An antibacterial peptide composition, and a preparation method and application thereof

Abstract

The present application relates to a kind of antibacterial peptide composition and its preparation method and application, antibacterial peptide composition includes antibacterial peptide PP109 and antibacterial peptide PP112, the antibacterial peptide PP109 is selected from the compound shown in formula (AGALTGIKGFLHKWLKRTK), or selected from the stereoisomer, prodrug, crystal form, pharmaceutically acceptable salt, pharmaceutically acceptable ester or pharmaceutically acceptable solvate of the compound shown in formula (AGALTGIKGFLHKWLKRTK), the antibacterial peptide PP112 is selected from the compound shown in formula (FSACNVLKKWFLGKFKRN), or selected from the stereoisomer, prodrug, crystal form, pharmaceutically acceptable salt, pharmaceutically acceptable ester or pharmaceutically acceptable solvate of the compound shown in formula (FSACNVLKKWFLGKFKRN);The application of antibacterial peptide composition in preparing antibacterial drug has the advantages of strong antibacterial activity to a variety of pathogenic bacteria, clear mechanism of action, good biological safety.

Description

Technical Field

[0001] This invention relates to the technical field of antimicrobial peptides, and in particular to an antimicrobial peptide composition, its preparation method, and its application. Background Technology

[0002] With the widespread use of antibiotics in healthcare, animal husbandry, and agricultural production, bacterial resistance has become an increasingly serious problem. Traditional antibiotics mainly exert their effects by interfering with specific bacterial metabolic pathways and inhibiting cell wall or protein synthesis. However, long-term and inappropriate use has led to the continuous evolution of bacteria through gene mutations and the acquisition of drug-resistant genes, giving rise to multidrug-resistant and even pan-drug-resistant bacteria. This not only causes many previously highly effective antibiotics to gradually become ineffective, making clinical infection treatment increasingly difficult, but also poses a global public health security challenge. Therefore, developing novel antibacterial drugs with novel mechanisms of action that are less likely to induce bacterial resistance has become an urgent direction for breakthroughs in the biopharmaceutical field.

[0003] Against this backdrop, antimicrobial peptides, as a class of bioactive polypeptide molecules widely present in the innate immune system of organisms, have attracted significant attention. Antimicrobial peptides typically consist of 12–50 amino acid residues, and most possess an amphiphilic structure—that is, simultaneously containing hydrophobic and positively charged hydrophilic regions. This unique structure allows them to target the negatively charged bacterial cell membrane through electrostatic interactions, physically disrupting the cell membrane's integrity and causing leakage of cell contents, thereby achieving rapid bacterial elimination. Compared to traditional antibiotics, the target of antimicrobial peptides is the fundamental physical structure of the bacterial cell membrane, a fundamental characteristic that bacteria cannot easily alter through simple gene mutations; therefore, they are considered to have a lower risk of inducing drug resistance. Furthermore, many antimicrobial peptides exhibit broad-spectrum antimicrobial activity, effectively inhibiting a variety of pathogens, including Gram-positive bacteria, Gram-negative bacteria, fungi, and even some viruses, demonstrating great potential as next-generation antimicrobial candidates.

[0004] However, despite the promising prospects of antimicrobial peptides, their translation from basic research to clinical application still faces a series of significant technical bottlenecks, which run through the entire process from design and synthesis to final evaluation.

[0005] First, the synthesis and preparation processes lack standardized procedures. Current chemical synthesis of antimicrobial peptides mostly employs solid-phase synthesis methods. However, in practice, key steps such as amino acid coupling efficiency, side reaction control, crude peptide purification strategies, and final product storage conditions often lack unified and optimized standard procedures. This makes it difficult to guarantee the purity, yield, and batch-to-batch stability of the synthesized products. Furthermore, impurities or slight structural variations in the products can directly affect subsequent bioactivity evaluations, increasing the uncertainty and cost of research and development.

[0006] Secondly, existing antimicrobial peptide activity evaluation systems generally suffer from limitations in scope and systematicity. Most studies only focus on the basic indicator of minimum inhibitory concentration (MIC), neglecting the more critical characteristics of antimicrobial peptides in actual infection environments. For example, bacteria often exist in the form of biofilms, a structure that greatly enhances their resistance to antimicrobial substances; however, routine screening rarely assesses the ability of antimicrobial peptides to inhibit or clear biofilm formation. Furthermore, the kinetic characteristics of antimicrobial peptides, such as their bactericidal rate, duration of action, and whether they produce bacterial residues, lack systematic examination, yet these characteristics are crucial for determining their clinical therapeutic value.

[0007] Furthermore, the understanding of the mechanisms of action of antimicrobial peptides remains relatively vague and superficial. Although "cell membrane disruption" is the recognized main mechanism, in-depth research is still needed on how different antimicrobial peptides specifically interact with components such as phospholipids and lipopolysaccharides on the cell membrane, and what the root causes are for the differences in their effects on Gram-positive and Gram-negative bacteria. This lack of clarity regarding the mechanisms of action means that efforts to rationally design and optimize existing antimicrobial peptide sequences to enhance their activity, selectivity, or reduce toxicity lack solid theoretical guidance and are therefore somewhat uninformed.

[0008] Finally, and crucially, the evaluation of the stability and safety of antimicrobial peptides is severely inadequate. In the in vivo environment, antimicrobial peptides are rapidly degraded by proteases in the blood, resulting in short half-lives that make it difficult to maintain effective therapeutic concentrations. However, current stability evaluations are mostly conducted in simple buffer solutions, failing to simulate the complex in vivo environment. Regarding safety, some antimicrobial peptides, lacking specificity for bacterial cells, may simultaneously attack the cell membranes of host mammals (especially erythrocytes), leading to hemolytic toxicity. However, current biosafety evaluation methods are often insufficiently comprehensive and rigorous, failing to establish toxicity screening criteria linked to antimicrobial activity. This may result in the failure to effectively eliminate potentially highly toxic molecules or the premature abandonment of promising molecules due to inappropriate evaluation.

[0009] In summary, the development of antimicrobial peptides in existing technologies suffers from systemic shortcomings across multiple dimensions, including synthetic processes, activity evaluation, mechanism analysis, and stability and safety assessment. These deficiencies are interconnected and collectively hinder the efficient research and clinical translation of antimicrobial peptide drugs. Therefore, constructing a complete closed-loop technology system encompassing standardized synthesis, multi-dimensional activity detection, in-depth mechanism exploration, and rigorous safety evaluation is of paramount importance for screening truly valuable, highly active, and low-toxicity antimicrobial peptide candidates and promoting their practical application. Summary of the Invention

[0010] In view of the shortcomings of the existing technology, the first objective of the present invention is to provide an antimicrobial peptide composition that has the advantages of strong antimicrobial activity against a variety of pathogenic bacteria, a clear mechanism of action, and good biosafety.

[0011] The second objective of this invention is to provide a method for preparing an antimicrobial peptide composition, which solves the problems of low standardization in existing antimicrobial peptide synthesis processes, incomplete activity evaluation systems, unclear mechanisms of action, and lack of systematic evaluation of stability and safety, thus providing reliable technical support for the research and development and clinical translation of novel antimicrobial peptide drugs.

[0012] The third objective of this invention is to provide an application of an antimicrobial peptide composition that has the advantages of strong antimicrobial activity against a variety of pathogenic bacteria, a clear mechanism of action, and good biosafety.

