Walnut meal-derived antibacterial polypeptide and its application

Abstract

The present invention discloses an antimicrobial polypeptide derived from walnut meal and its application, belonging to the field of biotechnology. The antimicrobial polypeptide derived from walnut meal of the present invention has an amino acid sequence as shown in any one of SEQ ID NO.1‑3. The present invention uses walnut meal as a raw material to extract and separate antimicrobial polypeptides, which can effectively improve the utilization rate of walnut meal, reduce resource waste, provide new polypeptide types for the field of antimicrobial polypeptides, and provide new resources for the development of new antimicrobial agents, bacterial lysates, food preservatives, and anti-infective drugs.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to an antibacterial polypeptide derived from walnut meal and application thereof. Background Art

[0002] Staphylococcus aureus is the main pathogen that causes food contamination, which can easily cause food poisoning and harm the human body. The more common method at present is chemical control, which can reduce food contamination and delay food corruption, but its drug residues also cause harm to the environment and humans and animals. In addition, with the extensive use of antibiotics, S. aureus has developed resistance to them, and the control effect of chemical methods has been significantly reduced. Therefore, it is very necessary to develop a natural, new and effective antibacterial substance to replace antibiotics.

[0003] Studies have found that antimicrobial peptides are a class of natural substances with antimicrobial activity. They are derived from plants or animals, are relatively safe, and have broad application prospects. Although the antimicrobial activity of antimicrobial peptides has been verified, their antimicrobial mechanism is not clear, and their application range is limited to inhibiting bacterial reproduction. In-depth research on the antimicrobial mechanism of antimicrobial peptides is of great significance for the development of more types of antimicrobial peptides, as well as the development and expansion of the application fields of antimicrobial peptides. Summary of the invention

[0004] The purpose of the present invention is to provide an antibacterial polypeptide derived from walnut meal and its application, so as to solve the problems existing in the above-mentioned prior art. The present invention uses walnut meal as a raw material to extract and separate antibacterial polypeptides, which can effectively improve the utilization rate of walnut meal, reduce resource waste, provide new polypeptide types for the field of antibacterial polypeptides, and provide new resources for the development of new antibacterial agents, bacterial lysates, food preservatives, and anti-infective drugs.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides an antibacterial polypeptide derived from walnut meal, wherein the amino acid sequence of the antibacterial polypeptide is shown in any one of SEQ ID NOs. 1-3.

[0007] SEQ ID NO.1: HAAPK;

[0008] SEQ ID NO.2: WVSK;

[0009] SEQ ID NO.3: TYFPH.

[0010] The present invention also provides the use of the above antibacterial polypeptide in the preparation of an antibacterial agent.

[0011] Preferably, the antibacterial agent has the effect of inhibiting and / or killing Staphylococcus aureus.

[0012] Furthermore, the minimum inhibitory concentration of the antibacterial polypeptide shown in SEQ ID NO.1 against Staphylococcus aureus is 15 mg / mL, and the inhibition zone reaches 13 mm±2.30 mm.

[0013] Furthermore, the minimum inhibitory concentration of the antibacterial polypeptide shown in SEQ ID NO.2 against Staphylococcus aureus is 15 mg / mL, and the inhibition zone reaches 10 mm±0.01 mm.

[0014] Furthermore, the minimum inhibitory concentration of the antibacterial polypeptide shown in SEQ ID NO.3 against Staphylococcus aureus is 15 mg / mL, and the inhibition zone reaches 10 mm±0.01 mm.

[0015] In the present invention, the antibacterial polypeptide has the effects of inhibiting the activities of MDH, SDH and ICDH of Staphylococcus aureus, hindering the production of ATP in bacterial cells, and interfering with cell energy metabolism; the antibacterial polypeptide also has the effects of increasing the LPS activity of Staphylococcus aureus and reducing the activity of β-galactosidase, destroying the bacterial cell membrane, causing the leakage of cell contents, and ultimately leading to the death of the bacteria.

[0016] The present invention also provides the use of the above antibacterial polypeptide in the preparation of drugs for treating bacterial infections.

[0017] Preferably, the drug for treating bacterial infection is a drug for treating Staphylococcus aureus infection.

[0018] The present invention also provides the use of the antibacterial polypeptide in preparing bacterial lysate.

[0019] Preferably, the bacterial lysate has the effect of destroying the cell membrane of Staphylococcus aureus and causing leakage of intracellular proteins, electrolytes and pyruvic acid.

[0020] The present invention also provides the use of the antibacterial polypeptide in the preparation of a food preservative.

[0021] The present invention also provides a food preservative, the main component of which is the above-mentioned antibacterial polypeptide.

[0022] Preferably, the food preservative further comprises food supplementary materials.

[0023] The present invention discloses the following technical effects:

[0024] The present invention separates three antibacterial peptides from fermented walnut meal, namely, HAAPK peptide, WVSK peptide and TYFPH peptide. It has been verified that all three peptides have significant inhibitory activity against Staphylococcus aureus, among which HAAPK peptide has the strongest antibacterial activity, with an MIC of 15 mg / mL and an inhibition zone of 13 mm±2.30, a MIC of 15 mg / mL for WVSK peptide and an inhibition zone of 10 mm±0.01, and a MIC of 15 mg / mL for TYFPH peptide and an inhibition zone of 10 mm±0.01.

