A hawthorn-derived polypeptide with antibacterial activity and a preparation method and application thereof

Abstract

The application belongs to the technical field of bioactive polypeptides, and specifically discloses a hawthorn-derived polypeptide with antibacterial activity, and a preparation method and application thereof. The amino acid sequence of the hawthorn-derived polypeptide is selected from SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3 and SEQ ID NO. 5. The polypeptide has antibacterial activity on Aeromonas sobria. The antibacterial activity of the polypeptide represented by SEQ ID NO. 3 is the strongest, with a minimum antibacterial concentration of 0.5 mg / mL and a minimum bactericidal concentration of 1.0 mg / mL. The polypeptide is obtained by taking hawthorn as raw material, and through crude protein extraction, enzymolysis, ultrafiltration, gel chromatography separation, antibacterial activity screening, polypeptide sequence identification and solid-phase synthesis, and can be used for preparing an antibacterial agent and an antibacterial preparation for aquaculture. The application provides a new active substance for the development and utilization of the hawthorn-derived antibacterial active polypeptide.

Description

Technical Field

[0001] This invention belongs to the field of bioactive polypeptide technology, and particularly relates to a hawthorn-derived polypeptide with antibacterial activity, its preparation method and application. Background Technology

[0002] Aeromonas tempera ( Aeromonas sobria Aeromonas hydrophila is a common aquatic pathogen and foodborne pathogen that can cause hemorrhagic septicemia in fish and intestinal infections in humans, posing a threat to the development of aquaculture and public health. Currently, the control of aquatic pathogens such as Aeromonas hydrophila primarily relies on antibiotics and other chemical antibacterial agents. However, long-term or improper use can easily lead to drug resistance in pathogens and may cause drug residues and environmental pollution. Therefore, developing alternative antibacterial substances that are naturally derived, have high safety, and possess good antibacterial activity is of great significance for the prevention and control of aquatic diseases and food safety.

[0003] hawthorn( Crataegus pinnatifida Hawthorn is rich in various active ingredients such as flavonoids, organic acids, triterpenoids, and proteins. Besides its high nutritional and medicinal value, studies have shown that hawthorn extract and some active substances derived from hawthorn have certain antibacterial effects, including inhibition of some aquatic pathogens. However, current research on the antibacterial activity of hawthorn mainly focuses on components such as whole hawthorn extract, flavonoids, polyphenols, or polysaccharides, with relatively little research on hawthorn protein and specific active peptides produced by its hydrolysis. In particular, the composition, activity, and characteristics of functional peptides with clear amino acid sequences that can directly exert antibacterial effects in hawthorn protease hydrolysis products lack systematic research.

[0004] Therefore, existing technologies lack hawthorn-derived peptides with well-defined amino acid sequences and strong antibacterial activity against Aeromonas hydrophila. How to screen and identify specific peptides with antibacterial activity from the complex composition of hawthorn protease hydrolysates, and to determine their amino acid sequences and antibacterial properties, is a pressing technical problem to be solved in this field.

[0005] The information disclosed in this background section is only intended to enhance the understanding of the background technology of this application and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0006] To address the limitations of existing technologies in which hawthorn has complex antibacterial active ingredients, specific amino acid sequences, and limited activity against Aeromonas hydrophila, this invention provides a hawthorn-derived polypeptide with antibacterial activity, its preparation method, and its application.

[0007] In a first aspect, the present invention provides a hawthorn-derived polypeptide with antibacterial activity, wherein the polypeptide is selected from any of the following: (1) The amino acid sequence is shown in SEQ ID NO.1; (2) The amino acid sequence is shown in SEQ ID NO.2; (3) The amino acid sequence is shown in SEQ ID NO.3; (4) The amino acid sequence is shown in SEQ ID NO.5.

[0008] Preferably, the amino acid sequence of the polypeptide is shown in SEQ ID NO.3.

[0009] Preferably, the polypeptide has antibacterial activity against Aeromonas hydrophila. Further, the polypeptide with the amino acid sequence shown in SEQ ID NO. 3 has a minimum inhibitory concentration of 0.5 mg / mL and a minimum bactericidal concentration of 1.0 mg / mL against Aeromonas hydrophila.