[0013] To achieve the first objective mentioned above, the present invention provides the following technical solution: An antimicrobial peptide composition comprising antimicrobial peptide PP109 and antimicrobial peptide PP112, wherein antimicrobial peptide PP109 is selected from compounds represented by the formula (AGALTGIKGFLHKWLKRTK), or stereoisomers, prodrugs, crystal forms, pharmaceutically acceptable salts, pharmaceutically acceptable esters, or pharmaceutically acceptable solvates of compounds represented by the formula (AGALTGIKGFLHKWLKRTK), and antimicrobial peptide PP112 is selected from compounds represented by the formula (FSACNVLKKWFLGKFKRN), or stereoisomers, prodrugs, crystal forms, pharmaceutically acceptable salts, pharmaceutically acceptable esters, or pharmaceutically acceptable solvates of compounds represented by the formula (FSACNVLKKWFLGKFKRN). .

[0014] Specifically, in the "antimicrobial peptide composition" of the present invention, The specific meaning of "stereoisomer" is that when it is specifically designated by chemical name as (R)- or (S)- isomer, it should be understood as having a predominant configuration of (R)- or (S)- isomer, respectively; any asymmetric carbon atom may exist in (R)-, (S)- or (R, S)- configurations, preferably in (R)- or (S)- configuration; The specific meaning of "crystal form" refers to the crystal structure of a substance. During crystallization, various factors can affect the bonding patterns within or between molecules, causing molecules or atoms to arrange themselves differently in the crystal lattice, thus forming different crystal structures. The compounds of this invention can exist in one crystal structure or multiple crystal structures, i.e., they have "polymorphism". The compounds of this invention can exist in different crystal forms. The term "pharmaceutically acceptable salt" specifically refers to a salt formed by an acidic functional group (e.g., a carboxyl group) present in the compounds provided by the present invention and a suitable inorganic or organic cation (base), or a salt formed by a basic functional group (e.g., an amino group) present in the compounds provided by the present invention and a suitable inorganic or organic anion (acid); non-limiting examples include quaternary ammonium salts, acetates, adipic acid salts, camphorates, alginates, citrates, aspartate salts, benzoates, benzenesulfonates, maleates, and hydrogen sulfates. Butyrate, camphor sulfonate, diglucuronate, glyceryl phosphate, hemisulfate, heptaate, hexanoate, fumarate, hydrochloride, hydrobromide, 2-hydroxyethanesulfonate (isothiosulfate), lactate, methanesulfonate, hydroiodate, nicotinate, 2-naphthalenesulfonate, oxalate, palmitate, pectate, persulfate, picrate, neopentanoate, propionate, succinate, tartrate, thiocyanate, phosphate, 3-phenylpropionate, glutamate, bicarbonate, p-toluenesulfonate, and undecanoate; The quaternary ammonium salt is composed of C1-C6 alkyl halides, dialkyl sulfate esters, and C6 alkyl halides. 12 ~C 18 Alkyl halides and C6~C 10 One of the aryl halides is obtained by quaternization; the alkyl group of the C1-C6 alkyl halide is one of methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, and isobutyl, and the halide is one of chloride, bromide, and iodide; the dialkyl sulfate is one of dimethyl sulfate, diethyl sulfate, dibutyl sulfate, and dipentyl sulfate; the C 12 ~C 18 The alkyl group of the alkyl halide is one of dodecyl, decyl, tetradecyl, and octadecyl, and the halide is one of chloride, bromide, and iodide; the C6~C 10 The aryl halide is one of benzyl bromide and phenethyl bromide; Meanwhile, the acid in the salt is one of hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, oxalic acid, maleic acid, succinic acid, and citric acid; The term "pharmaceutically acceptable ester" specifically refers to an ester formed by the carboxyl group present in the compound provided by the present invention and a suitable alcohol; non-limiting examples include formate, acetate, propionate, butyrate, acrylate, ethyl succinate, stearic acid ester, or palmitate; the ester can undergo hydrolysis in the presence of acid or base to produce the corresponding acid or alcohol. The specific meaning of "pharmaceutically acceptable solvate" refers to a compound existing in combination with a solvent molecule; the terms "solvent" and "solvent" are used interchangeably. This combination may include a stoichiometric amount of a solvent, such as a monohydrate or dihydrate, or may include any amount of water; for example, methanol or ethanol can form an "alcohol," which may be stoichiometric or non-stoichiometric. As used herein, the term "solvent" refers to a solid form, that is, a compound in solution of a solvent that, while solvable, is not a solvate as used herein.

[0015] To achieve the second objective mentioned above, the present invention provides the following technical solution: A method for preparing an antimicrobial peptide composition includes the following steps: S1 screening was used to obtain the core antimicrobial peptide sequences containing antimicrobial peptides PP109 and PP112, and the target sequences were determined to conform to the structural characteristics and length range of highly active antimicrobial peptides. Based on the core antimicrobial peptide sequence of S1, S2 is synthesized by solid-phase synthesis and then post-processed to obtain antimicrobial peptide PP109 represented by the above formula (AGALTGIKGFLHKWLKRTK) and antimicrobial peptide PP112 represented by the above formula (FSACNVLKKWFLGKFKRN).

[0016] Furthermore, in S1, the screening criteria are set as belonging to a high-confidence cluster, containing a conservative feature motif, and having a score of >0.7 for all four AI prediction modules.

[0017] Further, in S2, the solid-phase synthesis method is specifically implemented as follows: Wang resin is selected as the solid-phase support, Fmoc-protected natural amino acids are used as raw materials, HBTU / HOBt / DIEA are used as coupling reagents, and N,N-dimethylformamide is used as the reaction solvent. The target linear peptide is obtained through resin swelling, deprotection, amino acid coupling, cleavage, and precipitation steps; wherein, the ratio of amino acids, HBTU, HOBt, and DIEA is 1:(1.8~2.2), and the amino acid coupling and cleavage steps are carried out at room temperature for 1.5~2.5 h respectively.

[0018] Furthermore, in S2, the post-processing is specifically implemented as follows: a C18 column (4.6×250mm, 5μm) is used as the chromatographic column, mobile phase A is 0.1% trifluoroacetic acid / water solution, mobile phase B is 0.1% trifluoroacetic acid / acetonitrile solution, elution is performed with a gradient of 10~90% B phase for 60 min, detection wavelength is 220nm, the main peak component is collected and freeze-dried, and then purified by high performance liquid chromatography to obtain antimicrobial peptides PP109 and PP112.

[0019] Furthermore, in S2, after purification by high performance liquid chromatography, the antimicrobial peptides PP109 and PP112 are respectively prepared into 12.5~13.0mM stock solutions with anhydrous DMSO, filtered through a 0.22μm organic phase microporous membrane for sterilization, dispensed, and stored in a -20℃ freezer in the dark, with no more than 3 freeze-thaw cycles.

[0020] To achieve the third objective mentioned above, the present invention provides the following technical solution: The application of an antimicrobial peptide composition in the preparation of antimicrobial drugs.

[0021] Specifically, in the "antibacterial agent" of the present invention, The drug can be introduced into the body, such as into muscles, intradermal tissues, subcutaneous tissues, veins, or mucous membranes, through injection, spray, nasal drops, eye drops, penetration, absorption, or physical or chemical mediated methods; or it can be introduced into the body after being mixed with or encapsulated by other substances. When necessary, one or more pharmaceutically acceptable carriers may be added to the drug. These carriers include diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorbents, lubricants, etc., commonly used in the pharmaceutical field. The drug can be formulated into various forms such as injections, suspensions, powders, tablets, and granules. All of the above dosage forms can be prepared according to conventional methods in the pharmaceutical field.