[0025] The present invention also studied the antibacterial mechanism of HAAPK peptide, and found that when HAAPK peptide with a concentration of 1.0 MIC was used to act on S. aureus, compared with the blank control group, the protein concentration, intracellular electrolytes and pyruvate content decreased by 55.0%, 17.75% and 76.84%, respectively, indicating that HAAPK peptide caused leakage of cell contents; AKP content increased to about 9 times that of the control group, LPS content increased by 18.25%, and β-galactosidase content decreased by 68.57%, indicating that HAAPK peptide destroyed the bacterial cell membrane; ATP, ATPase, SDH, MDH content decreased by 93.28%, 8.08%, 82.63% and 86.89%, respectively, and ICDH activity in the cytoplasm increased to nearly 2 times that of the control group, indicating that HAAPK peptide interfered with intracellular energy metabolism. In summary, HAAPK peptide combines cell membrane damage and disrupts energy metabolism to exert antibacterial activity.

[0026] The invention adopts walnut meal as raw material to extract and separate antibacterial polypeptides, which can effectively improve the utilization rate of walnut meal, reduce resource waste, provide new polypeptide types for the field of antibacterial polypeptides, and provide new resources for the development of new antibacterial agents, bacterial lysates, food preservatives, and anti-infection drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0028] Figure 1 The results of the evaluation of the effect of 0-3 kDa fermented walnut meal on the extension of the shelf life of tomato juice; A is the change in the total number of colonies in tomato juice without S. aureus inoculation, and B is the change in the total number of colonies in tomato juice inoculated with S. aureus;

[0029] Figure 2are the results of peptide library analysis; A is the ratio of peptides with different molecular weights in the peptide library, B is the ratio of peptides with different net positive charge numbers in the peptide library, C is the ratio of peptides with different basic amino acid residue ratios in the peptide library, D is the ratio of peptides with different theoretical isoelectric points in the peptide library, E is the ratio of peptides with different hydrophobic amino acid residue contents in the peptide library, and F is the ratio of peptides with different hydrophobicity in the peptide library;

[0030] Figure 3 is the docking energy between the peptide library and penicillin binding protein;

[0031] Figure 4 The molecular docking results of HAAPK peptide, WVSK peptide, TYFPH peptide and penicillin-binding protein; A is the binding of HAAPK peptide, WVSK peptide, TYFPH peptide and 1dls, B is the binding of HAAPK peptide, WVSK peptide, TYFPH peptide and 1tvf, C is the binding of HAAPK peptide, WVSK peptide, TYFPH peptide and 1vqq, D is the binding of HAAPK peptide, WVSK peptide, TYFPH peptide and 3vsk, E is the binding of HAAPK peptide, WVSK peptide, TYFPH peptide and 7o61;

[0032] Figure 5 The molecular docking results of HAAPK peptide, WVSK peptide, TYFPH peptide and the main amino acids of cell membrane;

[0033] Figure 6 is the Fourier transform infrared spectroscopy (FTIR) analysis diagram of HAAPK peptide;

[0034] Figure 7 is the secondary structure fitting diagram of HAAPK peptide;

[0035] Figure 8 : is the circular dichroism (CD) analysis diagram of HAAPK peptide;

[0036] Fig. 9 The effects of HAAPK peptide treatment on protein concentration, relative conductivity and pyruvate content of S. aureus;

[0037] Fig.10 The effect of HAAPK peptide treatment on the activity of β-galactosidase and lipase (LPS) of S. aureus;

[0038] Fig.11 The effects of HAAPK peptide treatment on AKP activity, ATP content, ATPase activity, SDH enzyme activity, MDH enzyme activity and ICDH enzyme activity in the cytoplasm of S. aureus;

[0039] Fig.12The SEM results of the effect of HAAPK peptide treatment on the microscopic morphology of S. aureus;

[0040] Fig.13 TEM detection results of the effect of HAAPK peptide treatment on the microscopic morphology of S. aureus. DETAILED DESCRIPTION

[0041] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0042] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0043] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0044] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0045] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0046] The polypeptide of the present invention can be obtained by those skilled in the art through artificial synthesis or through fermentation and screening of walnut meal.

[0047] The plant lactobacillus used in the present invention is Lactobacillus plantarum CICC 23121, the Bacillus subtilis is Bacillus subtilis CICC 10002, and the Staphylococcus aureus is Staphylococcus aureus ATCC 25923.

