Bacteriocin and application thereof in removing methicillin-resistant staphylococcus aureus biofilm

Abstract

The invention discloses bacteriocin and application of the bacteriocin to removal of a methicillin-resistant staphylococcus aureus biological membrane. A novel bacteriocin PFB252 is obtained through separation and identification, the bacteriocin is composed of 102 amino acids, and the relative molecular weight of the bacteriocin is 11267.34 Da. Researches show that the bacteriocin PFB252 has good thermal stability and pH stability and is resistant to acid, and the antibacterial activity of the bacteriocin PFB252 is still kept at 76.23%-95.20% after the bacteriocin PFB252 is treated by different proteases. The bacteriocin PFB252 can inhibit the growth of methicillin-resistant staphylococcus aureus, reduce the biofilm formation amount of bacteria, the metabolic activity of bacteria in a biofilm and the content of extracellular polysaccharide, also can reduce the expression of related genes of the biofilm, can effectively remove the methicillin-resistant staphylococcus aureus biofilm, and can be used for treating the methicillin-resistant staphylococcus aureus. The drug resistance of the methicillin-resistant staphylococcus aureus is overcome, and the application has important significance for clinically preventing and treating the methicillin-resistant staphylococcus aureus.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and more specifically relates to a bacteriocin and its application in removing methicillin-resistant Staphylococcus aureus biofilm. Background Art

[0002] Staphylococcus aureus ( Staphylococcus aureus ) is a Gram-positive bacterium that is the main pathogen causing mastitis in dairy cows, posing a major threat to livestock health. In addition, Staphylococcus aureus is considered to be a prevalent and widely distributed foodborne pathogen that can be transmitted through contaminated food and produce toxins. The extensive use of antibiotics has led to the emergence of drug-resistant strains, such as methicillin-resistant Staphylococcus aureus (MRSA). Staphylococcus aureus , MRSA), thus posing a continuous challenge to animal husbandry, the food industry, and human health. Biofilms are membrane-like aggregates of bacteria that attach to living or non-living solid surfaces and secrete extracellular matrix to the extracellular space, wrapping themselves in the extracellular matrix, which enhances the bacteria's resistance to antibiotics. Due to the complex structure of MRSA biofilms and their high resistance to traditional antibiotics, current research uses new antibiotics and combination therapies to synergistically enhance the effects of inhibiting MRSA and clearing biofilms. However, there are still some adverse reactions to the use of kinase inhibitors and the combination of paroxetine (antidepressant) and benzylpenicillin. Therefore, it is of great significance to explore a new natural, safe and effective anti-biofilm drug to prevent and treat methicillin-resistant Staphylococcus aureus.

[0003] Bacteriocins are proteins or polypeptides synthesized by bacterial ribosomes that can effectively inhibit or kill pathogens. Various bacteriocins have been shown to inhibit pathogens by inhibiting biofilm formation. For example, bacteriocin Z057 can effectively reduce the biomass and cell viability of Vibrio parahaemolyticus biofilms, and bacteriocin HW01, produced by Pediococcus acidilactici, can inhibit biofilm formation and the production of virulence factors in Pseudomonas aeruginosa. However, there is still a lack of bacteriocins that can inhibit and eliminate methicillin-resistant Staphylococcus aureus biofilms. This is based on the present invention. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the shortcomings of existing bacteriocins that inhibit methicillin-resistant Staphylococcus aureus and can remove its biofilm, and to provide a bacteriocin and its application in removing methicillin-resistant Staphylococcus aureus biofilm.

[0005] The first object of the present invention is to provide a bacteriocin PFB252.

[0006] The second object of the present invention is to provide a method for preparing bacteriocin PFB252.

[0007] The third object of the present invention is to provide the application of bacteriocin PFB252.

[0008] A fourth object of the present invention is to provide a product.

[0009] The fifth object of the present invention is to provide a strain of Bacillus Velezii and its application.

