Application of cell wall lyase LysM15 in preparation of bacillus cereus inhibitor

Abstract

The invention belongs to the field of food safety, and discloses application of cell wall lyase LysM15 in preparation of a bacillus cereus inhibitor. The cell wall lyase LysM15 has the functions of specifically recognizing the cell wall of the bacillus cereus and efficiently cracking the peptidoglycan layer of the bacillus cereus. The LysM15 not only can obviously inhibit the germination of bacillus cereus spores and remove biofilms of the bacillus cereus spores, but also can keep higher enzyme activity in a temperature range of 4-50 DEG C and in a high-salt environment. The lyase has good biocompatibility, growth and pollution of bacillus cereus in fresh beef can be effectively reduced, the potential of the lyase serving as a food preservative is shown, and a safe and efficient solution can be provided for food preservation.

Description

Technical Field

[0001] This invention belongs to the field of food safety, specifically relating to the application of cell wall lysin LysM15 in the preparation of Bacillus cereus inhibitors. Background Technology

[0002] Food safety issues, especially those caused by microorganisms, are a significant public health concern that cannot be ignored. Bacillus cereus (… Bacillus cereus As a common foodborne pathogen, it is widely found in soil, water, and various food substrates, exhibiting strong environmental adaptability. Under adverse environmental conditions, this bacterium can form highly resistant spores and biofilms, producing enterotoxins and vomitoxins in food, thereby causing symptoms such as diarrhea and vomiting. Bacillus cereus Food intoxication and toxicoinfection. Comprehensive Reviews in Food Science and Food Safety, 20(4), 3719-3761. It is noteworthy that vomitoxin-producing strains pose a particular health threat due to their ability to synthesize the Bacillus cereus toxin Cereulide. This toxin is heat- and acid-resistant, and can cause severe poisoning symptoms even at low concentrations, potentially leading to liver failure or death (Sudden death of a young adult associated with...). Bacillus cereus Food poisoning. Journal of Clinical Microbiology, 49, 4379-4381. With the continuous expansion of the food industry and the widespread application of cold chain logistics, contamination incidents caused by Bacillus cereus are becoming increasingly frequent, posing a more severe challenge to the safety of food preservation, storage, and transportation.

[0003] Traditional methods (such as heat treatment, chemical disinfectants, and synthetic preservatives) are often ineffective in removing the biofilm and spores of Bacillus cereus, posing a significant challenge to its control. Furthermore, the excessive use of antibiotics and chemical preservatives has led to the emergence of multidrug-resistant strains of Bacillus cereus, and chemical preservatives can negatively impact human health. Therefore, developing a safe, non-toxic, highly effective, and stable novel antibacterial agent is of great importance.

[0004] The bacterial cell wall, primarily composed of peptidoglycan, is a crucial barrier for maintaining structural integrity, resisting osmotic pressure, and protecting intracellular components. Due to its highly conserved structure and vital role in growth, the cell wall is considered an ideal target for antimicrobial intervention. Cell wall lyases are a class of proteins that specifically recognize and efficiently hydrolyze the peptidoglycan layer of bacteria. These enzymes can originate from bacteriophages (called endolysins) or the bacteria themselves (called autolysins), and typically consist of a catalytically active domain and a targeting wall domain. Due to their high specificity and efficiency, cell wall lyases are considered promising candidates for novel food preservatives. However, current research mainly focuses on model strains such as Listeria and Staphylococcus aureus, while research on Bacillus cereus lyases is relatively limited, and mostly remains at the preliminary verification stage of lysing activity under buffer conditions. Especially in complex food systems, systematic and in-depth research is lacking on the inhibitory effects of lyases on Bacillus cereus, as well as their comprehensive control role in spore germination and biofilm formation.

[0005] Based on this, this invention screened and identified a novel cell wall lysin candidate protein, LysM15 (GenBank accession number WP_001199272). This protein had previously only been classified as a conserved protein of the DUF1202 family based on nucleotide sequence prediction in databases, with no experimental data reported. This invention provides the first systematic experimental verification of its function, confirming its potential application value in the control of Bacillus cereus. Summary of the Invention

[0006] The purpose of this invention is to overcome at least one deficiency of the prior art and to provide the application of cell wall lysin LysM15 in the preparation of Bacillus cereus inhibitors.

[0007] The technical solution adopted in this invention is: The first aspect of the present invention provides: Application of cell wall lysin LysM15 in the preparation of Bacillus cereus inhibitor, wherein the amino acid sequence of the cell wall lysin LysM15 is MQYHNRNVSNLNKLADNTKAAAFKWYQYCIDNGIEVLIYETIRTVEQQREYVRKGASQTMRSYHLVGQALDFVPIQSNGTEDWNGYNKEPWASAIRYAKQIGFEWGGDWKGFVDSPHLQYNYKGYGTDTFGKGAQNVVTPPPSNDSVGIAYINGSNVNLRKGPGTGYGVIRQLGKGESYKVFGQSNGWLNLGGDQWVYNDPSYIRYTGGNVPATSQSSNDGVGVVTIIADVLRVRTGPGTNYGIVKNVYQGEKYQSFGYKDGWYNVGGNQWVSGEYVTFVK (SEQ ID NO.1).