[0048] The research process of the present invention is as follows:

[0049] The invention prepares fermented walnut meal with B.subtilis and L.plantarum, obtains 0-3 kDa fermented walnut meal components through ultrafiltration separation, and applies the components to preserved fresh tomato juice; obtains polypeptide sequences closely docked with S.aureus penicillin binding protein (PBP, PDB ID: 1VQQ, 1TVF, 3VSK, 1DLS, 7061) from the 0-3 kDa fermented walnut meal components through virtual screening technology, then artificially synthesizes the corresponding polypeptides, detects their antibacterial activities against S.aureus, and obtains high antibacterial short peptide sequences HAAPK, WVSK and TYFPH; adopts molecular docking technology to dock the antibacterial peptides HAAPK, WVSK and TYFPH with PBPs and main amino acids of cell membrane, respectively, and the results show that the interaction forces mainly include hydrogen bonds, hydrophobic interactions and π-π conjugated interactions; FTIR spectrum and circular dichroism spectrum show that β-folding and random curling are the main secondary structures of HAAPK in solid and liquid states. The changes in spore morphology and ultrastructure, leakage of contents, and cell membrane-related enzyme content indicated that the antimicrobial peptide HAAPK destroyed the cell membrane of S. aureus; the changes in the activities of key enzymes in the ATP, ATPase, and TAC cycles indicated that HAAPK interfered with the energy metabolism of S. aureus.

[0050] Example 1 Preparation of antimicrobial peptides and preliminary verification of their function

[0051] 1. Preparation of fermented walnut meal

[0052] According to the method described in the literature (Hu Yue. Preparation of antibacterial fermentation broth, analysis of interaction mechanism and preservation of sea buckthorn [D]. Beijing Forestry University, 2022.), fermented walnut meal was prepared using Lactobacillus plantarum CICC23121 and Bacillus subtilis CICC 10002, and components with different molecular weight ranges (0-3kDa, 3-5kDa, 5-10kDa, >10kDa) were separated by ultrafiltration and freeze-dried for later use. The minimum inhibitory concentration (MIC) of fermented walnut meal components of each molecular weight against S. aureus was determined by the two-fold dilution method. Take 1mL (10 5 CFU / mL) of S. aureus spore suspension was evenly spread on LB agar medium for incubation. 100 μL of antibacterial ingredients were added to the first group, 1 / 2 of the concentration of the first group was added to the second group, and 1 / 2 of the concentration of the second group was added to the third group. A total of 5 groups were tested. Sterile water and natamycin were used as negative and positive controls, respectively, and the LB agar medium was cultured at 28°C for 5 days. The MIC value is the lowest concentration without bacterial growth. After testing, 0-3 kDa fermented walnut meal (FW) had the highest antibacterial activity, with a MIC of 9 mg / mL against S. aureus.

[0053] 2. Evaluation of the effect of 0-3 kDa fermented walnut meal on extending the shelf life of tomato juice

[0054] Tomatoes were crushed into a homogenate, mixed with water (1:3, w / v), and the supernatant was collected by filtration. In order to obtain sterile juice components and prevent interference from microorganisms, the juice was packaged and pasteurized (65°C, 30 min). S. aureus was added to tomato juice to prepare a final volume of 10 4 FW was added to sterile and non-sterile tomato juices at final concentrations of 0, 0.5 MIC (4.5 mg / mL) and 1.0 MIC (9 mg / mL), respectively, and stored at 4°C. The total number of colonies in the tomato juices was determined on days 0, 2, 4, 5, and 6.

[0055] The total colony count was determined according to GB 4789.2-2016. Take 1 mL of tomato juice and dilute it 10 times with sterile saline to prepare a series of 10-fold serial dilution samples. Take 1 mL of the sample and spread it on LB nutrient agar, incubate it at 37°C for 24 hours, and determine the total colony count.

[0056] Results: Figure 1As shown in A, the total number of colonies in the three groups of tomato juice without S. aureus added increased after the fourth day. Among them, the total number of colonies in the tomato juice with 0.5MIC and 1.0MIC FW was 90.71% and 92.14% lower than that in the blank group, respectively. On the sixth day, the total number of colonies in the 0.5MIC group and the 1.0MIC group was 86.67% and 90.0% lower than that in the blank group, respectively. Figure 1 As shown in B, on day 2, 0.5MIC and 1.0MIC FW containing 1.8×10 4 In tomato juice with Lg (CFU / mg) S. aureus, the total number of colonies decreased to 0. With the extension of storage time, on the 6th day, the total number of colonies in tomato juice with 0.5MIC and 1.0MIC FW was 56.36% and 75.45% lower than that in the blank group, respectively.

[0057] 3. Peptide identification and virtual screening

[0058] FW was purified by LC-MS / MS to identify the peptide sequence, PyRx was used to virtually screen the characteristic peptides with antibacterial activity, and artificial synthesis was used to verify its antibacterial activity.

[0059] (1) LC-MS / MS analysis

[0060] FW was pretreated with reductive alkylation and analyzed by liquid chromatography-tandem mass spectrometry using the Ultimate 3000 system with an Orbitrap Elite TM The analysis was performed using a hybrid ion trap-orbitrap mass spectrometer and an ESI nano-ray source. The peptides were separated using an Acclaim PepMap RPLC C18 analytical column (300 μm × 5 mm, 5 μm). Mobile phase A was 0.1% (v / v) formic acid solution, and mobile phase B was 0.1% (v / v) formic acid solution and 80% (v / v) acetonitrile. The elution gradient was: 4-8% B, 0-2 min; 8-28% B, 2-45 min; 28-40% B, 45-55 min; 40-95% B, 55-56 min; 95-95% B, 56-66 min, and the mobile phase flow rate was 600 nL / min. The parameters were as follows: MS scan range 100-1500 m / z; resolution 70000; AGC target 3e6; maximum injection time 40 ms; (2) MS / MS: resolution 1,7500; AGC target 1e5; maximum injection time 60 ms; Top N 20; NCE / steepedNCE 27. The peptide sequences were analyzed using the analysis software PEAKSStudio 8.5De Novo, and the peptide libraries were analyzed for the peptide sequences identified by LC-MS / MS.