[0010] The above-mentioned purpose of the present invention is achieved through the following technical solutions: The present invention provides a bacteriocin PFB252. The amino acid sequence of the bacteriocin is: KALDKEVSEGSVDQSSWSREFELDENDGAQVNFWISNNGSNDIKISIDGKVSRTIAPGKQGNISAEVKNGWFGWTYNYTFTASPTAQGGSVKMKYKIAQRNN.

[0011] The present invention purified and identified a novel bacteriocin, PFB252, from the cell-free supernatant of Bacillus velezensis isolated from seawater. This bacteriocin is composed of 102 amino acids and has a relative molecular weight of 11,267.34 Da. Sequencing homology analysis revealed that PFB252 is not homologous to other Class II bacteriocins and represents a novel bacterial bacteriocin. Studies have shown that PFB252 exhibits good thermal stability, retaining 67.07% of its antibacterial activity after treatment at 100°C for 1 hour. It also exhibits good pH stability and acid resistance, with its antibacterial activity remaining essentially unchanged at 4°C overnight at pH 2-6. Furthermore, its antibacterial activity remains at 76.23%-95.20% after treatment with pepsin, proteinase K, papain, and trypsin, demonstrating good stability and tolerance. Studies have shown that the bacteriocin PFB252 can inhibit the growth of methicillin-resistant Staphylococcus aureus, reduce the amount of bacterial biofilm formation, the metabolic activity of bacteria in the biofilm, and the content of exopolysaccharides, and can also reduce the expression of biofilm-related genes, eliminate methicillin-resistant Staphylococcus aureus biofilm, and overcome the drug resistance of methicillin-resistant Staphylococcus aureus. The bacteriocin provided by the present invention can effectively curb the spread of drug-resistant bacteria and reduce public health risks, and has important clinical and public health significance.

[0012] The present invention provides a method for preparing bacteriocin PFB252, comprising: Bacillus velezensis ) was inoculated into the fermentation medium, the supernatant was collected after centrifugation, and sterilized by filtration through a filter membrane to obtain a cell-free supernatant, which was then purified.

[0013] Preferably, the purification step is to sequentially perform acid precipitation, gel column chromatography, anion exchange chromatography and reverse phase high performance liquid chromatography.

[0014] Preferably, the Bacillus velezensis is the FB25-2 strain, which has been deposited in the Guangdong Provincial Microbiological Culture Collection Center on June 9, 2025, with the deposit number GDMCC No: 66485.

[0015] As a more preferred embodiment, the present invention provides a specific method for preparing bacteriocin PFB252. The steps include: inoculating Bacillus velezensis FB25-2 into LB liquid medium and culturing overnight in a 37°C constant-temperature incubator; inoculating the bacterial suspension into a fermentation medium at a concentration of 2% and incubating in a shaking incubator at 30°C and 180 rpm for 24 hours; then centrifuging at 4°C for 30 minutes, collecting the supernatant, and sterilizing it by filtration through a 0.22-μm filter to obtain a cell-free supernatant. A crude bacteriocin sample is obtained by acid precipitation: the pH of the cell-free supernatant is adjusted to 2.0 using 6 mol / L hydrochloric acid solution and allowed to stand at 4°C overnight. The resulting precipitate is collected by centrifugation (4°C, 8000 rpm, 30 minutes), redissolved in phosphate-buffered saline (PBS) at room temperature, and filtered again through a 0.22-μm membrane to remove bacterial cells. The bacteriocin was then purified by gel column chromatography: the crude bacteriocin extract was purified using TA-GF75 gel column chromatography. 1 mL of the crude extract was applied to a TA-GF75 gel column pre-equilibrated with deionized water at a flow rate of 1 mL / min, and fractions with antibacterial activity were collected using an automated collector. The bacteriocin was then purified by anion exchange chromatography: the fractions were applied to a Tiderose Q HP anion column equilibrated with 0.05 mol / L sodium chloride at a flow rate of 3 mL / min, and fractions with antibacterial activity were collected. The bacteriocin was then purified by reverse-phase high-performance liquid chromatography (RP-HPLC): the collected fractions with antibacterial activity were purified by reverse-phase high-performance liquid chromatography, using solvent A [0.1% (v / v) trifluoroacetic acid dissolved in water] and solvent B [0.1% (v / v) trifluoroacetic acid dissolved in acetonitrile] as eluents, configured with a C18 chromatographic column (4.6×150 mm, 5 μm), and elution at a flow rate of 1 mL / min. The elution process included: 0-10 min, 5% solvent B; 11-20 min, 5%-50% solvent B; 21-30 min, 50%-70% solvent B; 31-38 min, 70%-95% solvent B. Finally, the antibacterial active fractions were collected and concentrated, and then filtered and sterilized using a pore size of 0.22 μm. The final sample obtained was the bacteriocin PFB252.