[0008] Experimental data show that the cell wall lysin LysM15 can not only lyse the vegetative cells of Bacillus cereus, but also inhibit its spore germination and remove its biofilm. It also maintains high lysing activity in the range of 4℃ to 50℃ and under 300 mM NaCl conditions.

[0009] In some instances, the Bacillus cereus inhibitor is a Bacillus cereus inhibitor for food preservation.

[0010] In some instances, the food is selected from at least one of fresh meat, dairy products, fruits, vegetables, and cooked foods. Specifically, this includes, but is not limited to, beef, pork, lamb, poultry, milk, dairy products, fruits, vegetables, cooked food products, and ready-to-eat meals.

[0011] In some instances, the Bacillus cereus inhibitor is a Bacillus cereus inhibitor for fermentation.

[0012] A second aspect of the present invention provides: A food preservative additive comprising cell wall lyase LysM15, the amino acid sequence of which is shown in SEQ ID NO.1.

[0013] In some instances, the excipients are humectants and buffer salt solutions.

[0014] In some instances, the humectant is glycerol, and the buffer salt is Tris-HCl buffer. Preferably, the glycerol concentration is 10% and the Tris-HCl buffer concentration is 20 mM.

[0015] A third aspect of the present invention provides: A method for inhibiting foodborne Bacillus cereus includes dispersing an appropriate amount of cell wall lysin LysM15 on the surface of the food, the amino acid sequence of which is shown in SEQ ID NO.1.

[0016] In some instances, the food is selected from at least one of fresh meat, dairy products, fruits, vegetables, and cooked foods. Specifically, this includes, but is not limited to, beef, pork, lamb, poultry, milk, dairy products, fruits, vegetables, cooked food products, and ready-to-eat meals.

[0017] In some instances, the methods of dispersion include, but are not limited to, spraying, soaking, and coating. Surfaces include both outer and inner surfaces.

[0018] The beneficial effects of this invention are: The inventors have for the first time discovered and verified that the cell wall lysin LysM15 retains over 80% of its lytic activity within 60 days when stored at -20℃, indicating good long-term storage stability. Its lytic activity remains relatively stable between 4℃ and 40℃, and retains approximately 90% of its activity at 50℃, demonstrating good thermal stability. LysM15 retains approximately 90% of its activity at concentrations up to 300 mM NaCl, showing good salt stability. Under pH conditions of 7 to 8, the lysin activity is high, making it suitable for use in neutral and slightly alkaline environments. Taking fresh beef as an example, samples treated with LysM15 maintained a relatively uniform color and showed fewer signs of microbial contamination, while the control group showed significant color and texture changes after 6 days, accompanied by a strong odor. Furthermore, compared to the control group, LysM15 or lysozyme treatment significantly (p<0.01) reduced the number of Bacillus cereus colonies, indicating its effective inhibition of Bacillus cereus growth. These results demonstrate that LysM15 is as effective as lysozyme in inhibiting the growth of Bacillus cereus in beef, highlighting its potential as a food preservative.

[0019] Further research showed that LysM15 can disrupt the cellular structure of Bacillus cereus and induce intracellular DNA leakage, supporting its ability to disrupt cell wall integrity. Purified cell walls incubated with LysM15 (12.5 µg / mL) for 10 minutes showed an OD... 600 The value decreased significantly (p<0.05), further indicating that LysM15 effectively degraded the bacterial cell wall. These findings confirm that LysM15 can effectively degrade the bacterial cell wall, thereby exerting an antibacterial effect. Attached Figure Description

[0020] Figure 1This study focuses on the characterization and antibacterial activity of LysM15. (A) Domain composition and predicted three-dimensional structure of LysM15; (B) SDS-PAGE analysis of purified LysM15 protein; (C) Cleavage activity of LysM15 at different concentrations; (D) Disruption of pre-formed biofilms by LysM15 at different concentrations; (E) Inhibitory effect of LysM15 on spore germination. *p<0.05, **p<0.01.

[0021] Figure 2 This study evaluates the stability and biocompatibility of LysM15. (A) Relative lytic activity of LysM15 after incubation at different temperatures, assessing its thermal stability; (B) Salt tolerance of LysM15 tested at different NaCl concentrations; (C) Cleavage activity of LysM15 under different pH conditions; (D) Storage stability of LysM15 at -20℃; (E) Hemolytic activity of LysM15 against mouse erythrocytes; (F) Cytotoxicity of LysM15 against RAW264.7 cells. *p<0.05, **p<0.01, ***p<0.001.

[0022] Figure 3 This describes the preservation effect of LysM15 on fresh beef during storage. (A) Representative images of meat samples treated with LysM15, lysozyme, or untreated (control group and blank group) after 0 and 6 days of storage at 4°C; (B) Bacillus cereus count in beef samples after 6 days of storage. **p<0.01.

[0023] Figure 4 This study investigated the effect of LysM15 on the cell wall integrity of Bacillus cereus. (A) Scanning electron micrographs of Bacillus cereus after treatment with PBS (control) or LysM15; (B) Release of intracellular DNA after LysM15 treatment; (C) Macroscopic images of cell lysis after treatment with 12.5 µg / mL LysM15 for 0 min and 10 min; (D) Decrease in turbidity of purified Bacillus cereus cell walls after treatment with LysM15. *p<0.05, **p<0.01, ***p<0.001.