[0061] Results: Figure 2 As shown in Figure 2, polypeptides with a molecular weight of 0.5 to 1.0 kDa are the main components of FW, accounting for 68.5% of the total ( Figure 2 The proportion of peptides with a net positive charge greater than 0 in FW was 63.7% ( Figure 2 B), the proportion of polypeptides with basic amino acid residues greater than 10% was 73.5% ( Figure 2 C), the proportion of peptides with theoretical isoelectric points greater than 7 is 34.3% ( Figure 2 The proportion of peptides with hydrophobic amino acid residues greater than 60% was 21% ( Figure 2 E), hydrophilic peptides accounted for 64.08% and hydrophobic peptides accounted for 35.92% ( Figure 2 F).

[0062] (2) Virtual screening

[0063] KingDraw was used to draw the 3D structure diagram of the identified peptides, and the crystal structure of penicillin-binding protein (PBP, PDB ID: 1VQQ, 1TVF, 3VSK, 1DLS, 7061) involved in the biosynthesis of peptidoglycan in the bacterial cell wall of Staphylococcus aureus was obtained from the RCSB protein database (http: / / www.rcsb.org / pdb / 2020.03.12). PBPs were hydrogenated and dehydrated using Discovery Studio 2.5 software. PyRx 0.8 software was used to screen possible antimicrobial peptides, and AutodockVina was used as the docking software. Walnut protein sequences were downloaded from the Uniprot database (https: / / www.uniprot.org / proteomes), and genus6.1.6 software was used to compare whether the characteristic peptides belonged to the walnut protein sequence.

[0064] Results: Figure 3As shown in the figure, the docking energy of peptides and PBPs (1dls, 1vqq, 7o61, 3vsk, 1tvf) is mainly concentrated in [5.0, 9.0]. Among them, the docking energy is [5.0, 6.0), and the proportion of 1dls docking peptides is the highest (accounting for 67.19%); the docking energy is [6.0, 7.0), and the proportion of 1tvf docking peptides is the highest (accounting for 56.25%), followed by 1vqq docking peptides (accounting for 53.13%); the docking energy is [7.0, 8.0), and the proportion of 3vsk docking peptides is the highest (accounting for 57.03%), followed by 7o61 docking peptides (accounting for 53.91%); the docking energy is [8.0, 9.0), and the proportion of 7o61 docking peptides is the highest (accounting for 28.13%), followed by 3vsk docking peptides (accounting for 21.88%). The larger the absolute value of binding energy, the more stable the binding between the peptide and 1dls, 1vqq, 7o61, 3vsk, and 1tvf. Therefore, according to the binding ability with PBPs (1dls, 1vqq, 7o61, 3vsk, and 1tvf), three characteristic peptides HAAPK, WVSK, and TYFPH with high docking energy rankings and belonging to walnut protein sequences were screened out. The three characteristic peptides all bind stably to PBPs, and the order of binding energy from high to low is 1dls, 1tvf, 1vqq, 3vsk, and 7o61.

[0065] As shown in Table 1, the properties of the three characteristic peptides were analyzed. The proportions of basic and hydrophobic amino acid residues in HAAPK, WVSK and TYFPH decreased in sequence, which were 0.4, 0.25, 0.2 and 0.6, 0.5, 0.4, respectively. At the same time, the number of net positive charges of HAAPK, WVSK and TYFPH also showed the same pattern, which were 1.25, 0.98 and 0.25, respectively, which may be contributed by the presence of basic amino acid residues. Therefore, HAAPK has the largest number of net positive charges and the highest content of basic amino acid residues and hydrophobic amino acid residues. In addition, the antibacterial activity of antimicrobial peptides is also related to the position of amino acids in the sequence. The N-terminus of WVSK is the hydrophobic amino acid Trp, which can promote the initial interaction with the lipid bilayer, and the C-terminus is the basic amino acid Lys, which can promote the interaction with the negatively charged bacterial membrane.

[0066] Table 1 Properties of three characteristic peptides

[0067]

[0068] (3) Artificially synthesized peptides

[0069] HAAPK, WVSK and TYFPH peptides were artificially synthesized. The peptides were synthesized using the Fmoc solid phase synthesis method according to the amino acid sequence, and the powdered peptides were obtained after cutting, precipitation and purification. The synthesis process was entrusted to Nanjing Yuanpeptide Biotechnology Co., Ltd., and the purity was greater than 95%.

[0070] (4) Antibacterial activity of synthetic peptides

[0071] The minimum inhibitory concentration (MIC) of the synthetic peptides against S. aureus was determined by the two-fold dilution method, and the antibacterial activity of the synthetic peptides was evaluated by the diameter of the inhibition zone. Results: As shown in Table 1, HAAPK had the best antibacterial activity against S. aureus, with a MIC of 15 mg / mL and an inhibition zone of 13 mm ± 2.30. The antibacterial activities of TYFPH and WVSK were second, with a MIC of 15 mg / mL and an inhibition zone of 10 mm ± 0.01.