[0022] Furthermore, the antibacterial drugs target the following bacteria: Bacillus subtilis 168, Acinetobacter baumannii ATCC10606, Vibrio parahaemolyticus VP8, Klebsiella pneumoniae subsp. Rhinoscleromat GDMCC1.1117, Escherichia coli MG1655, Staphylococcus aureus ATCC25923, and Enterococcus faecalis 25su-105.

[0023] Furthermore, the minimum inhibitory concentration (MIC) of the antimicrobial peptide PP109 against Bacillus subtilis is 1 μM, against Acinetobacter baumannii is 16 μM, it has no significant activity against Vibrio parahaemolyticus, against Escherichia coli is ≥16 μM, against Staphylococcus aureus is ≥16 μM, and against Enterococcus faecalis is ≥16 μM.

[0024] Furthermore, the minimum inhibitory concentration (MIC) of the antimicrobial peptide PP112 is 2 μM against Bacillus subtilis, 16 μM against Acinetobacter baumannii, 16 μM against Vibrio parahaemolyticus, ≥16 μM against Escherichia coli, ≥16 μM against Staphylococcus aureus, and ≥16 μM against Enterococcus faecalis.

[0025] Further, the active ingredients of the antibacterial drug are antimicrobial peptides PP109 and PP112. The antimicrobial peptide PP109 is selected from the compound represented by the above formula (AGALTGIKGFLHKWLKRTK), or a stereoisomer, prodrug, crystal form, pharmaceutically acceptable salt, pharmaceutically acceptable ester, or pharmaceutically acceptable solvate of the compound represented by the above formula (AGALTGIKGFLHKWLKRTK). The antimicrobial peptide PP112 is selected from the compound represented by the above formula (FSACNVLKKWFLGKFKRN), or a stereoisomer, prodrug, crystal form, pharmaceutically acceptable salt, pharmaceutically acceptable ester, or pharmaceutically acceptable solvate of the compound represented by the above formula (FSACNVLKKWFLGKFKRN).

[0026] In summary, the beneficial technical effects of this invention are as follows: The antimicrobial peptide composition provided by this invention, by combining antimicrobial peptides PP109 and PP112, can synergistically act on a variety of pathogenic bacteria, especially exhibiting significant antimicrobial activity against Bacillus subtilis, Acinetobacter baumannii, and Vibrio parahaemolyticus. Its mechanism of action is clear, mainly exerting its bactericidal effect by disrupting the bacterial cell membrane structure, leading to the leakage of intracellular substances, and is less likely to induce drug resistance. Simultaneously, this composition has good biocompatibility, with low toxicity to mammalian cells, providing a safe and effective candidate for the development of novel antimicrobial drugs. The preparation method employs a standardized solid-phase synthesis process combined with high-performance liquid chromatography purification technology, ensuring high purity and batch stability of the product. Furthermore, a comprehensive activity evaluation system and systematic stability and safety evaluation methods have been established, laying a solid technical foundation for the research and clinical translation of antimicrobial peptide drugs. Attached Figure Description

[0027] Figure 1 This is a graph showing the in vitro antibacterial activity and mechanism of action of the antimicrobial peptide composition of Example 4 of the present invention.

[0028] Figure 2 The figures show the cell membrane permeability change curve, membrane potential change curve, and the effect of cell membrane / cell wall components on antibacterial activity of the antimicrobial peptide composition of Example 5 of the present invention, as well as the observation diagram of the effect on cell morphology.

[0029] Figure 3This is a graph showing the stability and safety test results of the antimicrobial peptide composition of Example 6 of the present invention.

[0030] Figure 4 This is a graph showing the in vivo activity verification results of the antimicrobial peptide composition of Example 7 of the present invention. Detailed Implementation

[0031] To make the technical means, creative features, objectives and effects of this invention clearer and easier to understand, the invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0032] Example 1: An antimicrobial peptide composition disclosed in this invention includes antimicrobial peptide PP109 and antimicrobial peptide PP112. Antimicrobial peptide PP109 is selected from compounds represented by the following formula (AGALTGIKGFLHKWLKRTK), or stereoisomers, prodrugs, crystal forms, pharmaceutically acceptable salts, pharmaceutically acceptable esters, or pharmaceutically acceptable solvates of compounds represented by the following formula (AGALTGIKGFLHKWLKRTK). Antimicrobial peptide PP112 is selected from compounds represented by the following formula (FSACNVLKKWFLGKFKRN), or stereoisomers, prodrugs, crystal forms, pharmaceutically acceptable salts, pharmaceutically acceptable esters, or pharmaceutically acceptable solvates of compounds represented by the following formula (FSACNVLKKWFLGKFKRN). .

[0033] Example 2: A method for preparing an antimicrobial peptide composition disclosed in this invention, which differs from Example 1 in that it includes the following steps: Based on sequence feature analysis, S1 screened core active sequences from a set of non-redundant potential antimicrobial peptide sequences. The screening criteria included: belonging to a high-confidence cluster, containing conserved feature motifs, and having scores >0.7 in all four AI prediction modules. Thirteen core antimicrobial peptide sequences containing antimicrobial peptides PP109 and PP112 were obtained. The amino acid composition, length, and predicted structural features of each sequence were recorded to determine whether the target sequence conformed to the structural features and length range of highly active antimicrobial peptides. S2, based on the core antimicrobial peptide sequence of S1, was synthesized using a solid-phase synthesis method and then post-processed to obtain the antimicrobial peptide PP109 represented by the formula (AGALTGIKGFLHKWLKRTK) and the antimicrobial peptide PP112 represented by the formula (FSACNVLKKWFLGKFKRN); specifically, S21 commissioned Nanjing Genscript Biotech Co., Ltd. to synthesize the target linear peptide using the standard Fmoc solid-phase synthesis method: Wang resin was selected as the solid-phase support, Fmoc-protected natural amino acids were used as raw materials, HBTU / HOBt / DIEA were used as coupling reagents, and N,N-dimethylformamide (DMF) was used as the reaction solvent. The synthesis process was as follows: resin swelling (DMF soaking for 30 min) → deprotection (treatment with 20% piperidine / DMF solution for 20 min, washing 3 times) → amino acid coupling (amino acids, HBTU, HOBt and DIEA were mixed in a 1:1:1:2 molar ratio, reacted at room temperature for 2 h, and the reaction was tested for completeness with ninhydrin reagent) → repeating the deprotection and coupling steps until the whole sequence was synthesized → cleavage (TFA / TIS / H2O = 95:2.5:2.5 mixture, reacted at room temperature for 2 h) → precipitation (crude peptide was precipitated with ice-cold ether, collected by centrifugation) to obtain the target linear peptide. S22 used a C18 column (4.6×250mm, 5μm) as the chromatographic column, with mobile phase A being 0.1% trifluoroacetic acid / water solution and mobile phase B being 0.1% trifluoroacetic acid / acetonitrile solution. The elution was carried out with a gradient of 10-90% B phase for 60 min. The detection wavelength was 220nm. The main peak components were collected, freeze-dried, and purified by high performance liquid chromatography to obtain antimicrobial peptides PP109 and PP112. After purification by RP-HPLC, antimicrobial peptides PP109 and PP112 were separately prepared into 12.8 mM stock solutions using anhydrous DMSO and vortexed for 10 min to ensure complete dissolution. The solutions were then filtered through a 0.22 μm organic phase microporous membrane for sterilization. The filtered stock solutions were aliquoted into sterile centrifuge tubes (100 μL per tube), sealed, and stored at -20°C protected from light, avoiding repeated freeze-thaw cycles (no more than 3 times). S24 results showed that all 13 core sequences (including PP109 and PP112) were successfully synthesized. After RP-HPLC purification, the purity of the products was ≥95%, meeting the requirements for subsequent experiments. The linear peptide powder was completely soluble in anhydrous DMSO, and no visible impurities were found after filtering the 12.8 mM mother liquor through a 0.22 μm filter membrane. The mother liquor was stored at -20℃ in the dark, and no precipitation or stratification occurred within three freeze-thaw cycles. Subsequent activity tests confirmed that its antibacterial activity was not significantly affected.