[0016] The present invention provides the use of bacteriocin PFB252 in inhibiting methicillin-resistant Staphylococcus aureus for non-disease treatment and diagnosis purposes.

[0017] The present invention provides application of bacteriocin PFB252 in clearing biofilm of methicillin-resistant Staphylococcus aureus.

[0018] The present invention provides the use of bacteriocin PFB252 in preparing antibacterial products or biofilm clearing products.

[0019] The present invention provides a product containing bacteriocin PFB252.

[0020] The present invention also provides a Bacillus velezensis FB25-2 strain, which has been deposited in the Guangdong Provincial Microbial Culture Collection Center on June 9, 2025, with a preservation number of GDMCC No: 66485.

[0021] The present invention provides the use of the Bacillus Velezii FB25-2 strain in preparing bacteriocins.

[0022] The present invention has the following beneficial effects: The present invention has obtained a new bacteriocin PFB252, which is composed of 102 amino acids and has a relative molecular weight of 11267.34Da. Sequencing homology analysis shows that bacteriocin PFB252 is not homologous to other Class II bacteriocins and is a new type of bacterial bacteriocin. Studies have shown that bacteriocin PFB252 has good thermal stability and protease and pH tolerance. In addition, bacteriocin PFB252 can inhibit the growth of methicillin-resistant Staphylococcus aureus, reduce the amount of bacterial biofilm formation, the metabolic activity of bacteria in the biofilm, and the content of extracellular polysaccharides. It can also reduce the expression of biofilm-related genes, eliminate methicillin-resistant Staphylococcus aureus biofilm, and overcome the drug resistance of methicillin-resistant Staphylococcus aureus. The bacteriocin provided by the present invention can effectively curb the spread of drug-resistant bacteria and reduce public health risks, and has important clinical and public health significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the phylogenetic tree of the FB25-2 strain.

[0024] Figure 2 Figure 2 shows the purification results of bacteriocin PFB252 (A: TA-GF75 gel chromatography; B: Tiderose Q HP anion chromatography; C: reversed-phase high-performance liquid chromatography (RP-HPLC)).

[0025] Figure 3 This is the result of SDS-PAGE analysis of bacteriocin PFB252.

[0026] Figure 4The diagram shows the homology comparison results of the amino acid sequence of bacteriocin PFB252 and the amino acid sequences of other bacteriocins (A: comparison between bacteriocin PFB252 and bacteriocin Cerecyclin (AP03198); B: comparison between bacteriocin PFB252 and bacteriocin Sil (AP02399); C: comparison between bacteriocin PFB252 and bacteriocin Microcin PDI (AP02015); D: comparison between bacteriocin PFB252 and bacteriocin Microcin S (AP02014); E: comparison between bacteriocin PFB252 and bacteriocin BM1122 (AP03290)).

[0027] Figure 5 Figure 2 shows the predicted structure of bacteriocin PFB252 (A: secondary structure prediction; B: tertiary structure prediction).

[0028] Figure 6 Stability results of bacteriocin PFB252 (A: temperature; B: pH; C: enzyme).

[0029] Figure 7 The effect of different concentrations of bacteriocins on the growth of methicillin-resistant Staphylococcus aureus.

[0030] Figure 8 The effect of bacteriocin PFB252 on the biofilm formation of methicillin-resistant Staphylococcus aureus.

[0031] Figure 9 The effect of bacteriocin PFB252 on the metabolic activity of bacteria in methicillin-resistant Staphylococcus aureus biofilm.