[0024] Figure 5 This is a transcriptome response analysis of Bacillus cereus to LysM15 treatment. (A) GO enrichment analysis of differentially expressed genes (DEGs); (B) KEGG pathway enrichment analysis of differentially expressed genes.

[0025] Figure 6This is a metabolomics analysis of Bacillus cereus treated with LysM15. (A) Hierarchical clustering heatmap of differentially expressed metabolites between the control and treatment groups; (B) Volcano plot of significantly changed metabolites; (C) KEGG pathway enrichment analysis of differentially expressed metabolites; (D–G) Heatmap of differentially expressed metabolites in representative key metabolic pathways: (D) Aminoacyl-tRNA synthesis; (E) Citrate cycle (TCA cycle); (F) ABC transporters; (G) mTOR signaling pathway.

[0026] Figure 7 This shows the effect of LysM15 on the citric acid cycle. The green arrows indicate downregulation of genes encoding key enzymes in the TCA cycle.

[0027] Figure 8 This shows the effect of LysM15 on bacterial chemotaxis. The green arrows indicate the downregulation of genes related to chemotactic signals and flagellar motility under LysM15 treatment.

[0028] Figure 9 This is the effect of LysM15 on the transcriptome expression profile of Bacillus cereus. (A) Correlation plot based on transcriptome data, showing the relationship between the treatment group and the control group; (B) Transcriptome heatmap constructed from differentially expressed genes (DEGs); (C) Principal component analysis (PCA) plot of the transcriptome of Bacillus cereus between the control group and the LysM15 treatment group; (D) Volcano plot based on differentially expressed genes.

[0029] Figure 10 This is an OPLS-DA analysis of metabolic changes in Bacillus cereus induced by LysM15 treatment. (A) OPLS-DA score plot showing a clear separation between the control and treatment samples; (B) Correlation plot showing the relationship between Q2 and R2 values ​​in the OPLS-DA model. Detailed Implementation

[0030] Bioinformatics Analysis The amino acid sequences of Bacillus cereus and its phage-derived lyases were obtained from the NCBI database. The genome of Bacillus cereus VD045 was used as a query sequence for comparison to screen for potential lyases. By improving existing algorithms, the inventors analyzed and obtained a series of potential cell wall lyases, one of which was named LysM15, whose amino acid sequence is shown in SEQ ID NO.1 (GenBank accession number WP_001199272).

[0031] Protein expression, purification and cleavage activity assay The LysM15 gene (WP_001199272), after codon optimization, was synthesized by Nanjing Genscript Biotech Co., Ltd. (Nanjing, China) and inserted into the pET-28a expression vector, containing an N-terminal His tag. The plasmid was transformed into *E. coli* BL21 for heterologous expression. The cells were cultured in LB medium until OD280. 600 After adjusting the concentration to 0.6, 1 mM IPTG was added at 16°C to induce expression for 24 hours. Cells were harvested and lysed by sonication. The His-tagged protein was purified by Ni-NTA affinity chromatography and eluted with imidazole-containing buffer. The purified protein was dialyzed against PBS and stored at -80°C for later use.

[0032] Lytic activity was assessed using a turbidimetric reduction assay. Logarithmic-phase Bacillus cereus NCTC 11143 cells were washed with 20 mM Tris-HCl buffer (pH 7.4) and adjusted to OD0.05. 600 Approximately 1.4. In 96-well plates, 100 μL of bacterial suspension was mixed with different concentrations of LysM15, incubated at 37°C, and OD was read every 5 minutes. 600 The concentration was maintained for 1 hour. To evaluate the lysis profile, the above experiment was repeated for other strains, using LysM15 at a final concentration of 32 µg / mL. Using Bacillus cereus NCTC11143 as a 100% lysis reference, the lysis rate of other strains was measured by OD0.05. 600 Compare the changes.

[0033] Removal of pre-formed biofilm Bacillus cereus NCTC 11143 was cultured in TSB medium at 37°C for 48 hours to form a biofilm. After removing planktonic bacteria, the culture was treated with 200 μL of LysM15 and incubated at 37°C for 1 hour. Following treatment, the lysin solution was removed, and the cells were gently washed twice with PBS. Then, 200 μL of 0.1% crystal violet solution was added to each well, and staining was performed at room temperature for 15 minutes. After staining, the dye was discarded, and the cells were washed three times again with PBS to remove unbound dye. Next, 200 μL of 95% ethanol solution was added to decolorize the crystal violet and fully dissolve the stained biofilm. The absorbance (OD) of each well was measured using a microplate reader. 575 The efficacy of lyases in removing pre-formed biofilms was evaluated.

[0034] Spore germination inhibition Bacillus cereus was cultured in LB medium for 48 hours, then transferred to sporulation medium and cultured for another 5 days. Spores were collected, washed with PBS, and resuspended in 20 mM Tris-HCl buffer. To remove vegetative cells, the spore suspension was treated at 80°C for 10 minutes. Then, it was incubated with different concentrations of LysM15 for 1 hour. After washing with PBS, the spores were diluted, plated, and incubated at 37°C for 12 hours to count the colonies. Germination rate = (number of colonies in the treatment group / number of colonies in the PBS control group) × 100%.