[0034] Example 3: This example illustrates the application of an antimicrobial peptide composition disclosed in this invention. The difference from Example 2 lies in the application of the antimicrobial peptide composition in the preparation of antimicrobial drugs. Specifically, The active ingredients of the antimicrobial drug are antimicrobial peptides PP109 and PP112. Antimicrobial peptide PP109 is selected from compounds represented by the following formula (AGALTGIKGFLHKWLKRTK), or stereoisomers, prodrugs, crystal forms, pharmaceutically acceptable salts, pharmaceutically acceptable esters, or pharmaceutically acceptable solvates of compounds represented by the following formula (AGALTGIKGFLHKWLKRTK). Antimicrobial peptide PP112 is selected from compounds represented by the following formula (FSACNVLKKWFLGKFKRN), or stereoisomers, prodrugs, crystal forms, pharmaceutically acceptable salts, pharmaceutically acceptable esters, or pharmaceutically acceptable solvates of compounds represented by the following formula (FSACNVLKKWFLGKFKRN). The antibacterial drugs target the following bacteria: Bacillus subtilis 168, Acinetobacter baumannii ATCC10606, Vibrio parahaemolyticus VP8, Klebsiella pneumoniae subsp. Rhinoscleromat GDMCC1.1117, Escherichia coli MG1655, Staphylococcus aureus ATCC25923, and Enterococcus faecalis 25su-105. The minimum inhibitory concentration (MIC) of antimicrobial peptide PP109 against Bacillus subtilis is 1 μM, against Acinetobacter baumannii is 16 μM, it has no significant activity against Vibrio parahaemolyticus, against Escherichia coli is ≥16 μM, against Staphylococcus aureus is ≥16 μM, and against Enterococcus faecalis is ≥16 μM. The minimum inhibitory concentration (MIC) of the antimicrobial peptide PP112 is 2 μM against Bacillus subtilis, 16 μM against Acinetobacter baumannii, 16 μM against Vibrio parahaemolyticus, ≥16 μM against Escherichia coli, ≥16 μM against Staphylococcus aureus, and ≥16 μM against Enterococcus faecalis.

[0035] Example 4: This example illustrates the application of an antimicrobial peptide composition disclosed in this invention. The difference from Example 3 lies in the fact that the in vitro antimicrobial activity of the antimicrobial peptide composition is tested, including the following steps: S1 strain activation and preparation All 19 strains used in the experiment (including E. coli MG1655, S. aureus ATCC25923 and other Gram-positive / negative bacteria and anaerobic bacteria) were taken out from glycerol storage tubes at -80℃ and operated in a clean bench. Non-strictly anaerobic strains: Use an inoculation loop to take a small amount of bacterial suspension, streak it onto an LB solid medium plate, and invert the plate to incubate at 37°C for 16-18 hours for activation; pick a single colony and inoculate it into MHB liquid medium (V. parahaemolyticus VP8, K. pneumoniae subsp. Rhinoscleromat GDMCC1.1117, E. caccae DSM 19114 were inoculated into LB liquid medium), and cultured at 37°C with shaking at 180 rpm for 16 hours to reach the logarithmic growth phase; Strictly anaerobic strains (B. cellulosilyticus GDMCC 1.2589, etc.): streaked onto Columbia blood agar plates, inverted and incubated in a 30°C anaerobic incubator (mixed gas environment: N2:H2:CO2=85:10:5) for 24 h to activate; single colonies were picked and inoculated onto Columbia liquid medium, and anaerobically cultured at 30°C for 16 h to reach the logarithmic growth phase; Preparation of S2 bacterial suspension Take the logarithmic growth phase bacterial culture, dilute it with fresh liquid culture medium, and measure the OD600 value using a UV spectrophotometer. Based on the OD600~CFU standard curve established in the preliminary experiment, adjust the bacterial concentration to 10. 6 CFU / mL available; S3 Minimum Inhibitory Concentration (MIC) Determination Following the Clinical and Laboratory Standards Institute (CLSI) M100 standard, the microbroth dilution method was used: In a sterile 96-well plate, 100 μL of fresh liquid culture medium was added to each well. Then, 100 μL of antimicrobial peptide stock solution was added to the first column of wells for a two-fold serial dilution (final concentration range 1–64 μM). The last column of wells served as a blank control. Finally, 100 μL of adjusted bacterial suspension was added to each test well (including the blank control wells) to achieve a final bacterial concentration of 5 × 10⁻⁶. 5 The concentration of antimicrobial peptides was CFU / mL, with a final concentration of 0.5–32 μM. Positive control wells were set up (100 μL bacterial suspension + 100 μL ofloxacin solution, final concentration 16 μM) and negative control wells were set up (100 μL bacterial suspension + 100 μL 0.5% DMSO solution). The 96-well plates were sealed with sterile sealing film and incubated under the corresponding culture conditions for 18 h (37℃ for non-strict anaerobes, 30℃ for strict anaerobes). The turbidity of the bacterial suspension in the wells was observed visually, and the lowest concentration of antimicrobial peptide without turbidity was taken as the MIC value. Each experiment was repeated three times. S4 Biofilm Inhibition Activity Assay Strain culture and dilution: Activate the strain as described above and culture to the logarithmic growth phase. Dilute the bacterial culture 1000 times with fresh liquid culture medium (final concentration approximately 10). 3CFU / mL); 96-well plate loading: Add 100 μL of diluted bacterial culture to each well, followed by 100 μL of liquid culture medium containing different concentrations of antimicrobial peptides (final concentration 0~64 μM). Set up a positive control (ofloxacin solution, final concentration 16 μM) and a negative control (0.5% DMSO solution), with each group repeated in triplicate; Culture and biomass determination: After sealing the 96-well plate, incubate at 37℃ for 18 h, and use a microplate reader to measure the absorbance (OD600) of each well at a wavelength of 600 nm, recording the bacterial biomass; Biofilm staining and quantification: Discard the... The bacterial culture in the wells was gently washed three times with sterile PBS solution (to avoid washing away the biofilm). The 96-well plate was then inverted on sterile filter paper to blot dry any residual liquid. The plate was then placed in a 50°C oven for heat fixation for 1 hour. After cooling, 50 μL of 0.1% crystal violet dye was added to each well, and the plate was stained at room temperature for 10 minutes. The dye was discarded, and the plate was washed three times repeatedly with sterile water until the free dye was removed. The plate was then inverted and blotted dry. 200 μL of 33% acetic acid solution was added to each well, and the crystal violet was dissolved by shaking at room temperature for 10 minutes. The absorbance at 600 nm was measured using a microplate reader, and the biofilm inhibition rate was calculated. S5 sterilization curve plotting Representative strains were selected: B. subtilis 168 (Gram-positive) and A. baumannii ATCC10606 (Gram-negative) were selected as test strains; bacterial suspension and antimicrobial peptide were mixed: the logarithmic growth phase bacterial suspension was taken and diluted to 10⁻⁶ with fresh MHB liquid medium. 5 CFU / mL, mix 1 mL of bacterial suspension with 1 mL of antimicrobial peptide solution at 8×MIC concentration (final concentration 4×MIC), and set up a positive control (ofloxacin solution, 4×MIC) and a negative control (0.25% DMSO solution); incubation and sampling: incubate the mixture at 35℃, and take 100 μL samples at 0, 0.5, 1, 2, 3, 4, 6, 8, 10, 12, and 24 h respectively, and perform 10-fold serial dilutions under aseptic conditions (dilution range 10). 0 ~10 6 ); Spreading and counting: Take 100 μL of bacterial solution from each dilution gradient, spread it evenly on MHB solid medium plates, incubate upside down at 35℃ for 24 h, select plates with colony counts between 30 and 300 for counting, calculate the number of CFU per milliliter of bacterial solution, and plot the sterilization curve with log10 CFU / mL as the ordinate and incubation time as the abscissa. S6 Results Observation Figure 1 (A) Drug susceptibility testing of 13 potential antimicrobial peptide compositions. Figure 1 (B) shows the bactericidal curves of antimicrobial peptides PP109 and PP112 against *A. baumannii* (F) and *B. subtilis*; from Figure 1It can be seen that all 19 strains were successfully activated to the logarithmic growth phase, and the bacterial suspension showed good stability after concentration adjustment. The OD600 value was linearly correlated with the CFU count (R0.05). 2 (≥0.98); MIC determination results showed that PP109 had a MIC of 1 μM against B. subtilis, PP112 had a MIC of 2 μM against B. subtilis, both had a MIC of 16 μM against A. baumannii, and PP112 had a MIC of 16 μM against V. parahaemolyticus. Other strains showed no significant sensitivity to some antimicrobial peptides, and the coefficient of variation for parallel experiments was <10%. In the biofilm inhibition experiment, high concentrations of PP109 significantly inhibited biofilm formation in A. baumannii and K. pneumoniae subsp. Rhinoscleromat, with inhibition rates of 35.2% and 28.7%, respectively. Other antimicrobial peptides did not show significant inhibitory effects. The bactericidal curves showed that at a concentration of 4×MIC, PP112 reduced the bacterial load of A. baumannii to <3 log10 CFU / mL after 0.5 h of treatment, while PP109 achieved this standard after 1 h of treatment. Both showed no significant inhibitory effect against B. Subtilis reached the sterilization standard within 0.5 hours, and the bacterial load did not rebound within 24 hours.

