Collagen-based antibacterial peptide and preparation method thereof

Abstract

The invention belongs to the technical field of polypeptide preparation, and provides collagen-based antibacterial peptide and a preparation method thereof. Connecting carboxyl of the collagen peptide with amino acid, dipeptide or tripeptide; the amino acid, the dipeptide or the tripeptide is composed of lysine and arginine. The antibacterial peptide can be used as an effective component of an antibacterial drug and can be prepared into an external application patch, a suppository, a surgical implant material and the like for preventing bacterial and fungal infection. The antibacterial peptide can also be used as a feed additive or a veterinary drug to be applied to animals. The raw materials are wide in source, the synthesis method is simple, and the cost is low; the prepared antibacterial peptide is good in salt tolerance, has an antibacterial effect under a wide pH range, and is good in biological safety; the antibacterial peptide has a good inhibition effect on gram-negative and gram-positive bacteria, especially has good biological activity on common vaginal malignant bacteria, and has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of polypeptide preparation, and particularly relates to a preparation method of an antibacterial peptide. BACKGROUND

[0002] The information disclosed in this background section is intended to provide an overview of the general background of the application and is not necessarily a recognition of the prior art of any kind.

[0003] Antimicrobial peptides (AMPs) are important molecules in the natural immune defense system of organisms, and have broad-spectrum antibacterial activity. They have a significant inhibitory effect on gram-positive bacteria, gram-negative bacteria, fungi and some viruses. AMPs are usually positively charged and have amphiphilic structures, which enable them to interact with the negatively charged microbial membrane, thereby destroying the integrity of the cell membrane or interfering with the biosynthesis process, ultimately leading to cell death. The diverse bactericidal mechanisms make them a focus of attention in the study of drug-resistant bacteria.

[0004] In recent years, antimicrobial peptides have gradually become a hot spot in the research of antibiotic substitutes. However, the preparation methods of natural antimicrobial peptides, such as extraction, synthesis and fermentation, have problems such as low yield, high cost and difficult purification, which cannot meet the actual demand. Although synthetic methods can synthesize short peptides through chemical routes, they are limited by the difficulty and cost of synthesizing long peptides. In view of this situation, it is of great significance to develop an efficient, economical and broad-spectrum antibacterial peptide preparation method.

[0005] Collagen is the most abundant structural protein in animals, and has good biocompatibility and degradability. Collagen molecules are composed of three α chains twisted into a triple helix, each α chain has more than 1000 amino acids, and the most typical feature of the α chain is its monotonous Gly-X-Y repeat sequence, in which X and Y are usually proline and 4-hydroxyproline (Hyp), respectively. After enzymatic, thermal or acid-base hydrolysis of collagen α chain, collagen is degraded into small molecular peptides, i.e. collagen polypeptides, and the conservative sequence makes the hydrolyzed peptides have high similarity. Currently, there have been studies on extracting collagen antimicrobial peptides from collagen hydrolysates. For example, Lima et al. obtained a hydrolyzed collagen polypeptide (GDHGETGEQGDR) from bovine collagen hydrolysate, which had antibacterial activity against Staphylococcus aureus, Escherichia coli and Bacillus subtilis. Ennaas et al. identified an antimicrobial peptide collagencin (GLPGPLGPAGPK) from mackerel collagen hydrolysate, which had inhibitory effect on a variety of strains and could completely inhibit the growth of Staphylococcus aureus at low concentration.

[0006] The existing antibacterial peptide is found accidentally, needs a large number of different experimental researches, and the cost of single synthesis of the antibacterial peptide is too high, thereby limiting the industrial application. SUMMARY

[0007] In view of the problems of difficulty in finding the antibacterial peptide and high synthesis cost, the application provides a collagen peptide-based antibacterial peptide synthesis method, which has wide raw material sources and simple synthesis steps.

[0008] Another object of the application is to provide the antibacterial peptide prepared by the above method, which has good inhibitory effect on common pathogenic bacteria and can be used for preparing antibacterial drugs.

[0009] To achieve the above object, the application adopts the following technical scheme.

[0010] A preparation method of an antibacterial peptide, comprising the following steps: connecting a carboxyl of a collagen peptide with a molecular weight of 800-20000 to an amino acid, a dipeptide or a tripeptide; The amino acid is lysine or arginine; the dipeptide is any one of lysyl lysine, lysyl arginine, arginyl arginine and arginyl lysine; and the tripeptide is any one of lysyl lysyl lysine, arginyl lysyl lysine, lysyl lysyl arginine, lysyl arginyl lysine, lysyl arginyl arginine, arginyl lysyl arginine, arginyl arginyl lysine and arginyl arginyl arginine.