[0010] Secondly, the present invention provides a method for preparing hawthorn-derived polypeptides with antibacterial activity, comprising the following steps: (1) Crude protein extraction: Select dried hawthorn, remove the pits and crush it, and pass it through a 60-mesh sieve to obtain hawthorn powder; add 0.1 mol / L NaOH solution to the hawthorn powder at a material-to-liquid ratio of 1:10, and stir and extract at room temperature for 2 h; after extraction, centrifuge at 4000 r / min for 20 min and collect the supernatant; adjust the pH of the supernatant to 4.5 with hydrochloric acid, let it stand for 2 h to precipitate, centrifuge at 4000 r / min for 20 min to collect the precipitate, wash it with distilled water until neutral, freeze dry to obtain hawthorn crude protein; (2) Enzymatic hydrolysis: The crude protein of hawthorn is hydrolyzed by alkaline protease. The amount of alkaline protease added is 3% of the crude protein of hawthorn. The hydrolysis is carried out at 50°C for 4 h to obtain the hydrolysate of hawthorn. (3) Separation: The hawthorn protein hydrolysate was subjected to ultrafiltration through a 3 kDa ultrafiltration membrane to collect the target components, and then further separated using a Sephadex G-25 gel column to obtain multiple polypeptide components; (4) Screening of active components: Screening the multiple polypeptide components for antibacterial activity to obtain active components with antibacterial activity; (5) Sequence identification: The active component was analyzed by LC-MS / MS to obtain the polypeptide sequence; (6) Peptide synthesis and activity screening: The identified peptide sequences are synthesized in a solid phase and the target peptides with antibacterial activity are screened to obtain the hawthorn-derived peptides with antibacterial activity.

[0011] Preferably, in step (2), the crude hawthorn protein is dissolved in 0.05 mol / L Tris-HCl buffer, and after the enzymatic hydrolysis is completed, the reaction system is treated at 95℃ for 10 min to inactivate the enzyme.

[0012] Preferably, in step (3), the Sephadex G-25 gel column has a size of 1.6 cm × 100 cm and uses ultrapure water as the mobile phase.

[0013] Preferably, in step (4), the antibacterial activity screening is performed using the agar diffusion method.

[0014] Preferably, in step (6), the solid-phase synthesis is the Fmoc solid-phase synthesis method.

[0015] Thirdly, the present invention provides an antibacterial composition comprising the hawthorn-derived polypeptide.

[0016] Preferably, the antibacterial composition is used to inhibit the growth of Aeromonas hydrophila.

[0017] Fourthly, the present invention provides the application of the hawthorn-derived polypeptide in the preparation of antibacterial agents or antimicrobial preparations for aquaculture.

[0018] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention uses hawthorn protein as raw material, and obtains active peptides through enzymatic hydrolysis, separation and purification, and antibacterial activity screening, and identifies their sequences by LC-MS / MS. Among the six candidate peptides obtained by screening, SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.5 all showed antibacterial activity, with inhibition zones of 12.6±0.4 mm, 10.2±0.3 mm, 18.8±0.5 mm and 11.5±0.5 mm, respectively, while SEQ ID NO.4 and SEQ ID NO.6 did not show antibacterial activity. This indicates that the antibacterial effect obtained by this invention is related to a specific amino acid sequence, providing new functional substances for the development and utilization of hawthorn-derived active peptides; (2) Among the six candidate peptides, the peptide shown in SEQ ID NO.3 exhibited the strongest antibacterial activity. Its amino acid sequence is IANAGELELVFDDM. At a concentration of 1 mg / mL, its inhibition zone reached 18.8 ± 0.5 mm, with a MIC of 0.5 mg / mL and an MBC of 1.0 mg / mL. In contrast, the MICs of SEQ ID NO.1 and SEQ ID NO.5 were both 2.0 mg / mL, and the MIC of SEQ ID NO.2 was 4.0 mg / mL. Therefore, SEQ ID NO.3 demonstrated superior antibacterial performance compared to the other candidate peptides and has significant potential for further development as a naturally sourced antibacterial active substance. (3) The hawthorn-derived polypeptide of the present invention can increase the leakage of contents of Aeromonas hydrophila cells and disrupt their cell wall integrity, thereby exerting an antibacterial effect, providing experimental evidence for its application as an antibacterial active ingredient. The DNS method detection results showed that, compared with the reducing sugar leakage of 25.6±2.1 μg / mL in the control group, the reducing sugar leakage of SEQ ID NO.3 under the conditions of 0.5×MIC, 1×MIC and 2×MIC reached 38.5±2.8 μg / mL, 52.3±3.5 μg / mL and 64.8±4.2 μg / mL, respectively, which were 1.50 times, 2.04 times and 2.53 times that of the control group, respectively, and the differences were statistically significant. Attached Figure Description