[0072] Example 2

[0073] (1) Molecular docking of antimicrobial peptides and penicillin-binding proteins

[0074] Antimicrobial peptides and penicillin binding proteins (PBPs, PDB ID: 1VQQ, 1TVF, 3VSK, 1DLS, 7061) were hydrogenated, optimized and minimized using Schrodinger Maestro 11.9 software, and the Glide module was used for molecular docking of peptides and PBPs. The default settings of the LigPrep module were used for preprocessing, optimization and minimization, and PBPs were imported. A size of The grid spacing is A grid box is used to surround the PBPs.

[0075] Results: Molecular docking was used to simulate the interaction between conformational antimicrobial peptides and PBPs. The docking results are as follows: Figure 4 As shown, hydrogen bonding, hydrophobic interactions and π-π stacking are the main interactions between the antimicrobial peptides HAAPK, TYFPH and WVSK and PBPs.

[0076] For the 1dls target protein, the HAAPK peptide can form 4 hydrogen bonds with its THR-146, SER-119, THR-56, and ILE-7 amino acid residues, and 2 hydrophobic interactions with LYS-55, ILE-16, and ALA-9. In particular, the protonated amino group of the peptide forms a salt bridge with GLU-30 and forms a π-π conjugated interaction with the benzene rings of PHE-31 and TYR-22. TYFPH forms 8 strong hydrogen bonds with the VAL-115, ILE-16, GLU-78, LEU-75, LYS-54, LYS-55, THR-56, and SER-59 amino acid residues at the 1dls protein site. The nitrogen heterocyclic ring of the peptide forms a π-π conjugated interaction with PHE-34, and the benzene ring forms a π-π conjugated interaction with SER-118. WVSK can form six hydrogen bonds with the active sites of 1dls protein, namely THR-56, TYR-121, SER-59, ALA-9, ILE-16, and THR-146, and five hydrophobic interactions with PHE-31, PHE-34, ILE-60, PRO-61, and LYS-55.

[0077] For the 1tvf target protein, the HAAPK peptide can form five hydrogen bonds with its LYS-292, SER-262, SER-116, TYR-268, and THR-260 amino acid residues, and form π-π conjugated interactions with LEU-294 and PHE-241. TYFPH forms four hydrogen bonds with the SER-116, TYR-291, SER-262, and GLU-183 amino acid residues at the 1tvf protein site, forms one hydrophobic interaction with ALA-74, and the nitrogen heterocyclic ring of the peptide forms a π-π conjugated interaction with the benzene ring of PHE and PHE-241. WVSK can form four hydrogen bonds with SER-116, SER-262, SER-75, and ASN-141 of 1tvf. In particular, the protonated amino group of the peptide forms a salt bridge with GLU-183 and forms a π-π conjugated interaction with the nitrogen heterocyclic ring of GLU-297.

[0078] For the 1vqq target protein, the HAAPK peptide can form two hydrogen bonds with its LYS-218 and GLY-374 amino acid residues, five hydrophobic interactions with LYS-219, TYR-223, VAL-217, LEU-224, and PRO-370, and one π-π conjugated interaction with GLU-379. TYFPH forms three hydrogen bonds with the 1vqq protein ASP-221, LYS-219, and ASP-367 amino acid residues, the benzene ring forms a π-π conjugated interaction with LYS-215, and four hydrophobic interactions with VAL-217, LEU-224, LEU-383, and PRO-370. WVSK can form three hydrogen bonds with ASP-221, TYR-223, and LYS-215 of 1vqq, three hydrophobic interactions with VAL-217, LEU-224, and PRO-370, and two π-π conjugated interactions with ASP-226 and GLU-189.

[0079] For the 3vsk target protein, the HAAPK peptide can form 5 hydrogen bonds with its MET-226, SER-225, THR-92, TYR-51, and ASN-223 amino acid residues, and 4 hydrophobic interactions with PRO-196, ALA-88, LYS-52, and PRO-248. TYFPH can form 4 strong hydrogen bonds with the 3vsk protein site THR-90, THR-224, MET-226, and TYR-209 amino acids, and 4 hydrophobic interactions with ALA-249, PRO-248, ALA-88, and LYS-52. In addition, the nitrogen heterocyclic ring of the peptide forms a π-π conjugated interaction with HIS-50. WVSK can form 5 hydrogen bonds with the 3vsk protein active site TYR-51, TYR-209, SER-225, ALA-88, and ALA-88, and 1 hydrophobic interaction with LYS-52.

[0080] For the 7o61 target protein, the HAAPK peptide can form 6 hydrogen bonds with its THR-659, THR-626, SER-664, ASP-662, GLY-698, and GLU-623, and 1 hydrophobic interaction with PRO-661. TYFPH forms 4 hydrogen bonds with the 7o61 protein sites LYS-610, GLY-628, ASP-662, and THR-626, and 3 hydrophobic interactions with ILE-614, LYS-697, and VAL-625. WVSK can form 5 hydrogen bonds with the 7o61 protein active sites THR-659, GLY-628, LYS-610, ASP-662, and THR-626, and 3 hydrophobic interactions with ILE-614, VAL-625, and LYS-697, and form a π-π conjugated interaction with the indole of GLU-623.