[0032] Figure 10 The effect of bacteriocin PFB252 on biofilm exopolysaccharides.

[0033] Figure 11 Effects of different concentrations of bacteriocin PFB252 on genes related to biofilm formation of methicillin-resistant Staphylococcus aureus.

[0034] Figure 12 Effects of different concentrations of bacteriocin PFB252 on methicillin-resistant Staphylococcus aureus biofilm (A: Control; B: 1 / 32MIC; C: 1 / 16MIC).

[0035] Note: The chart P value(* P <0.05,** P <0.01,*** P <0.001) indicated statistically significant differences. DETAILED DESCRIPTION

[0036] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0037] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0038] The methicillin-resistant Staphylococcus aureus CICC25138 (MRSA) used in the examples was purchased from the China Industrial Microbiological Culture Collection Center. The strain was cultured in tryptone soy agar (TSA) liquid medium at 37° C. overnight.

[0039] The fermentation medium used was made of glucose (4.0 g), beef extract (0.5 g), yeast extract powder (0.5 g), peptone (1.5 g), NaH2PO4·2H2O (0.02 g), Na2HPO4·12H2O (0.05 g), MgSO4·7H2O (0.05 g), CaCl2·6H2O (0.05 g) and MnSO4 (0.02 g) dissolved in distilled water with a volume of 100 mL.

[0040] LB liquid medium and LB solid medium were purchased from Beijing Luqiao Technology Co., Ltd.; TSA was purchased from Beijing Suolebao Technology Co., Ltd.

[0041] The results in the examples were statistically analyzed using GraphPad Prism 8, using one-way analysis of variance (ANOVA) followed by Tukey's test.

[0042] Example 1 Identification of strains and preparation of CFS 1. Screening and identification of strains In the early stage of this example, a bacterium FB25-2 was isolated from seawater. After bacterial DNA was extracted, the 16S rRNA gene sequence was amplified, and the PCR product was sent to Sangon Biotech (Shanghai) Co., Ltd. for subsequent testing.

[0043] The predicted sequences were aligned in the NCBI database and a phylogenetic tree was constructed, as shown in Figure 1 As shown, the FB25-2 strain is Bacillus velezensis The strain has a high homology with the strain, and combined with morphological identification, the taxonomic status of the strain was identified as Bacillus velezinis ( Bacillus velezensis ), and named it FB25-2 strain, GenBank accession number: PRJNA1242529, and deposited in Guangdong Provincial Microbiological Culture Collection on June 9, 2025, with the deposit number GDMCC No: 66485, and the deposit address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0044] 2. Preparation of Cell-Free Supernatant (CFS) Bacillus velez ( Bacillus velezensis FB25-2 was inoculated into LB liquid medium and cultured overnight in a 37°C incubator. The bacterial suspension was inoculated into the fermentation medium at a concentration of 2% and incubated in a shaking incubator at 30°C, 180 rpm, for 24 hours. The supernatant was then collected by centrifugation at 4°C for 30 minutes and sterilized by filtration through a 0.22-μm filter to obtain a cell-free supernatant.

[0045] Example 2 Purification of bacteriocin FFB252 The crude bacteriocin sample was obtained by acid precipitation and then purified by a series of steps including TA-GF75 gel column chromatography, Tiderose Q HP anion exchange chromatography, and reversed-phase high-performance liquid chromatography. The fractions from each purification step were collected and their activity against methicillin-resistant Staphylococcus aureus was determined by the solid agar punch method.

[0046] 1. Obtain crude bacteriocin sample by acid precipitation method The pH of the cell-free supernatant was adjusted to 2.0 using 6 mol / L hydrochloric acid solution and allowed to stand overnight at 4°C. The resulting precipitate was collected by centrifugation (8000 rpm, 30 min, 4°C), redissolved in phosphate-buffered saline (PBS) at room temperature, and filtered again through a 0.22 μm pore size membrane to remove bacterial cells, yielding a crude bacteriocin extract.