[0019] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the description of the specific embodiments will be briefly introduced below. Obviously, the following description is only a part of the embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 The chromatogram of hawthorn polypeptide gel filtration is shown.

[0021] Figure 2 The images show molecular docking models of hawthorn polypeptides and transpeptidase (E. coli PBP1b homologous model receptor, PDB ID: 5HL9). Specifically, A represents the molecular docking model of polypeptide SEQ ID NO.1 with transpeptidase (PDB ID: 5HL9), B represents the molecular docking model of SEQ ID NO.2 with transpeptidase (PDB ID: 5HL9), C represents the molecular docking model of SEQ ID NO.3 with transpeptidase (PDB ID: 5HL9), D represents the molecular docking model of SEQ ID NO.4 with transpeptidase (PDB ID: 5HL9), E represents the molecular docking model of SEQ ID NO.5 with transpeptidase (PDB ID: 5HL9), and F represents the molecular docking model of SEQ ID NO.6 with transpeptidase (PDB ID: 5HL9). Detailed Implementation

[0022] This invention proposes a hawthorn-derived polypeptide with antibacterial activity, its preparation method, and its application. To facilitate understanding of this invention by those skilled in the art, the specific embodiments of this invention are described below with reference to the accompanying drawings.

[0023] In this invention, unless otherwise specified, the equipment and raw materials used are commercially available or commonly used in the art. The methods in the following embodiments, unless otherwise specified, are conventional methods in the art. Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0024] Example 1: Extraction, enzymatic hydrolysis, and screening of active components of crude protein from hawthorn. 1.1 Experimental Methods 1.1.1 Extraction of crude protein from hawthorn Dried hawthorn berries were selected, pitted, pulverized, and passed through a 60-mesh sieve to obtain hawthorn powder. 0.1 mol / L NaOH solution was added to the hawthorn powder at a material-to-liquid ratio (mass:volume) of 1:10, and the mixture was extracted at room temperature with stirring for 2 h. After extraction, the mixture was centrifuged at 4000 r / min for 20 min, and the supernatant was collected. The pH of the supernatant was adjusted to 4.5 with hydrochloric acid, and the mixture was allowed to stand for 2 h to precipitate. The precipitate was then collected by centrifugation at 4000 r / min for 20 min, washed with distilled water until neutral, and freeze-dried to obtain crude hawthorn protein.

[0025] The crude protein yield was calculated as the ratio of the crude protein content of the obtained hawthorn to the mass of the original hawthorn powder.

[0026] 1.1.2 Enzymatic hydrolysis of crude protein from hawthorn The obtained crude hawthorn protein was dissolved in 0.05 mol / L Tris-HCl buffer (pH 8.0), and alkaline protease was added at 3% of the crude protein substrate mass. The mixture was incubated at 50°C with shaking for 4 h. After hydrolysis, the reaction system was treated at 95°C for 10 min to inactivate the enzyme, followed by centrifugation at 8000 r / min for 15 min. The supernatant was collected to obtain the crude polypeptide solution, which was then freeze-dried to obtain the crude hawthorn protease hydrolysate.

[0027] The yield of crude polypeptide was calculated as the ratio of the mass of the obtained crude polypeptide to the mass of crude protein used for enzymatic hydrolysis.