[0035] Stability assessment The relative cleavage activity of LysM15 was determined by turbidimetric assay to assess its stability. Thermal stability was determined by treating LysM15 protein at different temperatures (4–80 °C) for 30 minutes; salt tolerance was assessed by adding different concentrations of NaCl (0–300 mM) to the reaction system; pH stability was tested by reacting in buffer solutions at different pH values ​​(6–10); and storage stability was determined by measuring residual cleavage activity after storage at -20 °C for different numbers of days. Untreated samples were used as 100% activity controls in all tests.

[0036] In vitro safety evaluation The biosafety of LysM15 was assessed using hemolysis and cytotoxicity assays. In the hemolysis assay, fresh red blood cells from healthy mice were washed with PBS and diluted to a 3% concentration. These cells were then incubated with different concentrations of LysM15 (6.25-100 µg / mL) for 1 hour. The absorbance of hemoglobin in the supernatant at 540 nm was measured. PBS served as a negative control, and 0.1% Triton X-100 as a positive control. The hemolysis rate was calculated as 100% for the Triton X-100 group.

[0037] Cytotoxicity was assessed using the RAW264.7 macrophage cell line at a cell density of 1 × 10⁻⁶ cells. 4 Cells were incubated overnight, then different concentrations of LysM15 (6.25-200 µg / mL) were added, and the cells were incubated at 37°C and 5% CO2 for 24 hours. Cell viability was measured using a CCK8 assay kit, with the untreated group representing 100%.

[0038] Antibacterial effects in food To evaluate the preservative effect of LysM15 in food, a beef preservation model was established. Fresh lean beef (fat content <5%) was first washed twice with distilled water, cut into uniformly sized pieces (approximately 20 g), and the surface was inoculated with Bacillus cereus at a concentration of 1×10⁻⁶. 5CFU / g. After inoculation, the cells were placed at 30°C for 30 minutes, then sprayed with 2 mL of LysM15 solution (100 µg / mL), lysozyme (500 µg / mL), or PBS (control group), and stored at 4°C for 6 days. The control group was treated with sterile water, while the blank group contained no antibiotics or Bacillus cereus. Colony counts were performed using MYP medium, and visual changes were observed by photographing on day 0 and day 6.

[0039] Analysis of antibacterial mechanism Scanning electron microscope Logarithmic-phase Bacillus cereus NCTC 11143 was treated with different concentrations (6.25 or 12.5 μg / mL) of LysM15 and incubated at 37°C for 5 minutes. Subsequently, the bacteria were fixed in 1 mL of 2.5% glutaraldehyde solution and fixed at 4°C for 8 hours. After four gentle washes with deionized water, the cells were progressively dehydrated with different concentrations of ethanol (70%, 80%, 95%, and 100%). The dehydrated cells were then subjected to critical-point drying using liquid carbon dioxide. After drying, the cells were carefully coated with gold using a metal sputtering machine. Finally, the morphological changes of the bacteria were observed using scanning electron microscopy.

[0040] DNA leakage Bacillus cereus cells in the logarithmic growth phase were collected and washed three times, then adjusted to OD. 600 The value was 0.5. The cell suspension was treated with different concentrations of LysM15 at 37°C for 1 hour. After incubation for 10 minutes, the concentration of DNA released from the cells was quantitatively determined using a NanoDrop2000 spectrophotometer at a wavelength of 260 nm.

[0041] Disintegration of cell wall Bacillus cereus NCTC 11143 was cultured in LB medium to the logarithmic growth phase and washed twice with PBS. The bacteria were disrupted by sonication, and the cell lysate was centrifuged at 1500 ×g for 10 min, followed by centrifugation at 21,000 ×g for 5 min. Subsequently, 20 mL of 4% sodium dodecyl sulfate (SDS) was added to the coarse cell wall, and the mixture was boiled for 20 min. The precipitate was washed with 10 mL of deionized water (40 °C) and 1 M NaCl. Finally, the precipitate was washed 3–4 times with deionized water, initially centrifuged at 1500 ×g for 5 min, and then centrifuged at 21,000 ×g for 5 min. The OD of the Bacillus cereus NCTC 11143 cell wall extract was adjusted. 600 The value was 0.9, and then it was reacted with 12.5 μg / mL LysM15 at 37 °C for 10 minutes. The OD was measured... 600 The decrease in [something] was used to assess the pyrolysis effect.

[0042] Transcriptome analysis To further investigate the antibacterial mechanism of LysM15 against Bacillus cereus, we performed RNA sequencing (RNA-seq) analysis. After treatment with 1 µg / mL LysM15 for 20 minutes, cells were collected and total RNA was extracted using an RNA extraction kit. The quality and concentration of RNA were assessed using an Invitrogen Qubit 3.0 spectrophotometer and an Agilent 2100 bioanalyzer. The samples were then sent to the SMQ Group Medical Laboratory in Shenzhen for sequencing. During the experiment, rRNA was first removed, retaining mRNA and other non-coding RNAs. Next, the RNA was fragmented using high temperature and divalent cations to obtain fragments of 100–300 bp. cDNA was then synthesized via reverse transcription, with the second strand labeled with dUTP. After library construction, Illumina sequencing adapters were added, and excess adapters were removed using magnetic beads. PCR amplification was then performed to ensure sufficient library quantity. After PCR amplification, library concentration was determined using the Qubit spectrophotometer, and fragment size distribution was analyzed using an Agilent 2100 bioanalyzer. Finally, sequencing was performed on the Illumina high-throughput platform using a 2×150 bp paired-end strategy. The raw sequencing data obtained from the Illumina platform underwent rigorous quality control to ensure data integrity and reliability. First, the quality of the raw reads was assessed using FastQC (R package, v0.11.8), followed by removal of low-quality sequences, adapter contamination, and overexpressed sequences. Next, the data was processed using Trim Galore (v0.6.7) to obtain high-quality clean reads, which were then used for downstream analysis. To align these clean reads with the Bacillus cereus reference genome (NCTC11143 strain), efficient alignment was performed using the STAR (v2.7.10b) tool. After alignment, the data was sorted and indexed using SAMtools (v1.9). Subsequently, transcript assembly was performed using StringTie (v1.3.5), and gene expression levels were quantified using FPKM (number of fragments per kilobase transcript / number of aligned reads per million). Differentially expressed gene analysis was performed using the DESeq2 (version 1.32.0) R package. The criteria for screening differentially expressed genes (DEGs) were |log2(Fold Change)|>1 and p<0.05. Subsequently, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed to further classify differentially expressed genes.