[0047] 2. Purification of bacteriocin by TA-GF75 gel column chromatography The crude bacteriocin extract obtained by the acid precipitation method was purified using TA-GF75 gel column chromatography. The purification process involved applying 1 mL of the crude extract to a TA-GF75 gel column pre-equilibrated with deionized water at a flow rate of 1 mL / min. Subsequently, fractions were collected using an automated aspirator. Four peaks were separated by TA-GF75 gel column chromatography, collected, and assayed for antibacterial activity against methicillin-resistant Staphylococcus aureus.

[0048] The results are as follows Figure 2 As shown in A, only peak A3 had significant antibacterial activity.

[0049] 3. Purification of bacteriocins by Tiderose Q HP anion exchange chromatography The antibacterial fractions obtained from TA-GF75 gel chromatography were loaded onto a Tiderose Q HP anion column equilibrated with 0.05 mol / L sodium chloride at a flow rate of 3 mL / min. These fractions were then collected and evaluated for their antibacterial activity.

[0050] The A3 peak sample obtained above was further eluted through a Tiderose Q HP anion exchange chromatography column to obtain 5 peaks, among which the B5 peak showed an antibacterial effect, as shown in FIG. Figure 2 As shown in B.

[0051] 4. Purification of bacteriocin by reversed-phase high performance liquid chromatography (RP-HPLC) After purification by Tiderose Q HP anion exchange chromatography, fractions exhibiting antibacterial activity were collected and purified by reverse-phase high-performance liquid chromatography. Solvent A [0.1% (v / v) trifluoroacetic acid in water] and solvent B [0.1% (v / v) trifluoroacetic acid in acetonitrile] were used as eluents on a C18 column (4.6 × 150 mm, 5 μm) at a flow rate of 1 mL / min. The elution process was as follows: 0–10 min, 5% solvent B; 11–20 min, 5%–50% solvent B; 21–30 min, 50%–70% solvent B; and 31–38 min, 70%–95% solvent B. After concentration, the collected fractions were sterilized by filtration using a 0.22 μm pore size filter membrane and subsequently evaluated for antibacterial activity.

[0052] The B5 fraction obtained above was separated and purified by RP-HPLC to obtain 6 peaks, among which the highest peak showed antibacterial effect, such as Figure 2 As shown in C, the fractions of this peak were collected and named bacteriocin PFB252 for subsequent identification.

[0053] Example 3 Identification of bacteriocins 1. Determination of molecular weight of bacteriocin PFB252 and liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis A Tris-Tricine-SDS-PAGE gel kit was used to prepare the gel. The prestained ultralow molecular weight protein maker (3.3 kD to 31.0 kD) and the bacteriocin PFB252 obtained after the final purification in Example 2 were added to the pores and then electrophoresed. After the end, the staining and decolorization were completed, and the target band was cut out. The bacteriocin was commissioned to Beijing Biotechnology Company for LC-MS / MS analysis and de novo sequencing.

[0054] The electrophoresis results are as follows Figure 3As shown, the estimated molecular weight of the bacteriocin is 6.5-14.4 KDa. After LC-MS / MS analysis, the amino acid sequence of bacteriocin PFB252 was shown to be KALDKEVSEGSVDQSSWSREFELDENDGAQVNFWISNNGSNDIKISIDGKVSRTIAPGKQGNISAEVKNGWFGWTYNYTFTASPTAQGGSVKMKYKIAQRNN. The calculated molecular weight of the bacteriocin was 11267.34 Da, which is consistent with the SDS-PAGE results. Compared with other class II bacteriocins, the results are as follows Figure 4 As shown, no homology was found, indicating that it is a new type of bacteriocin.

[0055] 2. Physicochemical properties and protein structure prediction of bacteriocin PFB252 The physicochemical properties of bacteriocin PFB252 were analyzed using the ExPASy-ProtParam tool (https: / / web.expasy.org / protparam / ). The secondary structure of the bacteriocin was predicted using the SOPMA online website for protein secondary structure determination (https: / / npsa.lyon.inserm.fr / cgi-bin / npsa.cgi / ), while the tertiary structure was predicted using the SWISS-MODEL platform (https: / / swissmodel.expasy.org / ).