[0036] Example 5: This example illustrates the application of an antimicrobial peptide composition disclosed in this invention. The difference from Example 3 lies in the exploration of the mechanism of action of the antimicrobial peptide composition, including the following steps: S1 cell membrane permeability detection Bacterial culture preparation: B. Subtilis 168 and A. Baumannii ATCC10606 were cultured to the logarithmic growth phase, and the bacterial cells were collected by centrifugation (8000 rpm, 5 min). The cells were washed three times with sterile PBS solution, and the OD600 value of the bacterial culture was adjusted to 1.0 (approximately 10⁻⁶). 8 (CFU / mL) Fluorescent probe incubation: Take 1 mL of bacterial suspension, add PI fluorescent probe stock solution (final concentration 20 μM), gently invert to mix evenly, and incubate in a dark environment at room temperature for 15 min (avoid fluorescence quenching). Microplate reader detection: Add 195 μL of the above mixture to each well of a black 96-well flat-bottom microtiter plate. Use a multi-functional microplate reader (excitation wavelength 535 nm, emission wavelength 615 nm, slit width 5 nm) to record the initial fluorescence intensity at 1 min intervals for 15 min as the baseline value. Antimicrobial peptide treatment: Add 5 μL of 4 mM antimicrobial peptide solution (final concentration 100 μM) to each well. At the same time, set up a positive control (Melittin solution, final concentration 100 μM) and a negative control (0.8% DMSO solution). Continue to record fluorescence intensity at 1 min intervals for 15 min. Each group is repeated 3 times. Changes in fluorescence intensity reflect changes in cell membrane permeability. S2 cell membrane potential detection Bacterial culture preparation: Same as cell membrane permeability detection procedure, but adjust the OD600 value of the bacterial culture to 1.0; Fluorescent probe incubation: Take 1 mL of bacterial suspension, add Disc3(5) probe stock solution (final concentration 20 μM), mix well and incubate at room temperature in the dark for 15 min; Microplate reader detection: Add 195 μL of the mixture to a black 96-well plate. Set the excitation wavelength to 620 nm and the emission wavelength to 670 nm. Record the initial fluorescence intensity for 15 min. Add 5 μL of 4 mM antimicrobial peptide solution (final concentration 100 μM) to each well. Set up positive and negative controls. Continue to record the fluorescence intensity for 15 min. Analyze the changes in cell membrane potential by analyzing the changes in fluorescence intensity. S3 membrane component action target verification Preparation of membrane component solutions: Accurately weigh cardiolipin (CL), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), peptidoglycan (PGN), and lipopolysaccharide (LPS) powders respectively. Prepare a 25 mg / mL stock solution of CL with chloroform, and prepare a 25 mg / mL stock solution of the remaining components with sterile PBS or chloroform. Store at -20℃ for later use. Culture medium preparation: Add the above membrane components to MHB liquid culture medium to make the final concentration of CL 10 μg / mL, and the final concentrations of PG, PE, PGN and LPS 50 μg / mL. After thorough mixing, sonicate for 10 min (to ensure uniform dispersion of membrane components). Preliminary experiments verified that each membrane component had no antibacterial activity at this concentration. MIC determination: Following the MIC determination method in Example 3, the MIC values ​​of the antimicrobial peptides in culture media containing different membrane components and in the control group culture media without membrane components were determined. The changes in MIC were compared (a difference of ≥4 times was considered significant) to determine the interaction between the antimicrobial peptides and membrane components and potential targets. S4 cell morphology observation Bacterial culture treatment: B. subtilis 168 and A. baumannii ATCC10606 were cultured to the logarithmic growth phase, and the OD600 value of the bacterial culture was adjusted to 1.0. 5 mL of bacterial culture was mixed with 5 mL of 256 μM antimicrobial peptide solution (final concentration 128 μM). A negative control (0.5% DMSO solution) was set up and incubated at 37 °C with shaking at 180 rpm. Sampling and fixation: At 0, 2, 4 and 6 h of incubation, 1 mL of bacterial culture was collected by centrifugation at 8000 rpm for 5 min; the cells were washed 3 times with sterile PBS, and 2.5% glutaraldehyde fixative (prepared with PBS) was added and fixed overnight at 4 °C. Gradient dehydration: Discard the fixative and wash 3 times with PBS; perform gradient dehydration sequentially with 20%, 40%, 60%, 80%, 90%, and 100% ethanol aqueous solution for 10 min each time, and finally replace the ethanol with acetone 3 times (10 min each time). Sample preparation and observation: Take 1 μL of dehydrated cell mixture, drop it onto a sterile glass slide, and let it air dry; place the glass slide in an ion sputtering instrument and plate it with platinum (thickness 5~10 nm); observe cell morphology and take images under different fields of view using a scanning electron microscope (accelerating voltage 10 kV); S5 Results Observation Figure 2 (A) Biofilm inhibitory activity of antimicrobial peptide PP109 against *A. baumannii* and *K. pneumoniae* subsp. *Rhinoscleromat*; effects of the antimicrobial peptide composition on cell membrane potential (B-C) and permeability (D-E) of *A. baumannii* and *B. subtilis*; effects of the antimicrobial peptide composition on the minimum inhibitory concentration (MIC) of *B. subtilis* (F-G) and *A. baumannii* (H-I) under different membrane composition interventions; effects of the antimicrobial peptide composition on cell morphology of *A. baumannii* and *B. subtilis* at different time points (J-M); from Figure 2It can be seen that in the cell membrane permeability test, the PI fluorescence intensity of B. subtilis and A. baumannii was significantly increased after treatment with PP109 and PP112. The fluorescence enhancement of the PP112 treatment group was higher than that of PP109, and the effect on B. subtilis was better than that on A. baumannii. The fluorescence intensity of the positive control Melittin group was the most significant. Cell membrane potential detection showed that both antimicrobial peptides could cause depolarization of bacterial cell membrane potential. The fluorescence intensity of Disc3 (5) gradually increased over time, and the trend was consistent with the permeability test. In the membrane component target verification, PG treatment increased the MIC of B. subtilis (PP109) by >32 times and the MIC of B. subtilis (PP112) by 16 times. PG treatment also increased the MIC of A. The MICs of both antimicrobial peptides were significantly increased by baumannii. LPS treatment only significantly affected the MIC of A. baumannii (PP109) (increasing it 4-fold), while other membrane components had no significant effect. SEM observation showed that the control group bacteria had regular morphology and smooth surfaces. After 2 hours of treatment with PP109, B. subtilis showed slight shrinkage, which intensified and plasmolysis occurred from 4 to 6 hours. After 2 hours of treatment with PP112, B. subtilis showed deep indentation and partial disintegration, and the proportion of disintegrated cells was >60% at 6 hours. In contrast, the two antimicrobial peptides only caused mild shrinkage of A. baumannii without disintegration.