[0081] In summary, the main interactions between the antimicrobial peptides HAAPK, TYFPH and WVSK and PBPs are hydrogen bonds, hydrophobic interactions and π-π stacking, which are tightly bound to each other and have a high degree of matching.

[0082] (2) Molecular docking of antimicrobial peptides and major amino acids in cell membranes

[0083] HAAPK, WVSK, and TYFPH were used as ligands, tryptophan (Trp), phenylalanine (Phe), and tyrosine (Tyr) were used as receptors, and the receptor mass center was used as the center position of the box, and the box size was set to 40×40×40. Molecular docking was performed using AutoDock software, and the binding effect visualization was performed using Pymol 2.1 software. The Lamarckian genetic algorithm was used for calculation, and the algorithm was as follows: the population size was 150, the energy evaluation was up to 25 million times, the maximum was 2000 times, the crossover rate was 0.8 times, the mutation was 0.02 times, and the independent docking operation was 10 times.

[0084] Results: The present invention carried out molecular docking of HAAPK, WVSK, and TYFPH with the target Phe, Trp, and Tyr, respectively. The three peptides were stably bound to Phe, Trp, and Tyr (Table 2). The molecular docking was used to simulate the conformation, and the docking results were as follows: Figure 5 As shown, hydrogen bonding, hydrophobic interactions, and π-π stacking are the main interactions between HAAPK, TYFPH, and WVSK and Phe, Trp, and Tyr.

[0085] Phe forms two hydrogen bonds with Pro of HAAPK (the bond lengths are ), forming a π-π conjugated interaction (bond length ); Phe forms two hydrogen bonds with Tyr and Phe of TYFPH (the bond lengths are ), forming a π-π conjugated interaction with the five-membered ring of TYFPH (bond length is ), Phe forms two hydrogen bonds with Val and Trp of WVSK (the bond lengths are ), forming a π-π conjugated interaction with the six-membered ring of WVSK (bond length ).

[0086] Trp forms two hydrogen bonds with Lys and Pro of HAAPK (the bond lengths are ), forming a π-π conjugated interaction with the five-membered ring of HAAPK (bond length is ), Trp forms two hydrogen bonds with Phe and Tyr of TYFPH (the bond lengths are ), forming two π-π conjugated interactions with the five-membered ring and the six-membered ring of TYFPH (the bond lengths are ), Trp forms two hydrogen bonds with Val and Lys of WVSK (the bond lengths are ), forming a π-π conjugated interaction with the indole of WVSK (bond length is ).

[0087] Tyr and Phe residues differ by only one phenolic hydroxyl group, have similar structures, and have similar binding modes with peptides, which can form hydrogen bonds and π-π conjugated interactions, allowing peptides and small molecules to bind effectively. Tyr forms two hydrogen bonds with Pro of HAAPK (the bond lengths are ), forming a π-π conjugated interaction (bond length is ); Tyr forms two hydrogen bonds with Thr and Phe of TYFPH (the bond lengths are ), forming a π-π conjugated interaction with the six-membered ring of TYFPH (bond length is ); Tyr forms three hydrogen bonds with Trp, Val, and Lys of WVSK (the bond lengths are ), no π-π conjugated interaction is formed.

[0088] In summary, the hydrogen bond distances between Phe, Trp, Tyr and HAAPK, WVSK, and TYFPH targets are short, less than or equal to the traditional hydrogen bond distances. Strong binding force. In addition, the π-π conjugated interaction formed by the benzene ring of the amino acid and the five-membered ring or six-membered ring of the polypeptide also makes an important contribution to the binding of the two. Therefore, hydrogen bonding, hydrophobic interaction, and π-π conjugated interaction are the main interaction forces between HAAPK, TYFPH, WVSK and Phe, Trp, and Tyr.

[0089] Table 2 Binding energy of characteristic peptides and main amino acids of cell membrane

[0090]

[0091] Example 3 Structural Characterization of HAAPK Peptide

[0092] (1) Fourier transform infrared spectroscopy (FTIR) analysis

[0093] The secondary structure of solid-state HAAPK was determined by FTIR spectroscopy in ATR mode. The spectra were recorded in transmittance mode from 400 to 4000 cm-1 with 32 scans and a resolution of ±4 cm -1 The experimental results were analyzed using OMNIC 8.2 software. The positions and numbers of sub-peaks were estimated after Gaussian peak fitting. Based on the correspondence between each sub-peak and the secondary structure, the relative content of each secondary structure was calculated using the comprehensive area.

[0094] Results: Figure 6 As shown, HAAPK is at 3211.5 cm -1 The absorption peak at 3067.1cm belongs to the -OH stretching vibration. -1 The absorption peak at 1073.05cm belongs to CH stretching vibration. -1 The absorption peak at 1540.58 cm belongs to the amide I band, which is mainly caused by the stretching vibration of C=O. -1 The absorption peak at belongs to the amide II band, which is mainly caused by the NH bending vibration. Figure 7 As shown, select 1600cm -1 ~1700cm -1 The bands were fitted for protein secondary structure, and in the solid HAAPK, β-sheets accounted for 45.0%, random coils accounted for 21.55%, β-turns accounted for 19.91%, and α-helices accounted for 13.54% (Table 3). In the solid state, β-sheets are the main secondary structure, which indicates that the β-sheet motif may be related to the activity of HAAPK.