[0056] The physicochemical parameters of bacteriocin PFB252 are shown in Table 1, which shows that the number of amino acids in bacteriocin PFB252 is 102, the molecular weight is 11267.34 Da, the theoretical isoelectric point is 6.45, and the molecular formula is C 494 H 757 N 139 O 162 S1. The structural prediction results of bacteriocin PFB252 are shown in Figure 2. Figure 5 shown.

[0057] Table 1 Physicochemical parameters of bacteriocin PFB252

[0058] Example 4 Determination of the stability and antibacterial properties of bacteriocins 1. Stability of bacteriocin PFB252 To evaluate the temperature stability, bacteriocin PFB252 was treated at different temperatures (40°C, 60°C, 80°C, 100°C, and 120°C) for 20 min and then cooled to room temperature.

[0059] To evaluate the effect of pH on bacteriocins, bacteriocin PFB252 samples were adjusted to pH levels of 2, 4, 6, 8, 10, and 12 and readjusted to their original pH after incubation at 37 °C for 3 h.

[0060] To evaluate the effects of different proteases on bacteriocin activity, proteinase K, pepsin, trypsin, and papain were added to bacteriocin PFB252 at a final concentration of 1 mg / mL and incubated at 37°C for 1 h.

[0061] In all the above tests, untreated bacteriocin was used as a control, and the antibacterial activity of bacteriocin against methicillin-resistant Staphylococcus aureus was determined by the solid agar punch method.

[0062] The results of the effects of different temperatures, pH, and enzymes on the activity of bacteriocin PFB252 are as follows: Figure 6 As shown in the results, the bacteriocin PFB252 has good thermal stability and still maintains 67.07% of antibacterial activity after being treated at 100°C for 1 h. It also has good pH stability and acid resistance. Its antibacterial activity remains basically unchanged when placed at 4°C overnight under pH 2-6 conditions. The antibacterial activity after treatment with pepsin, proteinase K, papain, and trypsin remains at 76.23%-95.20%, showing good stability and tolerance.

[0063] 2. Minimum inhibitory concentration (MIC) determination The minimum inhibitory concentration of bacteriocin PFB252 against methicillin-resistant Staphylococcus aureus was determined by the broth microdilution method. Methicillin-resistant Staphylococcus aureus cultured overnight was inoculated into TSA liquid medium at a 1% (V / V) inoculum. The final concentrations of bacteriocin added were 2×MIC, MIC, 1 / 2MIC, 1 / 4MIC, 1 / 8MIC, 1 / 16MIC, and 1 / 32MIC. The culture was incubated at 37°C for 24 h, and the OD was recorded at different time points. 600 value.

[0064] The results showed that the MIC of bacteriocin PFB252 against methicillin-resistant Staphylococcus aureus was 14.61 μg / mL. The growth curve of methicillin-resistant Staphylococcus aureus after treatment with bacteriocin PFB252 was as follows: Figure 7 As shown, bacterial growth stopped at concentrations of 2×MIC and 1×MIC, and bacterial growth was inhibited at concentrations of 1 / 2×MIC, 1 / 4×MIC, and 1 / 8×MIC. Concentrations of 1 / 16×MIC and 1 / 32×MIC had no effect on bacterial growth, so subsequent experiments selected concentrations of 1 / 16×MIC and 1 / 32×MIC for study.

[0065] Example 5 Anti-biofilm effect of bacteriocin PFB252 1. Determination of anti-biofilm activity The crystal violet staining method was used to evaluate the effect of bacteriocin PFB252 on the biofilm formation of methicillin-resistant Staphylococcus aureus. Overnight cultured methicillin-resistant Staphylococcus aureus was inoculated into TSA liquid medium at a 1% (V / V) inoculum. Bacteriocin PFB252 was added to the bacterial suspension to a final concentration of 1 / 32×MIC and 1 / 16×MIC. 200 μL was transferred to a 96-well plate, and the 96-well plate was then placed in a 37°C incubator for 24 h. After the incubation, the supernatant was discarded, the plate was washed three times with PBS buffer, dried, and 100 μL of 1% (V / V) crystal violet was added for staining for 30 min. The staining solution was discarded, and the plate was washed three times with PBS again. After drying, 200 μL of 33% (V / V) acetic acid solution was added, and the plate was incubated at 37°C for 20 min. The OD was measured using a microplate reader. 595 The group without bacteriocin was used as the positive control.