[0043] Metabolomics analysis To investigate the effects of LysM15 treatment on the metabolism of Bacillus cereus, metabolomics analysis was performed. Samples were prepared according to the method described above. Metabolite extraction was performed by homogenizing each sample with pre-chilled 80% methanol and stainless steel beads, followed by incubation at -20°C for 30 min to promote protein precipitation. The mixture was centrifuged at 20,000 × g for 15 min at 4°C, and the supernatant was collected, vacuum dried, and redissolved in 100 μL of 80% methanol. After a second centrifugation under the same conditions, the final supernatant was analyzed by UPLC-HRMS.

[0044] Chromatographic separation was performed using an ACQUITY UPLC HSS T3 column at a flow rate of 0.35 mL / min. Mobile phase A consisted of water containing 5 mmol / L ammonium acetate and 5 mmol / L acetic acid, while mobile phase B was acetonitrile (LC-MS grade). The gradient elution program was as follows: 0.0–0.3 min, 1% B; 0.3–1.9 min, linearly increasing to 60% B; 1.9–4.6 min, increasing to 99% B; 4.6–7.3 min, maintaining 99% B; 7.3–7.8 min, returning to 1% B; 7.8–10.0 min, equilibrating at 1% B.

[0045] Mass spectrometry analysis was performed using a Q-Exactive high-resolution mass spectrometer in both positive and negative ion modes with data-dependent acquisition (DDA). The spray voltage was set to +3.8 kV in positive mode and -3.5 kV in negative mode, and the capillary temperature was maintained at 350 °C. Full MS scans covered the m / z range of 70–1050, with a resolution of 70,000 (at m / z 200), an AGC target of 3e6, and a maximum injection time of 100 ms. The top five strongest precursor ions (intensity >100,000) were selected for MS / MS fragmentation at a resolution of 17,500, with a dynamic exclusion time of 6 seconds.

[0046] Raw mass spectrometry data were converted to mzXML format and peak detection, alignment, and quantification were performed in R using the XCMS package (version 3.2). Metabolite characteristics were annotated by comparison with the HMDB, KEGG, and Meltlin databases. Principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were used to assess the separation between different treatment groups and identify key differentially expressed metabolites. Significant metabolites were selected based on a Variable Importance in Projection (VIP) score >1 and p < 0.05 (Student's t-test). Finally, pathway enrichment analysis was performed on the identified differentially expressed metabolites using the KEGG database to reveal key metabolic pathways involved in the LysM15 antibacterial mechanism.

[0047] Statistical analysis All experiments were analyzed using SPSS 22.0 software, and all experiments were performed in triplicate. Data are expressed as mean ± standard deviation (Mean ± SD), and Student's t-test was used. A p-value < 0.05 was considered statistically significant.

[0048] Results and Discussion Antibacterial activity LysM15 has been identified as a potential lyase with two domains: an N-terminal catalytic domain belonging to the M15 superfamily, and a C-terminal SH3 cell wall binding domain. Figure 1 A). The recombinant protein was successfully expressed in *E. coli* and purified by affinity chromatography. SDS-PAGE analysis showed a distinct band at approximately 35 kDa, consistent with the predicted molecular weight (A). Figure 1 B). The lytic activity of LysM15 against Bacillus cereus NCTC 11143 was evaluated using a turbidimetric reduction method. Figure 1 As shown in Figure C, LysM15 exhibits a strong dose-dependent cleavage effect. Even at low concentrations (1 µg / mL), the enzyme can reduce OD by 60 minutes. 600 Significantly reduced. At higher concentrations (16 µg / mL and 32 µg / mL), turbidity decreased rapidly, indicating its strong lytic potential against Bacillus cereus.

[0049] To evaluate the lytic spectrum of LysM15, it was tested against various bacterial strains at a concentration of 32 µg / mL, and the results are summarized in Table 1. LysM15 exhibited strong lytic activity against Bacillus cereus NCTC 11143 (vomiting type strain). Conversely, no lytic effect was observed against other pathogenic bacteria such as Staphylococcus aureus, Pseudomonas aeruginosa, Vibrio parahaemolyticus, and Salmonella typhi, as well as several probiotic strains such as Lactobacillus acidophilus, Lactobacillus rhamnosus, and Bifidobacterium. These results indicate that LysM15 has a narrow lytic spectrum and specifically targets certain Bacillus cereus strains. Furthermore, the lack of activity against probiotic strains further supports its potential to target Bacillus cereus without affecting beneficial bacteria.