[0095] (2) Circular dichroism (CD) analysis

[0096] The secondary structure content of HAAPK in liquid environment was determined by CD method. HAAPK with a concentration of 0.2 mg / mL was dissolved in 10 mM HCl to simulate the acidic environment of the stomach; and in 10 mM sodium phosphate buffer, pH 7.2, to simulate the water environment. CD spectrum measurement was performed using the Peltier module of a JASCO J-1500 spectropolarimeter, and the temperature was maintained at 25°C. The quartz disk had a diameter of 1 mm, a slit of 2 nm, and a scanning speed of 60 nm / min, and the CD spectrum was recorded in the range of 195 to 250 nm.

[0097] Results: Figure 8As shown in the figure, the most abundant secondary structure of HAAPK in acidic and aqueous environments is random coil (61.4% and 51.2%), followed by β-fold (19.0% and 19.4%), indicating that the secondary structure of HAAPK changes when it changes from solid to liquid (Table 3). Therefore, it can be inferred that β-fold and random coil are the main secondary structures of HAAPK in solid and liquid states.

[0098] Table 3 Secondary structure analysis results

[0099]

[0100] Example 4 Antibacterial Mechanism of HAAPK Peptide

[0101] (1) Leakage of cell contents

[0102] The concentration was 1×10 5 cfu / mL of S. aureus was suspended in LB medium and cultured at 28°C for 18h. The precipitate was obtained by centrifugation at 8000×g for 5min and suspended in phosphate buffer (PB, pH 7.2) to obtain a culture solution. At the same time, HAAPK peptide was added to the culture solution to make the final concentrations of 0.5MIC and 1MIC, respectively. Anhydrous ethanol was used as a negative control. After constant temperature culture at 28°C for 8h, centrifugation was performed at 8000×g for 2min at 4°C. The supernatant was collected and the relative conductivity was measured using a conductivity meter. The bacterial precipitate was washed 3 times with PBS, freeze-dried, and the changes in cell components were analyzed by FTIR.

[0103] The bacterial pellet was ground with liquid nitrogen, and 9 volumes of PBS buffer were added to prepare a slurry, which was centrifuged at 2000 rpm for 10 minutes at 4° C. Protein and pyruvate detection kits (Nanjing Jiancheng Bioengineering Institute) were used to detect protein and pyruvate content.

[0104] Results: The changes in membrane permeability can be indirectly reflected by measuring protein concentration, relative conductivity (used to characterize intracellular electrolyte leakage) and pyruvate content, which can be used to examine the integrity of the cell membrane. Fig. 9 As shown in the figure, compared with the control group, after the HAAPK peptide with a concentration of 0.5 MIC was used to act on S. aureus, the intracellular protein concentration, relative conductivity and pyruvate content decreased by 45.91%, 10.75% and 23.39% respectively; after the HAAPK peptide with a concentration of 1.0 MIC was used to act on S. aureus, they decreased by 55.0%, 17.75% and 76.84% respectively. This shows that the HAAPK peptide caused the leakage of protein, electrolytes and pyruvate in S. aureus cells in a concentration-dependent manner, thus leading to cell death.

[0105] (2) Determination of β-galactosidase activity and lipase activity

[0106] The concentration was 1×10 5 cfu / mL of S. aureus was suspended in LB medium and cultured at 28°C for 18h. The precipitate was obtained by centrifugation at 8000×g for 5min, and it was suspended in phosphate buffer (PB, pH 7.2) to obtain a culture solution. At the same time, HAAPK peptide was added to the culture solution to make the final concentrations of 0.5MIC and 1MIC, respectively. Anhydrous ethanol was used as a negative control. After constant temperature culture at 28°C for 8h, the bacterial precipitate was obtained by centrifugation at 8000×g for 2min at 4°C. The bacterial precipitate was ground with liquid nitrogen, and 9 volumes of PBS buffer were added to prepare a slurry, which was centrifuged at 2000rpm at 4°C for 10min. The activity of β-galactosidase and lipase (LPS) was detected using the kit provided by Nanjing Jiancheng Bioengineering Research Institute.

[0107] Results: Fig.10 As shown, after S. aureus was treated with 0.5MIC and 1.0MIC HAAPK peptides, LPS activity increased by 17.85% and 18.25% respectively compared with the control group, and β-galactosidase activity decreased by 55.71% and 68.57% respectively, indicating that HAAPK peptide increased cell membrane permeability, promoted phospholipid degradation, and destroyed cell membranes.