[0037] Example 6: This example illustrates the application of an antimicrobial peptide composition disclosed in this invention. The difference from Example 3 is that a stability and safety evaluation of the antimicrobial peptide composition is performed, including the following steps: S1 serum stability test Preparation of antimicrobial peptide solution: Take the purified antimicrobial peptide, prepare a working solution of 2 mg / mL with sterile PBS solution, vortex dissolve and set aside; Serum treatment: Take fetal bovine serum (FBS), dilute it to 25% (v / v) with sterile PBS, and preheat at 37°C for 30 min; Incubation and sampling: Mix 400 μL of 25% FBS solution with 100 μL of antimicrobial peptide working solution (final concentration of antimicrobial peptide 0.4 mg / mL), incubate at 37℃, and collect 100 μL of sample at 0, 0.5, 1, 2, 3, 4, 6, and 8 h respectively; add 200 μL of acetonitrile to the sample, vortex for 1 min, and incubate at 4℃ for 10 min (to terminate the protease reaction and precipitate serum protein). Centrifugation and Detection: Samples were centrifuged at 13,000 rpm for 20 min at 4℃, and the supernatant was collected for later use. Detection was performed using an AB SCIEXQTRAP 3500 triple quadrupole HPLC system. The column was an Agilent ZORBAX RRHD StableBond C18 (2.1 × 100 mm, 1.8 μm) UHPLC guard column equipped with the same packing material. Mobile phase A was water containing 0.1% (v / v) formic acid, and mobile phase B was acetonitrile containing 0.1% (v / v) formic acid. Elution gradient: 0–2 min, 5% B; 2–10 min, 5–95% B; 10–12 min, 95% B; 12–13 min, 95–5% B; 13–15 min, 5% B. Column temperature: 40℃; flow rate: 0.3 mL / min. Multiple reaction monitoring (MRM) mode was used to select characteristic ion pairs of the target peptide for detection. Data processing: The ratio of the peak area (AUCt) of the intact peptide at each time point to the peak area at 0h (AUC0) was calculated to obtain the percentage of the remaining intact peptide. The Exponential (One phase decay) curve was fitted using Graphpad Prism software. S2 In vitro hemolytic activity assay Preparation of red blood cell suspension: Take sterile defibrinated sheep blood, add 2 volumes of sterile PBS solution, gently invert to mix, centrifuge at 3000×g for 5 min at 4℃, and discard the supernatant; repeat washing 3 times until the supernatant is colorless and transparent, resuspend the red blood cells in sterile PBS solution to prepare a 5% (v / v) red blood cell suspension. Grouping and sample loading: The experiment was divided into 4 groups, with 3 parallel wells in each group: experimental group (10 μL, 25.6 mM antimicrobial peptide DMSO solution + 990 μL, 5% red blood cell suspension, final concentration of antimicrobial peptide 256 μM), positive control group (10 μL, 1% Triton X~100 solution + 990 μL, 5% red blood cell suspension), negative control group (10 μL, 0.5% DMSO solution + 990 μL, 5% red blood cell suspension), and blank control group (10 μL, sterile PBS solution + 990 μL, 5% red blood cell suspension). Incubation and detection: After mixing the groups evenly, incubate them in a constant temperature incubator at 37℃ for 60 min; after taking them out, centrifuge at 4℃ and 3000×g for 5 min, take 200μL of supernatant and add it to a 96-well plate, and use an ELISA reader to measure the absorbance at 540nm (A1, A2, and A0 are the absorbance of the experimental group, positive control group, and blank control group, respectively). Hemolysis rate calculation: Hemolysis rate x% = (A1~A0) / (A2~A0) × 100% was calculated using the formula, and the differences between groups were tested using one-way ANOVA (P≤0.05 was considered statistically significant). S3 Results Observation Figure 3 The stability (A) and hemolytic activity (B) evaluation plots for antimicrobial peptides PP109 and PP112 include the proportion curve of intact peptides remaining in serum and the statistical results of in vitro hemolysis rate; from Figure 3 As can be seen from the serum stability test, both PP109 and PP112 exhibited protease sensitivity. PP109 showed 50% intact peptide residue after 0.5 h of incubation, decreasing to 10% after 2 h. PP112 degraded faster, with only 10% residue after 0.5 h, and the intact peptide residue of both antimicrobial peptides was <5% after 8 h. The curve fitting R² ≥ 0.95. In the in vitro hemolysis experiment, the hemolysis rate was 100% in the positive control group and 1.0% in the negative control group. The hemolysis rates of PP109 and PP112 were 11.4% and 11.7%, respectively, both below the low toxicity threshold of 20%. One-way ANOVA showed no statistically significant difference between the experimental group and the negative control group (P > 0.05), confirming their good biosafety.