[0066] The results of the test are as follows Figure 8 As shown in the data, at concentrations of 1 / 32×MIC and 1 / 16×MIC, the biofilm formation was reduced by 36.13% and 52.51%, respectively. The results showed that bacteriocin PFB252 could significantly affect the formation of biofilm in a concentration-dependent manner.

[0067] 2. Determination of bacterial activity in biofilms The activity of bacteria within biofilms was determined using a CCK8 kit. Overnight methicillin-resistant Staphylococcus aureus was inoculated into TSA liquid medium at a 1% (v / v) inoculum. The bacteriocin PFB252 was added to the bacterial suspension to a final concentration of 1 / 32×MIC and 1 / 16×MIC. 200 μL was transferred to a 96-well plate. The 96-well plate was then placed in a 37°C incubator and incubated for 24 hours. The supernatant was removed, the plate was washed three times with PBS buffer, and 100 μL of PBS and 10 μL of CCK8 reagent were added. The plate was incubated at 37°C for 1 hour. OD was measured using a microplate reader. 450 The group without bacteriocin was used as the positive control.

[0068] The results of the effect of bacteriocin PFB252 on the metabolic activity of bacteria in biofilms are as follows Figure 9 As shown, at concentrations of 1 / 32×MIC and 1 / 16×MIC, the metabolic activity of bacteria in the biofilm was reduced by 27.37% and 37.92%, respectively. The results showed that bacteriocin PFB252 can significantly affect the formation of metabolic activity of bacteria in the biofilm in a concentration-dependent manner.

[0069] 3. Determination of exopolysaccharide and protein content in biofilm The phenol-sulfuric acid method was used to study the effect of bacteriocin PFB252 on exopolysaccharides. Methicillin-resistant Staphylococcus aureus cultured overnight was inoculated into LB liquid culture medium at a 1% (V / V) inoculum, and bacteriocin PFB252 was added to the bacterial suspension to a final concentration of 1 / 32×MIC and 1 / 16×MIC. A sterile glass slide (18 mm) was placed in a 24-well plate, 1 mL of sample was added to the 24-well plate, and cultured at 37°C for 24 h. The supernatant was removed and washed three times with PBS, 100 μL of PBS buffer was added, and then frozen for 1 min, then sonicated for 1 min, and finally phenol-sulfuric acid solution was added. The plate was heated in water at 100°C for 20 min, and the OD was measured. 490 The treatment group without bacteriocin was used as the control.

[0070] The results of the effect of bacteriocin PFB252 on biofilm extracellular polysaccharides (EPS) and proteins are as follows Figure 10 As shown in the data, exopolysaccharide (EPS) is an important component of extracellular polymers in biofilms. It was shown that at concentrations of 1 / 32×MIC and 1 / 16×MIC, the EPS content decreased by 30.26% and 39.32%, respectively. The results showed that bacteriocin PFB252 could significantly affect the content of exopolysaccharides in biofilms.

[0071] 4. RT-qPCR The effect of bacteriocin PFB252 on biofilm-related genes was evaluated by reverse transcription quantitative PCR (RT-qPCR). Methicillin-resistant Staphylococcus aureus cultured overnight was re-inoculated into TSA medium at a concentration of 1% (V / V). After culturing for 8 hours, bacteriocin PFB252 was added to the bacterial suspension to a final concentration of 1 / 16×MIC and 1 / 32×MIC, and then cultured at 37°C and 180rpm / min for 16 hours. No bacteriocin was added to the control group. Total RNA was extracted using RNAiso Plus reagent and then reverse transcribed into cDNA using PrimeScript™ RT reagent kit. According to TB Green ® The PCR amplification reaction system was prepared according to the instructions of Premix Ex Taq™ II, using 16S rRNA as the internal reference gene. The primers used for gene detection are shown in the table. The relative gene expression levels were calculated by comparing the Ct values ​​(2- ΔΔ Ct method) to determine the expression level.