[0108] (3) Enzyme activities related to cellular energy metabolism

[0109] The concentration was 1×10 5 cfu / mL of S. aureus was suspended in LB medium and cultured at 28°C for 18h. The precipitate was obtained by centrifugation at 8000×g for 5min and suspended in phosphate buffer (PB, pH 7.2) to obtain a culture solution. At the same time, HAAPK peptide was added to the culture solution to a final concentration of 0.5MIC and 1MIC, respectively. Anhydrous ethanol was used as a negative control. After constant temperature culture at 28°C for 8h, the bacterial precipitate was obtained by centrifugation at 8000×g for 2min at 4°C. The bacterial precipitate was ground with liquid nitrogen, and 9 volumes of PBS buffer were added to prepare a slurry, which was centrifuged at 2000rpm at 4°C for 10min. The alkaline phosphatase activity (AKP), ATP content, ATPase activity, succinate dehydrogenase (SDH) activity, malate dehydrogenase (MDH) activity and mitochondrial isocitrate dehydrogenase (ICDH) activity in the cytoplasm were determined using the kit provided by Nanjing Jiancheng Bioengineering Research Institute.

[0110] Results: Fig.11As shown, after S. aureus was treated with 0.5 and 1.0 MIC HAAPK peptide, the content of AKP increased by about 6 times and 8 times. After S. aureus was treated with 0.5 MIC HAAPK peptide, the ATP content and ATPase activity decreased by 79.16% and 2.16%, respectively. After S. aureus was treated with 1.0 MIC HAAPK peptide, the ATP content and ATPase activity decreased by 93.28% and 8.08%, respectively. After S. aureus was treated with 0.5 MIC HAAPK peptide, the SDH and MDH activities decreased by 38.12% and 72.13%, respectively, and the ICDH activity in the cytoplasm was about 1 times that of the control group. When treated with 1.0 MIC HAAPK peptide, the SDH and MDH activities decreased by 82.63% and 86.89%, respectively, and the ICDH activity in the cytoplasm was about 2 times that of the control group. These results suggest that HAAPK peptide may inhibit the production of ATP in S. aureus by inhibiting the activities of MDH, SDH, and ICDH in S. aureus in a concentration-dependent manner, thereby preventing cell death.

[0111] (4) Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) analysis

[0112] The concentration was 1×10 5 cfu / mL of S. aureus was suspended in LB medium and cultured at 28°C for 18h. The precipitate was obtained by centrifugation at 8000×g for 5min and suspended in phosphate buffer (PB, pH 7.2) to obtain a culture solution. At the same time, HAAPK peptide was added to the culture solution to a final concentration of 0.5MIC and 1MIC, respectively. Anhydrous ethanol was used as a negative control. After constant temperature culture at 28°C for 8h, the bacterial precipitate was obtained by centrifugation at 8000×g for 2min at 4°C. The bacterial precipitate was diluted in 0.1M phosphate buffer (pH 7.0), then 2.5% glutaraldehyde was added and vacuum cultured for 10 minutes, and rinsed with the same buffer at 4°C for 8 hours. The sample was further dehydrated in a graded ethanol series (50%, 70%, 90%), critical point dried with CO2, and finally mounted on a specimen stub, coated with gold, and examined with a scanning electron microscope.

[0113] TEM samples were dehydrated in graded dilutions of ethanol (50%, 70%, 90%) for 15 min, acetone: embedding solution (3:1) for 2-3 h, acetone: embedding solution (1:1) for 2-3 h, and then immersed in 100% embedding solution overnight, and finally fixed in a graded oven (37°C overnight, 45°C for 12 h, and 60°C for 24 h). Ultrathin sections (thickness of about 50-60 nm) were stained with 3% uranyl acetate and lead citrate, and then examined by TEM.

[0114] Results: Fig.12 As shown in the figure, after S. aureus was treated with anhydrous ethanol, slight wrinkles appeared on the cell membrane surface, and the cells grew normally with the expected size and distribution. After treatment with 1MIC HAAPK peptide, large bubbles appeared on the surface of S. aureus cells and the wrinkles increased, and the cell membrane was concave and obviously ruptured. Fig.13 As shown, after S. aureus was treated with anhydrous ethanol, the cells maintained normal cell morphology and the intracellular substances were evenly distributed. After being treated with 1MIC HAAPK peptide, the cell wall of S. aureus became thinner, the cytoplasmic matrix decreased, a large number of vacuoles were fused and formed in the cytoplasm, and the internal structure of the cell became blurred.

[0115] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. An antimicrobial polypeptide derived from walnut meal, characterized in that: The amino acid sequence of the antibacterial polypeptide is shown in any one of SEQ ID NOs. 1-3.

2. Use of the antibacterial polypeptide according to claim 1 in the preparation of an antibacterial agent, characterized in that: The antibacterial agent has the function of inhibiting and / or killing Staphylococcus aureus.

3. Use of the antibacterial polypeptide according to claim 1 in the preparation of a drug for treating bacterial infection, characterized in that: The drug for treating bacterial infection is a drug for treating Staphylococcus aureus infection.

4. Use of the antibacterial polypeptide according to claim 1 in preparing bacterial lysate, characterized in that: The bacterial lysis solution is Staphylococcus aureus lysis solution.

5. The use according to claim 4, characterized in that: The bacterial lysate has the effect of destroying the cell membrane of Staphylococcus aureus and causing the leakage of intracellular proteins, electrolytes and pyruvic acid.

6. Use of the antimicrobial polypeptide according to claim 1 in the preparation of a food preservative.

7. A food preservative, characterized in that: The main component is the antibacterial polypeptide according to claim 1.

8. The food preservative according to claim 7, characterized in that: Also contains food ingredients.

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