[0050] Table 1. Fragmentation spectra of LysM15

[0051] a By measuring OD within 1 hour 600 The percentage reduction is used to determine the relative cleavage activity of LysM15: -, no cleavage; +, limited cleavage; ++, moderate cleavage; +++, rapid cleavage.

[0052] Disruption of biofilm and inhibition of spore germination The effects of LysM15 on Bacillus cereus biofilm and spore germination were further evaluated. Figure 1 As shown in Figure D, LysM15 treatment significantly (p<0.05) reduced the biomass of pre-formed Bacillus cereus biofilms. At concentrations of 12.5–50 µg / mL, OD... 575 The value was significantly lower than that of the untreated control group, indicating that it effectively removed biofilm. The effect of LysM15 on Bacillus cereus spore germination showed that at concentrations of 12.5, 25, and 50 μg / mL, the spore germination rate was lower than that of the control group, while there was no significant difference at a concentration of 6.25 μg / mL. These results indicate that LysM15 can effectively inhibit spore germination at higher concentrations.

[0053] Stability assessment To assess the potential of LysM15 in food-related applications, its stability and tolerance under common food processing and storage conditions must be evaluated. Figure 2 As shown in Figure A, LysM15 maintained relatively stable cleavage activity between 4°C and 40°C, retaining approximately 90% of its activity at 50°C. However, when the temperature increased to 60°C, its activity decreased significantly (p<0.001), dropping to 50%. In the salt tolerance test (… Figure 2 (B) LysM15 retains approximately 90% of its activity at NaCl concentrations up to 300 mM, demonstrating good salt stability. Under conditions of pH 7 to 8, the enzyme activity is high ( Figure 2 LysM15 exhibits significantly reduced activity (p<0.001) under acidic (pH 6) or alkaline (pH 10) conditions, indicating that it is best suited for use in neutral and slightly alkaline environments. Figure 2 The D-distribution model shows that LysM15 maintained more than 80% of its pyrolysis activity for 60 days when stored at -20°C, indicating that it has good long-term storage stability.

[0054] In vitro safety assessment The hemolytic activity of LysM15 was assessed using mouse erythrocytes. Figure 2 As shown in Figure E, LysM15 did not exhibit significant hemolytic activity at a concentration of 100 μg / mL. Furthermore, LysM15 maintained over 95% cell viability at concentrations up to 200 μg / mL. Figure 2 F) further confirmed its good biocompatibility.

[0055] Determination of antibacterial activity in beef Beef is a common food, rich in fat, protein, vitamins, and minerals. However, its high protein / moisture content and suitable pH level also provide an ideal environment for the growth and reproduction of microorganisms such as Bacillus cereus, potentially leading to foodborne infections. To assess the antimicrobial activity of LysM15 in beef, samples were processed and stored at 4°C. Figure 3 As shown in Figure A, the samples treated with LysM15 or lysozyme maintained a relatively uniform color and showed fewer signs of microbial contamination, while the control group showed obvious color and texture changes after 6 days, accompanied by a strong odor. Figure 3 B shows the Bacillus cereus count in beef samples over a 6-day period. Compared to the control group, treatment with LysM15 or lysozyme significantly (p<0.01) reduced the number of Bacillus cereus colonies, indicating effective inhibition of bacterial growth. These results demonstrate that LysM15 is as effective as lysozyme in inhibiting the growth of Bacillus cereus in beef, highlighting its potential as a food preservative.

[0056] The effect of LysM15 on cell wall integrity The morphological changes of Bacillus cereus NCTC 11143 cells after LysM15 treatment were evaluated using scanning electron microscopy. Figure 4 As shown in Figure A, the control group appeared as intact rod-shaped bacteria with a smooth surface. However, after treatment with LysM15, the cells underwent morphological changes, including membrane rupture and fragmentation. These observations indicate that LysM15 disrupts the integrity of the bacterial cell wall, leading to significant cell damage.

[0057] To further investigate the impact of LysM15 on cell wall integrity, DNA leakage was assessed, and the DNA concentration in the supernatant of bacterial samples from the treatment and control groups was measured. Figure 4 As shown in Figure B, the DNA concentration in the supernatant of the LysM15-treated group increased significantly (p < 0.05), indicating that LysM15 induced cell membrane disruption, which subsequently led to leakage of intracellular DNA, further supporting its ability to disrupt cell wall integrity.

[0058] In addition, the direct effect of LysM15 on the purified Bacillus cereus cell wall was evaluated. The purified cell wall was incubated with LysM15 (12.5 µg / mL) for 10 minutes. Figure 4 As shown in C and 4D, OD 600 The value decreased significantly (p<0.05), indicating that LysM15 effectively degraded the bacterial cell wall. These findings confirm that LysM15 can effectively degrade the bacterial cell wall, leading to the loss of its structural integrity.