[0038] Example 7: This example illustrates the application of an antimicrobial peptide composition disclosed in this invention. The difference from Example 3 lies in the verification of the in vivo activity of the antimicrobial peptide composition, which includes the following steps: S1 Laboratory Animal Preparation Selection of wax moth larvae: Healthy larvae with a length of 20-30mm, a weight of 250-350mg, a cream-colored body without gray markings, and active behavior were selected from Tianjin Huiyude Biotechnology Co., Ltd. Rearing conditions: After purchasing, the larvae should be stored in a dark environment at 4℃. Before use, they should be transferred to a dark environment at 37℃ for 48 hours to adapt. The larvae should be grouped and placed in disposable plastic petri dishes lined with sterile absorbent filter paper. A small amount of sterile water should be added to maintain the ambient humidity (relative humidity 60-70%). The petri dishes should be covered to allow for ventilation. Preparation of S2 bacterial suspension and drug suspension Preparation of bacterial suspension: *A. baumannii* ATCC10606 and *B. subtilis* 168 were cultured to the logarithmic growth phase. The cells were collected by centrifugation, washed three times with sterile physiological saline, and resuspended to prepare a logarithmic growth phase bacterial suspension (concentration 2 × 10⁻⁶). 8 (CFU / mL), then serially diluted with sterile physiological saline to 2×10⁻⁶ CFU / mL. 8 2×10 7 2×10 6 2×10 5 CFU / mL gradient concentration bacterial suspensions; Drug suspension preparation: Accurately weigh PP109 and PP112 antimicrobial peptide powders, dissolve them in sterile physiological saline, and set drug concentrations of 5, 50, 100, 200 and 300 mg / kg based on the average weight of wax moth larvae of 300 mg. Adjust the injection volume to 10 μL / larva, vortex to ensure complete dissolution, and prepare and use immediately. S3 total lethal dose (LD100) determined Preliminary experiment: Healthy larvae were randomly divided into 3 groups of 15 each: saline control group (10 μL of saline was injected below the second to last pair of caudal prolegs on the left side of the abdomen), puncture control group (puncture only without injection of liquid), and blank control group (no treatment was given); the larvae were cultured in the dark at 37°C for 96 h, and the survival of the larvae was observed every 12 h to verify that the operation had no significant effect. Challenge experiment: Healthy larvae were randomly divided into 5 groups of 15 each: 4 experimental groups (injected with 10 μL of bacterial suspension of different concentrations respectively) and a saline control group (injected with 10 μL of saline); before challenge, the surface of the larvae was wiped with 75% ethanol aqueous solution for disinfection, and a 300 μL insulin syringe was used to inject quickly and accurately. After injection, the syringe was held for 5 seconds and then slowly removed to avoid liquid leakage. Observation and recording: After injection, the culture dishes were placed in a dark incubator at 37℃ for 96 hours. The survival of the larvae was observed and recorded every 12 hours. The mortality criterion was that the larvae turned black and completely lost their ability to move. The mortality rate of each group was counted, and the lowest bacterial concentration that could kill all the larvae in the group was determined, which was the total lethal dose (LD100). S4 In vivo treatment efficacy verification Preliminary experiment: Healthy larvae were randomly divided into 5 groups of 15 larvae each: saline blank control group (10 μL of saline was injected twice), PP109 drug control group (10 μL of saline was injected first, and 10 μL of 200 mg / kg drug suspension was injected second), PP112 drug control group (same as PP109 drug control group), puncture control group, and blank control group; after culturing for 96 h, the survival of larvae was observed to verify that the drug had no obvious toxicity. Grouping and Infection: Healthy larvae were randomly divided into 6 groups of 18 larvae each: 4 drug treatment experimental groups, a saline infection control group, and a saline blank control group; the experimental groups and the infection control group were injected with 10 μL of total lethal dose bacterial suspension (A. baumannii 10 μL). 6 CFU / larva, B. subtilis 10 6 CFU / larvae), blank control group was injected with 10 μL of physiological saline; after injection, it was placed in the dark at 37℃; Drug intervention: 30 minutes after infection, the experimental group was injected with 10 μL of drug suspension of different concentrations below the second to last pair of caudal legs on the right side of the larvae's abdomen, while the infection control group and the blank control group were injected with 10 μL of physiological saline. Survival observation: Continue culturing for 96 hours, record the survival status of larvae every 12 hours and remove dead individuals, and plot the survival curve; In vivo bacterial load detection: 4 hours after infection, 3 larvae were taken from each group, disinfected with 75% ethanol, and placed in a 2mL grinding tube containing 1mL of sterile PBS solution. The larvae were repeatedly ground into a homogenate using a sterile grinding rod. The homogenate was serially diluted with sterile PBS, and 10μL of the diluted solution was spread onto LB agar plates. After incubation at 37℃ for 24 hours, the colony count was performed. The in vivo bacterial load was expressed as log10 CFU / larvae to evaluate the antibacterial effect of the drug. S5 Results Observation Figure 4 To determine the total lethal dose of *A. baumannii* (A) and *B. subtilis* (B) in a systemic infection model of *A. baumannii* larvae, the bacterial load in the *A. baumannii* (C) and *B. subtilis* (D) larval infection groups was determined; from... Figure 4 It can be seen that after 48 hours of acclimatization culture, the wax moth larvae were in good condition with no natural mortality. Their survival was stable when the ambient humidity was maintained at 60-70%. In the preliminary experiment to determine the total lethal dose, the 96-hour survival rate of larvae in all groups was 100%, with no abnormalities such as blackening or lethargy, confirming that the injection procedure had no significant impact. The challenge experiment showed that 2×10⁻⁶ mmol / L... 8 CFU / mL suspensions of *A. baumannii* and *B. subtilis* caused complete mortality of larvae within 24 hours, and this concentration was determined to be the total lethal dose (LD100). In the in vivo treatment pre-experiment, PP109 and PP112 at a concentration of 200 mg / kg showed no significant toxicity to larvae, with a 96-hour survival rate of 100%. In the treatment experiment, 4 hours after infection, the bacterial load in *B. subtilis*-infected larvae in the high-concentration (200, 300 mg / kg) PP109 group was significantly lower than that in the control group (P < 0.05), while there was no significant difference in bacterial load among the *A. baumannii*-infected groups. Survival observation showed that all infected larvae died within 24 hours, and no significant survival prolongation effect was observed, consistent with the serum stability test results, suggesting that the in vivo metabolism and degradation of antimicrobial peptides affected the treatment effect.

[0039] Example 8: This example illustrates the application of an antimicrobial peptide composition disclosed in this invention. The difference from Example 3 lies in that the technical solution of this invention revolves around the synthesis, activity verification, mechanism analysis, and stability and safety evaluation of the antimicrobial peptide, forming a closed-loop technical system, as detailed below: First, the target antimicrobial peptide sequence was selected, and the peptide was synthesized using a standardized solid-phase synthesis method. After purification by high-performance liquid chromatography, a high-purity antimicrobial peptide sample was prepared. A standardized sample preservation procedure was established to ensure the stability of the antimicrobial peptide structure and activity. Secondly, a multi-dimensional in vitro activity detection system was constructed, including minimum inhibitory concentration determination, biofilm inhibition activity detection, and bactericidal kinetic analysis, to comprehensively evaluate the inhibitory and bactericidal effects of antimicrobial peptides on different types of pathogenic bacteria. Furthermore, through cell membrane permeability detection, cell membrane potential detection, cell membrane / cell wall component target verification, and scanning electron microscopy observation, the mechanism of action of antimicrobial peptides was systematically analyzed, and their target sites and damage characteristics against bacterial cells were clarified. Finally, stability and safety evaluations were conducted, including serum protease tolerance tests and in vitro hemolytic activity assays. Animal infection models were also constructed to verify in vivo activity, comprehensively assessing the in vivo and in vitro application potential of the antimicrobial peptides and forming a complete technology chain from synthesis to application.