[0011] Specifically, the method comprises the following steps: (1) degrading collagen to obtain collagen peptides with a molecular weight of 800-20000 Da; (2) reacting the collagen peptides with Boc to obtain Boc-collagen peptides, and then activating the Boc-collagen peptides with HoBt and DIC; (3) coupling Fomc-modified amino acid protected amino acids on a Wang resin by using an Fmoc solid-phase synthesis method, and then removing the Fmoc protecting group; (4) adding the activated Boc-collagen peptides in step (2) to the product in step (3) to perform a coupling reaction under N2, washing the resin, then adding a cleavage solution to perform a reaction, and removing the resin by filtration; and separating and purifying the filtrate to obtain the antibacterial peptide.

[0012] The method for degrading collagen is not limited, and can be an enzymatic method or a physical method, such as alkaline protease, trypsin, pepsin, papain, bromelain, acid / alkali degradation, thermal degradation, etc. The alkaline protease is obtained by fermentation of Bacillus, such as strains of Bacillus subtilis and Bacillus licheniformis, and common alkaline proteases include Subtilisin, Alcalase, 2709 alkaline protease, etc.

[0013] Preferably, the collagen peptide has a molecular weight of 1000-10000 Da; more preferably, the collagen peptide has a molecular weight of 1000-5000 Da.

[0014] An antibacterial peptide obtained by the above preparation method.

[0015] The above antibacterial peptide can be used as an effective component of an antibacterial product.

[0016] The above antibacterial peptide can be prepared into a topical patch, an adhesive bandage, a suppository, a gel, a surgical implant material, and the like, for preventing bacterial and fungal infections.

[0017] The above antibacterial peptide can also be used as a food additive.

[0018] The above antibacterial peptide can also be used as a cosmetic additive.

[0019] The above antibacterial peptide can also be used as a feed additive or a veterinary drug for animals.

[0020] The present application has the following advantages: The present application uses a semi-synthetic method to obtain an antibacterial peptide from a collagen peptide, which has a wide source of raw materials, a simple synthesis method, and a low cost. The prepared antibacterial peptide has good salt resistance, an inhibitory effect in a wide pH range, and good biological safety. The antibacterial peptide has a good inhibitory effect on both gram-negative and gram-positive bacteria, especially on harmful bacteria commonly found in the vagina, and can be applied in the medical field to prepare patches, gels, and other drugs or implant materials. Based on its low cost and broad-spectrum antibacterial characteristics, the antibacterial peptide can also be applied in the field of veterinary drugs or feed additives. The antibacterial peptide has a wide application prospect in the field of inhibiting harmful microorganisms. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 Amino acid analysis spectrum of collagen-based antibacterial peptides CP-K and CP-2K; Figure 2 Amino acid analysis spectrum of collagen-based antibacterial peptides CP-R and CP-2R; Figure 3 Amino acid analysis spectrum of collagen-based antibacterial peptides CP-KR and CP-RK; Figure 4 Growth curve of Escherichia coli and Staphylococcus aureus under the action of collagen-based antibacterial peptides CP-K and CP-2K; Figure 5 Growth curve of Escherichia coli and Staphylococcus aureus under the action of collagen-based antibacterial peptides CP-R and CP-2R; Figure 6The growth amount of E. coli and S. aureus under the action of collagen-based antibacterial peptides CP-RK and CP-KR; Figure 7 The ion exchange chromatogram of collagen peptide; Figure 8 The ultraviolet spectrum of cationic components; Figure 9 The effect of co-incubation of CP and CP-K, CP-2K on the fluorescence intensity of P. luminescens T3; Figure 10 The growth amount of E. coli (a) and S. aureus (b) under the action of collagen-based antibacterial peptide CP-3R; Figure 11 The broad-spectrum antibacterial ability diagram of collagen-based antibacterial peptides CP-K and CP-2K, wherein (a) and (b) are the antibacterial ability of CP-K and CP-2K on P. fluorescens; (c) and (d) are the antibacterial ability of CP-K and CP-2K on B. licheniformis; Figure 12 The broad-spectrum antibacterial ability diagram of collagen-based antibacterial peptides CP-R and CP-2R, wherein (a) and (b) are the antibacterial ability of CP-R and CP-2R on P. fluorescens; (c) and (d) are the antibacterial ability of CP-R and CP-2R on B. licheniformis; Figure 13 The effect of different salt ion concentrations on the antibacterial ability of collagen-based antibacterial peptides, wherein (a) is the effect on the antibacterial ability of CP-K; (b) is the effect on the antibacterial ability of CP-2K; Figure 14 The effect of different salt ion concentrations on the antibacterial ability of collagen-based antibacterial peptides, wherein (a) is the effect on the antibacterial ability of CP-R; (b) is the effect on the antibacterial ability of CP-2R; Figure 15 The effect of different pH on the antibacterial ability of collagen-based antibacterial peptides, wherein (a) is the effect on the antibacterial ability of CP-K; (b) is the effect on the antibacterial ability of CP-2K; Figure 16 The effect of different pH on the antibacterial ability of collagen-based antibacterial peptides, wherein (a) is the effect on the antibacterial ability of CP-R; (b) is the effect on the antibacterial ability of CP-2R; Figure 17 The cytotoxicity result diagram of collagen-based antibacterial peptides CP-K and CP-2K on liver cancer cells HepG2, wherein (a) is the cytotoxicity of CP-K; (b) is the cytotoxicity of CP-2K; Figure 18 The cytotoxicity result diagram of collagen-based antibacterial peptides CP-R and CP-2R on liver cancer cells HepG2, wherein (a) is the cytotoxicity of CP-R; (b) is the cytotoxicity of CP-2R; Figure 19 The hemolytic toxicity results of collagen-based antibacterial peptides CP-K and CP-2K, wherein (a) is the hemolytic toxicity of CP-K; (b) is the hemolytic toxicity of CP-2K; Figure 20 The hemolytic toxicity results of collagen-based antibacterial peptides CP-R and CP-2R, wherein (a) is the hemolytic toxicity of CP-R; (b) is the hemolytic toxicity of CP-2R; Figure 21 The effect of co-incubation of antibacterial peptides with different molecular weights on the fluorescence intensity of Photobacterium leiognathi T3; Figure 22 The effect of CP-K prepared from different enzymatic collagen peptides on the growth of Escherichia coli (a) and Staphylococcus aureus (b); Figure 23 The antibacterial ability of collagen-based antibacterial peptide CP-2K against vaginal pathogenic bacteria. DETAILED DESCRIPTION