[0028] 1.1.3 Ultrafiltration and gel chromatography separation of hawthorn protein hydrolysate The obtained crude peptide solution was ultrafiltered through a 3 kDa ultrafiltration membrane to collect the target component. Further separation was then performed using a Sephadex G-25 gel column (1.6 cm × 100 cm), with ultrapure water as the mobile phase at a flow rate of 0.5 mL / min. 2 mL of eluent was collected from each tube, and the absorbance of the eluent in each tube was measured at 220 nm.

[0029] Based on the absorption peak at 220 nm, the eluted components were divided into four components: F1, F2, F3, and F4. These components were collected and freeze-dried for later use.

[0030] 1.1.4 Screening of antibacterial activity of each component The antibacterial activity of fractions F1–F4 was evaluated using the agar diffusion method. Aeromonas tempera bacterial suspensions (1×10⁻⁶) were then used. 8 The CFU / mL solution was evenly spread on LB plates, and after punching wells, 0.2 mL of each component solution (1 mg / mL) was added. Sterile water was used as a negative control. The plates were incubated at 37°C for 24 h, and the diameter of the inhibition zone was measured.

[0031] Meanwhile, the antibacterial activity of crude hawthorn peptides that were not separated by gel chromatography was determined under the same conditions to compare the antibacterial effects of the samples before and after separation.

[0032] 1.2 Experimental Results and Analysis Crude protein was extracted from hawthorn using an alkali-soluble acid-precipitation method, with a yield of 12.5%. After enzymatic hydrolysis with alkaline protease, the crude polypeptide yield was 45.6%, indicating that the above extraction and enzymatic hydrolysis methods can effectively convert hawthorn protein into soluble polypeptide components.

[0033] The crude peptides were further separated using a 3 kDa ultrafiltration membrane and Sephadex G-25 gel chromatography. Four fractions, F1, F2, F3, and F4, were obtained based on the elution curves at 220 nm. Figure 1 As shown in Table 1, the antibacterial activity of each component was screened using the agar diffusion method.

[0034] Table 1. Antibacterial activity of various hawthorn polypeptide components against Aeromonas hydrophila.

[0035] As shown in Table 1, the crude polypeptide exhibited certain antibacterial activity against Aeromonas hydrophila, with an inhibition zone of 12.3 ± 0.5 mm. After further separation by ultrafiltration and gel chromatography, different components showed significantly different antibacterial activities. F1 showed no antibacterial activity, while the inhibition zones of F2 and F3 were 6.8 ± 0.3 mm and 9.2 ± 0.4 mm, respectively. The inhibition zone of F4 reached 17.1 ± 0.5 mm, significantly higher than that of the crude polypeptide and other separated components. Therefore, F4 was identified as the main antibacterial active component in the hawthorn protein hydrolysate and used for subsequent polypeptide sequence identification and active polypeptide screening.

[0036] Example 2: LC-MS / MS identification and candidate sequence screening of peptides in F4 2.1 Experimental Methods 2.1.1 LC-MS / MS analysis of F4 component The F4 active component obtained in Example 1 was analyzed by LC-MS / MS to identify the peptide sequences. Database searches and sequence matching were performed on the obtained mass spectrometry data, and a false discovery rate (FDR) of no more than 1% was used as the quality control standard for peptide identification, thereby obtaining highly reliable peptide sequences. A total of 21 peptide sequences meeting the FDR ≤ 1% requirement were identified from the F4 component.

[0037] 2.1.2 Screening and Molecular Docking Analysis of Candidate Peptides Among the 21 peptides identified by LC-MS / MS, the six peptides with the highest scores were selected as candidate peptides based on the mass spectrometry identification results. They are denoted as SEQ ID NO.1 to SEQ ID NO.6, and their amino acid sequences are shown in Table 2.

