by further stress was reduced, forming a “cross-protection” phenomenon. Ultrasound combined with electrolyzed water treatment has a broad application prospect in microbial inactivation. This study revealed the potential molecular mechanism of cross-protection of spores under stress. These findings established a theoretical foundation and provided new ideas for the application of ultrasound and electrolyzed water in microbial inactivation and promoted its application in food processing.
CRediT authorship contribution statement
Zixuan Jia: Data curation, Writing – original draft. Jianwei Zhou: Investigation, Methodology, Visualization. Jingzeng Han: Investigation. Donghong Liu: Conceptualization, Supervision. Ruiling Lv: Methodology, Writing – review & editing, Project administration.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
This study was supported by the National Natural Science Foundation of China (grant number 32202221) and the Natural Science Foundation of Ningbo (grant number 2022J166).
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