[0059] Transcriptome analysis To investigate the antibacterial mechanism of LysM15 and its impact on the bacterial transcriptome, RNA sequencing was performed on *Bacillus cereus* NCTC 11143 treated with LysM15 using the Illumina NovaSeq platform. Figure 9 As shown in Figure A, correlation analysis of gene expression revealed the distribution patterns of differentially expressed genes. Figure 9 The heatmap in section B shows the gene expression profiles between the different groups. Consistent with the heatmap analysis, principal component analysis (PCA) revealed a clear separation between the control group and the LysM15 treatment group. Figure 9 (C) further confirmed that LysM15 treatment caused significant changes in gene expression. Figure 9 The volcano plot of D shows the distribution of differentially expressed genes, with 666 downregulated genes and 777 upregulated genes compared to the control group. These results confirm that LysM15 induces significant changes in gene expression at the transcriptomic level.

[0060] To gain a deeper understanding of the biological significance of differentially expressed genes, GO enrichment analysis was performed. For example... Figure 5 As shown in Figure A, differentially expressed genes in the Molecular Function (MF) category were significantly enriched in entries related to transport activities, including transmembrane transporter activity, active transmembrane transporter activity, and ion transmembrane transporter activity, suggesting that LysM15 may affect membrane transport systems. In the Cellular Composition (CC) category, differentially expressed genes were mainly associated with membrane-related components, such as the plasma membrane, integrative components of the plasma membrane, and organelle membranes. In the Biological Processes (BP) category, enriched entries primarily involved various metabolic processes, including organic acid metabolism, carboxylic acid metabolism, and nitrogen transport, indicating that LysM15 may interfere with bacterial metabolic pathways and nutrient transport.

[0061] KEGG pathway enrichment analysis further revealed functional pathway changes induced by LysM15 treatment. For example... Figure 5 As shown in Figure B, differentially expressed genes were significantly enriched in multiple metabolic pathways, including the biosynthesis of secondary metabolites, microbial metabolism in various environments, carbon metabolism, 2-oxygen metabolite metabolism, and several amino acid metabolic pathways. Furthermore, differentially expressed genes involved in ABC transporters, the TCA cycle, and bacterial chemotaxis were also significantly downregulated, suggesting that LysM15 may impair central energy production, nutrient transport, and environmental sensing in bacteria.

[0062] Metabolomics analysis To better understand the metabolic changes induced by LysM15 treatment, non-targeted metabolomics analysis was performed. For example... Figure 10As shown in Figure A, the OPLS-DA analysis plot reveals a clear separation between the control and treatment groups, indicating that LysM15 caused a significant change in the overall metabolic profile. This model was further validated by a displacement test. Figure 10 B). Consistently, the heatmaps showed a significant difference in metabolite abundance between the treatment and control groups ( Figure 6 A). Furthermore, the volcano plot revealed that 96 metabolites were significantly upregulated after LysM15 treatment, while 154 metabolites were significantly downregulated (A). Figure 6 B).

[0063] Further KEGG pathway enrichment analysis revealed that several key metabolic pathways were affected. Figure 6 C). These pathways include the TCA cycle, oxidative phosphorylation, the mTOR signaling pathway, purine metabolism, ABC transporters, branched-chain amino acid degradation, and amino acid biosynthesis. These results indicate that LysM15 disrupts central energy metabolism and amino acid homeostasis, and may also interfere with membrane transport, cell signaling, and nucleotide biosynthesis. To further investigate the metabolic pathways most affected by LysM15, heatmaps were generated to visualize differentially involved metabolites in key enriched pathways, including amino acid synthesis pathways, the TCA cycle, ABC transport, and the mTOR signaling pathway. Figure 6 (DG). Decreased levels of several amino acids, including valine and isoleucine, were observed in the amino acid synthesis pathway, indicating disruption of protein synthesis. Furthermore, changes in mTOR pathway-related metabolites suggest that LysM15 may affect bacterial growth regulation and cell signaling. These pathway-specific metabolic changes further support the broad-based influence of LysM15 on bacterial physiology.

[0064] Effects on membrane transport LysM15 treatment significantly downregulated multiple differentially expressed genes involved in the ABC transporter pathway, particularly those encoding oligopeptide binding and transporters such as oppA, oppB, oppC, oppD, and oppF. The Opp system plays a crucial role in the acquisition of short peptides, essential amino acids, nitrogen, and carbon sources for bacterial growth, especially under nutrient-limited conditions. Inhibition of this key nutrient acquisition pathway severely hinders cellular metabolic and synthetic processes. Furthermore, genes associated with the transport of arginine (artP, artQ), nucleosides (rbsC), and cobalt ions (cbiQ) were also downregulated, suggesting that LysM15 may cause widespread disruption of membrane permeability. ABC transporters are essential for maintaining bacterial metabolism, osmotic homeostasis, and stress resistance. The downregulation of these genes indicates that LysM15 may interfere with membrane transport pathways, limiting the uptake and expulsion of small molecules such as amino acids, peptides, and ions.

[0065] also, yycF ,citS , ycbA and ycbB The gene was significantly upregulated after treatment with LysM15. yycF yes yycFG A component of the two-component system, which is essential for maintaining cell wall homeostasis and regulating membrane integrity. citS It is involved in the regulation of citric acid metabolism and may respond to changes in carbon source availability. ycbA and ycbB Each gene encodes a two-component sensing kinase and a response regulator, both involved in cell signaling and regulation. Therefore, the upregulation of these differentially expressed genes suggests that LysM15 activates the two-component signaling pathway as part of an adaptive response to membrane damage or capsule stress.