[0038] Using the Escherichia coli penicillin-binding protein PBP1b (PDB ID: 5HL9) as a homologous model receptor for Aeromonas tempera transpeptidase, and the full-length structure of PBP1b as a potential target, molecular docking was performed using MOE software to analyze the binding mode between peptides and transpeptidase. Energy calculations were performed using the Amber10:EHT force field, the Triangle Matcher was used as the ligand conformation matching method, GBVI / WSA dG was used as the scoring function, and an all-atom flexible docking mode was used for analysis. 30 docking conformations were generated for each candidate peptide.

[0039] The docking score S-value and the root mean square deviation (rmsd_refine) value of each candidate peptide were recorded to analyze the potential binding characteristics between the candidate peptide and the target protein and to provide candidate targets for subsequent antibacterial activity verification.

[0040] 2.2 Experimental Results and Analysis LC-MS / MS analysis results showed that the F4 active component obtained in Example 1 contained multiple peptides. After quality control screening with an FDR ≤ 1%, a total of 21 high-confidence peptide sequences were identified, indicating that the F4 active component has a relatively complex peptide composition. Further, based on the mass spectrometry identification scores, 6 candidate peptides were selected from the 21 peptides for subsequent analysis. Their sequences and molecular docking results are shown in Table 2. The molecular docking model diagrams of each peptide with transpeptidase are shown in the figure below. Figure 2 As shown.

[0041] Table 2. Comprehensive Score Table for Molecular Docking of Hawthorn Peptides and Peptidase

[0042] As shown in Table 2, all six candidate peptides were able to form a certain degree of potential binding with the target protein transpeptidase (PDB ID: 5HL9). Among them, SEQ ID NO.3 had the lowest docking score S value of -11.7805, indicating that its binding affinity to transpeptidase was the strongest among the six candidate sequences. However, it is worth noting that the rmsd_refine value of this sequence was 4.3341 Å, the largest among the six sequences, indicating that this conformation was not as stable as the binding of other sequences. SEQ ID NO.4 had the lowest rmsd_refine value of 1.7630 Å, indicating that its calculated binding conformation with the target protein was relatively stable.

[0043] Further analysis revealed differences in docking scores and conformational parameters among the different candidate peptides, making it difficult to directly determine their actual antibacterial activity based solely on molecular docking parameters. Therefore, experimental antibacterial activity evaluations were conducted on the aforementioned six candidate peptides to identify the hawthorn-derived peptides with actual antibacterial activity.

[0044] Example 3 Evaluation of the antibacterial activity of 6 candidate peptides 3.1 Experimental Methods 3.1.1 Determination of antibacterial activity of 6 candidate peptides First, the polypeptide sequences SEQ ID NO.1 to SEQ ID NO.6 obtained in Example 2 were prepared using the Fmoc solid-phase synthesis method.

[0045] The specific process of the Fmoc solid-phase synthesis method is as follows: using Rink Amide resin as a solid-phase support, Fmoc-protected amino acids are sequentially coupled from the C-terminus to the N-terminus of the target polypeptide sequence. After each round of coupling reaction, the Fmoc protecting group is removed using a deprotecting reagent, which is 20% piperidine / DMF (v / v). The amino acid coupling is repeated until the target sequence is synthesized. After the reaction, the crude polypeptide is obtained by lysis, precipitation, centrifugation, and lyophilization. The synthesized product is purified by reversed-phase high-performance liquid chromatography (RP-HPLC), and its molecular weight is confirmed by mass spectrometry to be consistent with the theoretical molecular weight. Finally, polypeptide samples with a purity >95% are obtained, namely SEQ ID NO.1 to SEQ ID NO.6.

[0046] Then, the antibacterial activity of different peptides was compared using the agar diffusion method, with 1×10⁻⁶ Aeromonas hydrophila suspensions. 8 The peptide solution (1 mg / mL) was evenly spread onto LB agar plates, and 0.2 mL of peptide solution (1 mg / mL) was added to each well after punching. Sterile water was used as a negative control, and ampicillin (1 mg / mL) was used as a positive control. The plates were incubated at 37°C for 24 h, and the diameter of the inhibition zone was measured.