[0072] The results of the effect of bacteriocin PFB252 on genes related to biofilm formation are as follows Figure 11As shown, at concentrations of 1 / 32×MIC and 1 / 16×MIC, bacteriocin PFB252 had a significant effect on biofilm-related genes, significantly reducing the expression of biofilm-related genes, including quorum sensing genes, virulence genes, etc., and was concentration-dependent. The results showed that bacteriocin PFB252 could significantly reduce the expression of genes related to biofilm formation of methicillin-resistant Staphylococcus aureus.

[0073] 5. Scanning electron microscope (SEM) The effect of bacteriocin PFB252 on methicillin-resistant Staphylococcus aureus biofilm formation was investigated using scanning electron microscopy (SEM). A sterile coverslip (9 mm) was placed in a 48-well plate. 1 mL of MRSA culture medium containing different concentrations of PFB252 (1 / 32×MIC and 1 / 16×MIC) was added to the plate. After incubation at 37°C for 48 h, the supernatant was discarded, the plates were washed three times with PBS, and fixed overnight at 4°C with 2.5% glutaraldehyde. The plates were then washed three times with PBS and dehydrated with different concentrations of ethanol (30%, 50%, 70%, 85%, and 100%), dried with carbon dioxide, and coated with gold. Finally, the samples were observed using a scanning electron microscope (TESCAN MIRA3 LMU, Czech Republic).

[0074] Scanning electron microscopy results Figure 12 As shown, the bacteria in the control group were in an aggregated state, with a large amount of extracellular polymers wrapping the bacteria. The bacterial aggregation of the treatment group with 1 / 32×MIC concentration of bacteriocin PFB252 was significantly reduced. There was only a small amount of bacterial aggregation in the treatment group with 1 / 16×MIC concentration, and most of the bacteria were in a scattered state. This shows that bacteriocin PFB252 can effectively clear the biofilm of methicillin-resistant Staphylococcus aureus.

[0075] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A bacteriocin PFB252, characterized in that The amino acid sequence of the bacteriocin is: KALDKEVSEGSVDQSSWSREFELDENDGAQVNFWISNNGSNDIKISIDGKVSRTIAPGKQGNISAEVKNGWFGWTYNYTFTASPTAQGGSVKMKYKIAQRNN.

2. The method for preparing the bacteriocin PFB252 according to claim 1, characterized in that: Bacillus velez ( Bacillus velezensis ) was inoculated into the fermentation medium, the supernatant was collected after centrifugation, and sterilized by filtration through a filter membrane to obtain a cell-free supernatant, which was then purified.

3. The preparation method according to claim 2, characterized in that: The purification steps are acid precipitation method, gel column chromatography, anion exchange chromatography and reverse phase high performance liquid chromatography in sequence.

4. The preparation method according to claim 2, characterized in that The Bacillus Velez is the FB25-2 strain, which was deposited in the Guangdong Provincial Microbial Culture Collection Center on June 9, 2025, with the deposit number GDMCC No: 66485.

5. Use of the bacteriocin PFB252 according to claim 1 in inhibiting methicillin-resistant Staphylococcus aureus for non-disease treatment and diagnosis purposes.

6. Use of the bacteriocin PFB252 according to claim 1 in clearing biofilms of methicillin-resistant Staphylococcus aureus.

7. Use of the bacteriocin PFB252 according to claim 1 in the preparation of antibacterial products or products for removing biofilms.

8. A product, characterized in that Containing the bacteriocin PFB252 according to claim 1.

9. A strain of Bacillus velezinoffii ( Bacillus velezensis ) FB25-2 strain, characterized in that This strain was deposited in Guangdong Provincial Microbiological Culture Collection Center on June 9, 2025, with the collection number GDMCC No: 66485.

10. Use of the FB25-2 strain according to claim 9 in the preparation of bacteriocins.

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