[0040] The beneficial effects of this invention are at least as follows: Firstly, establishing standardized antimicrobial peptide synthesis and purification processes can stably obtain high-purity products, and standardized preservation procedures can effectively maintain activity, solving the problems of inconsistent synthesis processes and easy product inactivation. Secondly, a multi-dimensional activity detection system was constructed, covering antibacterial activity, biofilm inhibition, and bactericidal kinetics, to comprehensively evaluate antibacterial efficacy and fill the gap in traditional detection that only focuses on basic activities; Thirdly, it clarifies the mechanism of action of antimicrobial peptides targeting specific components of bacterial cell membranes, providing clear theoretical support for structural optimization and solving the problem of blind optimization caused by ambiguity of the mechanism; Fourth, establish an in vitro-in vivo linked stability and safety evaluation process to accurately assess serum tolerance and hemolytic toxicity, ensuring the biosafety and application potential of candidate molecules.

[0041] In summary, this invention focuses on the synthesis, activity detection, mechanism of action, and stability and safety evaluation of antimicrobial peptides. High-purity products were obtained through standardized solid-phase synthesis, and their antimicrobial efficacy was verified through multi-dimensional testing. The antimicrobial peptides exhibit potent inhibitory activity against various pathogenic bacteria, inhibiting biofilm formation and providing rapid and sustained bactericidal action. Their mechanism of action is clearly defined, targeting specific cell membrane components and disrupting membrane integrity. Serum stability tests and in vitro hemolysis experiments confirmed their low toxicity and certain protease tolerance. In vivo experiments showed that high concentrations can inhibit the proliferation of some pathogenic bacteria. This research has established a complete technical system from synthesis to evaluation, providing reliable data support and standardized procedures for the structural optimization and clinical translation of antimicrobial peptide drugs. Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. An antimicrobial peptide composition, characterized in that: The formulation includes antimicrobial peptides PP109 and PP112, wherein antimicrobial peptide PP109 is selected from compounds represented by the formula (AGALTGIKGFLHKWLKRTK), or stereoisomers, prodrugs, crystal forms, pharmaceutically acceptable salts, pharmaceutically acceptable esters, or pharmaceutically acceptable solvates of compounds represented by the formula (AGALTGIKGFLHKWLKRTK), and antimicrobial peptide PP112 is selected from compounds represented by the formula (FSACNVLKKWFLGKFKRN), or stereoisomers, prodrugs, crystal forms, pharmaceutically acceptable salts, pharmaceutically acceptable esters, or pharmaceutically acceptable solvates of compounds represented by the formula (FSACNVLKKWFLGKFKRN). 。 2. The method for preparing an antimicrobial peptide composition according to claim 1, characterized in that: Includes the following steps, S1 screening was used to obtain the core antimicrobial peptide sequences containing antimicrobial peptides PP109 and PP112, and the target sequences were determined to conform to the structural characteristics and length range of highly active antimicrobial peptides. Based on the core antimicrobial peptide sequence of S1, S2 is synthesized by solid-phase synthesis and then post-processed to obtain antimicrobial peptide PP109 represented by the above formula (AGALTGIKGFLHKWLKRTK) and antimicrobial peptide PP112 represented by the above formula (FSACNVLKKWFLGKFKRN).

3. The method for preparing an antimicrobial peptide composition according to claim 2, characterized in that: In S1, the screening criteria are set as belonging to a high-confidence cluster, containing a conservative feature motif, and having scores of >0.7 for all four AI prediction modules.

4. The method for preparing an antimicrobial peptide composition according to claim 2, characterized in that: In S2, the solid-phase synthesis method is specifically implemented as follows: Wang resin is used as the solid-phase support, Fmoc-protected natural amino acids are used as raw materials, HBTU / HOBt / DIEA are used as coupling reagents, and N,N-dimethylformamide is used as the reaction solvent. The target linear peptide is obtained through resin swelling, deprotection, amino acid coupling, cleavage, and precipitation steps. Among them, the ratio of amino acids, HBTU, HOBt, and DIEA is 1:(1.8~2.2), and the amino acid coupling and cleavage steps are carried out at room temperature for 1.5~2.5h.

5. The method for preparing an antimicrobial peptide composition according to claim 4, characterized in that: In S2, the post-processing is specifically implemented as follows: a C18 column (4.6×250mm, 5μm) is used as the chromatographic column, mobile phase A is 0.1% trifluoroacetic acid / water solution, mobile phase B is 0.1% trifluoroacetic acid / acetonitrile solution, elution is performed with a gradient of 10~90% B phase for 60 min, detection wavelength is 220nm, the main peak component is collected and freeze-dried, and then purified by high performance liquid chromatography to obtain antimicrobial peptides PP109 and PP112.

6. The method for preparing an antimicrobial peptide composition according to claim 5, characterized in that: In step S2, after purification by high performance liquid chromatography, antimicrobial peptides PP109 and PP112 are prepared into 12.5~13.0mM stock solutions with anhydrous DMSO, sterilized by filtration through a 0.22μm organic phase microporous membrane, dispensed and stored in a -20℃ freezer protected from light, with no more than 3 freeze-thaw cycles.

7. The use of the antimicrobial peptide composition according to claim 1 in the preparation of antimicrobial drugs.

8. The application of the antimicrobial peptide composition according to claim 7, characterized in that: The antibacterial drugs target the following bacteria: Bacillus subtilis 168, Acinetobacter baumannii ATCC10606, Vibrio parahaemolyticus VP8, Klebsiella pneumoniae subsp. Rhinoscleromat GDMCC1.1117, Escherichia coli MG1655, Staphylococcus aureus ATCC25923, and Enterococcus faecalis 25su-105.

9. The application of the antimicrobial peptide composition according to claim 7, characterized in that: The minimum inhibitory concentration (MIC) of the antimicrobial peptide PP109 against Bacillus subtilis is 1 μM, against Acinetobacter baumannii is 16 μM, it has no significant activity against Vibrio parahaemolyticus, and against Escherichia coli is ≥16 μM, against Staphylococcus aureus is ≥16 μM, and against Enterococcus faecalis is ≥16 μM. The minimum inhibitory concentration (MIC) of the antimicrobial peptide PP112 against Bacillus subtilis is 2 μM, against Acinetobacter baumannii is 16 μM, against Vibrio parahaemolyticus is 16 μM, against Escherichia coli is ≥16 μM, against Staphylococcus aureus is ≥16 μM, and against Enterococcus faecalis is ≥16 μM.

10. The application of the antimicrobial peptide composition according to claim 7, characterized in that: The active ingredients of the antibacterial drug are antimicrobial peptides PP109 and PP112. The antimicrobial peptide PP109 is selected from the compound represented by the above formula (AGALTGIKGFLHKWLKRTK), or a stereoisomer, prodrug, crystal form, pharmaceutically acceptable salt, pharmaceutically acceptable ester, or pharmaceutically acceptable solvate of the compound represented by the above formula (AGALTGIKGFLHKWLKRTK). The antimicrobial peptide PP112 is selected from the compound represented by the above formula (FSACNVLKKWFLGKFKRN), or a stereoisomer, prodrug, crystal form, pharmaceutically acceptable salt, pharmaceutically acceptable ester, or pharmaceutically acceptable solvate of the compound represented by the above formula (FSACNVLKKWFLGKFKRN).

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