[0047] The minimum inhibitory concentration (MIC) of each candidate peptide was further determined using the microbroth dilution method. Each candidate peptide was prepared into different concentration gradients (0.0625–4 mg / mL) and placed in 96-well plates. A suspension of *Aeromonas tempera* (1 × 10⁻⁶ mg / mL) was added to each well. 6 The concentration (CFU / mL) was incubated at 37°C for 24 h, and the minimum concentration at which no bacterial growth was observed to be visible to the naked eye was defined as the MIC.

[0048] 3.1.2 Correlation analysis between molecular docking parameters and actual antibacterial activity The molecular docking results of the six candidate peptides (Table 2) were compared with the actual antibacterial activities obtained in 3.1.1. The six candidate peptides were ranked according to the molecular docking S-value, rmsd_refine value, and actual MIC, respectively, and the correspondence between molecular docking parameters and actual antibacterial activities was analyzed.

[0049] The S-value characterizes the scoring result of molecular docking between the candidate peptide and the target protein. A lower S-value indicates a more favorable calculated and predicted binding free energy, and stronger binding ability. The rmsd_refine value characterizes the conformational deviation after docking conformation refinement. A smaller rmsd_refine value indicates a smaller deviation of the refined conformation from the reference conformation. By comparing the above parameters of different candidate peptides with the actual MIC, the effectiveness of screening candidate peptides based on molecular docking results is evaluated.

[0050] 3.2 Experimental Results and Analysis 3.2.1 Comparison of antibacterial activities of 6 candidate peptides The results of the antibacterial activity assays of the six candidate peptides against Aeromonas tempera are shown in Table 3. The actual antibacterial activities of the six candidate peptides showed significant differences. SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.5 all exhibited antibacterial activity, while SEQ ID NO.4 and SEQ ID NO.6 did not show detectable antibacterial activity. Among the six candidate peptides, SEQ ID NO.3 had the largest inhibition zone diameter (18.8 ± 0.5 mm) and the lowest MIC (0.5 mg / mL), lower than the 2.0 mg / mL of SEQ ID NO.1 and SEQ ID NO.5, and the 4.0 mg / mL of SEQ ID NO.2. This indicates that SEQ ID NO.3 possessed the strongest actual antibacterial activity among the six candidate peptides.

[0051] Table 3. Antibacterial activity of 6 candidate peptides against Aeromonas tempera.

[0052] 3.2.2 Comparison of molecular docking results with actual antibacterial activity The molecular docking parameters of the six candidate peptides were further compared with their actual antibacterial activities.

[0053] Sorted by molecular docking binding ability from strongest to weakest (S value from lowest to highest): SEQ ID NO.3 > SEQ ID NO.1 > SEQ ID NO.5 > SEQ ID NO.4 > SEQ ID NO.6 > SEQ ID NO.2; Sort by conformational deviation from smallest to largest (rmsd_refine value from lowest to highest): SEQ ID NO.4 > SEQ ID NO.6 > SEQ ID NO.2 > SEQ ID NO.5 > SEQ ID NO.1 > SEQ ID NO.3; The actual antibacterial activity is sorted from strongest to weakest (MIC value from lowest to highest): SEQ ID NO.3 > SEQ ID NO.1 ≈ SEQ ID NO.5 > SEQ ID NO.2 > SEQ ID NO.4 ≈ SEQ ID NO.6.

[0054] The results show that SEQ ID NO.3, SEQ ID NO.1, and SEQ ID NO.5, which have strong predicted binding ability, also rank among the top three in actual antibacterial activity, indicating that the S value has some reference value for screening candidate peptides. The relatively large rmsd_refine values ​​of these three sequences suggest significant conformational changes before and after refinement. However, molecular docking parameters do not perfectly correlate with actual antibacterial activity. For example, the S values ​​and rmsd_refine values ​​of SEQ ID NO.4 and SEQ ID NO.6 are lower than those of SEQ ID NO.2, but neither showed detectable antibacterial activity, while the MIC of SEQ ID NO.2 is 4.0 mg / mL. In conclusion, molecular docking parameters can provide some reference for screening candidate peptides, but they cannot accurately predict the actual antibacterial activity and its specific intensity on their own. In particular, the outstanding antibacterial activity of SEQ ID NO.3 still needs to be verified through actual experiments.