[0066] LysM15 interferes with TCA cycle Transcriptome analysis revealed that LysM15 treatment significantly downregulated multiple genes encoding key TCA cycle enzymes. Figure 7 ).in, PDHA1 and PDHB The E1 subunit, which encodes the pyruvate dehydrogenase complex, initiates the conversion of pyruvate to acetyl-CoA via decarboxylation, serving as a key pathway for entry into the TCA cycle. ACO2 Catalytic conversion of citric acid to isocitrate is an early step in the cycle. IDH2 It encodes a mitochondrial NADP⁺-dependent enzyme responsible for converting isocitrate to α-ketoglutarate, producing NADH, which is a key factor in maintaining intracellular redox balance. SUCLA2 The β subunit encodes succinyl-CoA synthase in the TCA cycle, an enzyme that converts succinyl-CoA to succinate. Later in the TCA cycle... SDHC , SDHA , SDHB , FH and MDH1 It was also significantly downregulated, indicating that the entire cycle was widely suppressed.

[0067] Metabolomics analysis showed a significant decrease in succinic acid, further indicating impaired circulating flux. Figure 6 B). Furthermore, the level of phosphoenolate (PEP) was also reduced, reflecting widespread perturbation of central carbon metabolism. Simultaneously, the level of thiamine pyrophosphate (TPP), an essential cofactor for several TCA-related enzymes such as the pyruvate dehydrogenase complex, changed significantly, further supporting the hypothesis that LysM15 disrupts mitochondrial carbon metabolism and redox homeostasis.

[0068] LysM15 inhibits bacterial chemotaxis Bacterial chemotaxis refers to the directional movement of bacteria towards or away from harmful chemicals along favorable chemical gradients. This chemotactic behavior plays an important role in various biological functions, including biofilm formation, bacterial signal transduction, virulence factor expression, and host infection. Transcriptome analysis showed that several key genes related to chemotaxis were significantly downregulated, including... CheA , CheY , CheV , MCP , MotB , FliG , FliM , FliF , FliP and FlgC ( Figure 8 ).

[0069] MCPs (methyl receptor chemokines) are key receptors that enable bacteria to sense chemical gradients and initiate chemotactic responses. CheA Encoding a histidine kinase, CheY is a key initiating enzyme in the bacterial chemotactic signaling pathway. As a response regulator, phosphorylation of CheA by CheY activates the reverse rotation of the flagellar motor, thereby altering bacterial motility. Meanwhile, CheV is a core coupling-adaptive protein in the chemotactic signaling system, working in conjunction with CheW to mediate the interaction between MCPs and CheA. Furthermore, MotB Encoding components of the flagellated motor, responsible for driving the rotation of the flagellum driven by the proton gradient, enabling directional movement of bacteria. FliG and FliM are key structural proteins of the flagellated motor, involved in regulating the rotational switching mechanism of the flagellum. Therefore, under LysM15 treatment, cheV , cheA , cheY and motB Downregulation of genes may weaken the chemotaxis and motility of Bacillus cereus.

[0070] in conclusion In summary, LysM15 is a novel and promising cell wall lysin exhibiting potent antibacterial activity against Bacillus cereus. It effectively disrupts the bacterial cell wall, removes biofilms, and inhibits spore germination, demonstrating promising application potential in controlling Bacillus cereus contamination in food systems. Transcriptomic and metabolomic analyses revealed that LysM15 significantly interferes with several key bacterial physiological processes, including energy metabolism, amino acid biosynthesis, and chemotaxis. These findings suggest that the antibacterial effect of LysM15 stems not only from its direct lysis of the cell wall but also from its inhibitory effect achieved by disrupting bacterial physiological functions. Furthermore, LysM15 exhibits good stability under various environmental conditions, further highlighting its potential as a food preservative.

[0071] The above is a further detailed description of the present invention and should not be considered as a limitation on the specific implementation of the present invention. For those skilled in the art, simple deductions or substitutions without departing from the concept of the present invention are all within the protection scope of the present invention.

Claims

1. Application of cell wall lysin LysM15 in the preparation of Bacillus cereus inhibitor, wherein the amino acid sequence of the cell wall lysin LysM15 is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The Bacillus cereus inhibitor is a Bacillus cereus inhibitor for food preservation.

3. The application according to claim 2, characterized in that, The food is selected from at least one of fresh meat, dairy products, fruits, vegetables, and cooked food.

4. The application according to claim 1, characterized in that, The Bacillus cereus inhibitor is a Bacillus cereus inhibitor for fermentation.

5. A food preservative additive, characterized in that, It includes the cell wall lysin LysM15, the amino acid sequence of which is shown in SEQ ID NO.

1.

6. The food preservative additive according to claim 5, characterized in that, Its excipients are humectants and buffer salt solutions.

7. The food preservative additive according to claim 6, characterized in that, The moisturizer is glycerin.

8. The food preservative additive according to claim 6, characterized in that, The buffer salt solution is a Tris-HCl buffer solution.

9. A method for inhibiting foodborne Bacillus cereus, comprising dispersing an appropriate amount of cell wall lysin LysM15 on the surface of the food, the amino acid sequence of said cell wall lysin LysM15 being shown in SEQ ID NO.

1.

10. The method according to claim 9, characterized in that, The food is selected from at least one of fresh meat, dairy products, fruits, vegetables, and cooked food.

Images