[0055] Example 4: Relationship between antibacterial activity and concentration of SEQ ID NO.3 4.1 Experimental Methods Based on the antibacterial activity evaluation results of the six candidate peptides in Example 3 (Table 3), the peptide shown in SEQ ID NO.3 was selected for further antibacterial activity study. SEQ ID NO.3 was prepared into five concentrations: 0.125 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 1.0 mg / mL, and 2.0 mg / mL. The antibacterial activity of SEQ ID NO.3 against Aeromonas tempera at different concentrations was determined by agar diffusion method. Sterile water was used as a negative control, and ampicillin (1 mg / mL) was used as a positive control.

[0056] After determining the MIC of each candidate peptide using the microbroth dilution method, the contents of the well plate at or above the MIC concentration were spread onto LB plates and incubated at 37°C for 24 h. The lowest concentration at which no colony growth was observed was taken as the minimum bactericidal concentration (MBC).

[0057] 4.2 Experimental Results and Analysis The antibacterial activity of SEQ ID NO.3 against Aeromonas hydrophila at different concentrations is shown in Table 4. As the concentration of SEQ ID NO.3 increased, the diameter of its inhibition zone gradually increased. When the concentration of SEQ ID NO.3 increased from 0.125 mg / mL to 2.0 mg / mL, the diameter of the inhibition zone increased from 8.2 ± 0.3 mm to 20.3 ± 0.5 mm, indicating that SEQ ID NO.3 has a significant concentration-dependent antibacterial effect against Aeromonas hydrophila within the tested concentration range. Its MBC was further determined to be 1.0 mg / mL using the plate culture method.

[0058] Table 4. Antibacterial activity of different concentrations of SEQ ID NO.3 peptide against Aeromonas tempera.

[0059] Example 5: Effect of SEQ ID NO.3 on the cell wall integrity of Aeromonas tempera. 5.1 Experimental Methods The concentration of reducing sugars in the supernatant was determined using the DNS (3,5-dinitrosalicylic acid) colorimetric method to evaluate cell wall integrity. After bacterial cell wall disruption, intracellular reducing sugars leak into the extracellular space. Aeromonas tempera was co-incubated with different concentrations of hawthorn polypeptide (0, 0.5×MIC, 1×MIC, 2×MIC) for 2 h. The supernatant was collected by centrifugation, DNS reagent was added, and the mixture was heated in a boiling water bath for 5 min. After cooling, the absorbance at 540 nm was measured, and the reducing sugar concentration was calculated based on the glucose standard curve. The control group did not contain SEQ ID NO. 3.

[0060] 5.2 Experimental Results and Analysis As shown in Table 5, with the increase of the concentration of SEQ ID NO.3 treatment, the leakage of reducing sugar in the Aeromonas hydrophila culture system gradually increased, indicating that SEQ ID NO.3 treatment can increase the release of bacterial extracellular reducing sugar, and this effect has a certain concentration dependence, indicating that SEQ ID NO.3 can affect the cell structure integrity of Aeromonas hydrophila.

[0061] Based on the molecular docking results in Example 2, SEQ ID NO.3 may potentially interact with the transpeptidase associated with Aeromonas tempera. Key interaction analysis shows that: the His residue in the polypeptide forms a hydrogen bond with Ser62 of the transpeptidase; the Asp residue forms an electrostatic interaction with Lys65; the Phe residue forms a π-π stacking interaction with Trp89; and the Leu and Val residues form hydrophobic interactions with Val48 and Ile52. Combined with the reducing sugar leakage experiment results, it is speculated that SEQ ID NO.3 may interact with cell wall synthesis-related proteins, affecting cell wall-related processes, thereby disrupting the cellular structural integrity of Aeromonas tempera and causing intracellular substances to leak to the extracellular space, thus exerting an antibacterial effect.

[0062] Table 5. Effect of SEQ ID NO.3 peptide on reducing sugar leakage from Aeromonas tempera.

[0063] Note: * indicates P<0.05 compared with the control group, ** indicates P<0.01.

[0064] In summary, hawthorn-derived polypeptides with antibacterial activity were obtained through protein extraction, enzymatic hydrolysis, separation, and activity screening. Among them, SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.5 showed inhibitory activity against Aeromonas tempera, with SEQ ID NO.3 exhibiting the strongest antibacterial activity, with a MIC of 0.5 mg / mL and an MBC of 1.0 mg / mL. Based on the results of molecular docking and reducing sugar leakage experiments, it is speculated that SEQ ID NO.3 exerts its antibacterial effect by affecting the integrity of cell structure.

[0065] The embodiments of the present invention described above do not constitute a limitation on the scope of protection of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A hawthorn-derived polypeptide with antibacterial activity, characterized in that, The polypeptide is selected from any of the following: (1) The amino acid sequence is shown in SEQ ID NO.1; (2) The amino acid sequence is shown in SEQ ID NO.2; (3) The amino acid sequence is shown in SEQ ID NO.3; (4) The amino acid sequence is shown in SEQ ID NO.

5.

2. The hawthorn-derived polypeptide with antibacterial activity according to claim 1, characterized in that: The amino acid sequence of the polypeptide is shown in SEQ ID NO.

3.

3. The hawthorn-derived polypeptide with antibacterial activity according to claim 2, characterized in that: The peptide has a minimum inhibitory concentration of 0.5 mg / mL and a minimum bactericidal concentration of 1.0 mg / mL against Aeromonas hydrophila.

4. A method for preparing a hawthorn-derived polypeptide with antibacterial activity, characterized in that, Includes the following steps: (1) Crude protein extraction: Select dried hawthorn, remove the pits and crush it, and pass it through a 60-mesh sieve to obtain hawthorn powder; add 0.1 mol / L NaOH solution to the hawthorn powder at a material-to-liquid ratio of 1:10, and stir and extract at room temperature for 2 h; After extraction, centrifuge at 4000 r / min for 20 min and collect the supernatant; The pH of the supernatant was adjusted to 4.5 with hydrochloric acid, and the precipitate was allowed to stand for 2 h. The precipitate was collected by centrifugation at 4000 r / min for 20 min, washed with distilled water until neutral, and then freeze-dried to obtain crude hawthorn protein. (2) Enzymatic hydrolysis: The crude protein of hawthorn is hydrolyzed by alkaline protease. The amount of alkaline protease added is 3% of the crude protein of hawthorn. The hydrolysis is carried out at 50°C for 4 h to obtain the hydrolysate of hawthorn. (3) Separation: The hawthorn protein hydrolysate was subjected to ultrafiltration through a 3 kDa ultrafiltration membrane to collect the target components, and then further separated using a Sephadex G-25 gel column to obtain multiple polypeptide components; (4) Screening of active components: Screening the multiple polypeptide components for antibacterial activity to obtain active components with antibacterial activity; (5) Sequence identification: The active component was analyzed by LC-MS / MS to obtain the polypeptide sequence; (6) Peptide synthesis and activity screening: The identified peptide sequences are synthesized in a solid phase and the target peptides with antibacterial activity are screened to obtain the hawthorn-derived peptides with antibacterial activity.

5. The preparation method according to claim 4, characterized in that: In step (2), the crude hawthorn protein is dissolved in 0.05 mol / L Tris-HCl buffer. After the enzymatic hydrolysis is completed, the reaction system is treated at 95℃ for 10 min to inactivate the enzyme.

6. The preparation method according to claim 4, characterized in that: In step (3), the Sephadex G-25 gel column has a size of 1.6 cm × 100 cm and uses ultrapure water as the mobile phase.

7. The preparation method according to claim 4, characterized in that: In step (4), the antibacterial activity screening is performed using the agar diffusion method.

8. The preparation method according to claim 7, characterized in that: In step (6), the solid-phase synthesis is the Fmoc solid-phase synthesis method.

9. An antibacterial composition, characterized in that: Includes the hawthorn-derived polypeptide as described in claim 1.

10. The application of the hawthorn-derived polypeptide as described in claim 1 in the preparation of antibacterial agents or antimicrobial preparations for aquaculture.

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