[0022] The application will be further described below in conjunction with examples and drawings, but the application is not limited by the following examples.

[0023] Example 1 Preparation of antibacterial peptides 1. Preparation of collagen peptides 100 g of gelatin was weighed into a beaker, 400 mL of deionized water was added, and the beaker was placed in a 60 °C constant temperature water bath to heat until completely dissolved. The pH was adjusted to 7.0. Subtilisin enzyme 0.5 mL was added to the gelatin solution, and the solution was magnetically stirred at 55 °C for 2-10 h. After the reaction was completed, the enzyme was completely inactivated by water bath at 100 °C for 5 min, and the collagen peptides (molecular weight range 800-20000) sample was obtained by freeze-drying, which was ready for use.

[0024] 2. Activation of collagen peptides 10 g of collagen peptides and 1 g of sodium bicarbonate were weighed and dissolved in 50 mL of deionized water to prepare a polypeptide solution. 2 g of di-tert-butyl dicarbonate (Boc) was weighed and dissolved in 50 mL of tetrahydrofuran (THF), and the Boc solution was mixed with the polypeptide solution, and the mixture was reacted at room temperature for 12 h. After the reaction was completed, THF was removed by rotary evaporation, and water was added for dissolution. After freeze-drying, Boc-polypeptide was obtained. 5 g of Boc-polypeptide was weighed and dissolved in 30 mL of N,N-dimethylformamide (DMF), and 1 g of 1-hydroxybenzotriazole (HoBt) and 1.5 mL of N,N'-diisopropylcarbodiimide (DIC) were added for pre-activation.

[0025] 3. Suffix amino acid / dipeptide / tripeptide pretreatment (1) Lys(Boc)-OH resin Lys(Boc)-OH resin can be prepared by one of the following steps: A. Take 2 g Wang resin and place it in a solid-phase synthesis tube, add 20 mL dichloromethane (DCM) and swell for 30 min. Filter. Take 6 g Fmoc-Lys (Boc)-OH and dissolve it in 20 mL N,N-dimethylacetamide (DMA), add 1.5 g HoBt and 2 mL DIC, and place it in a shaker at 20°C, 140 rpm for 0.5-4 h. Add the above mixture to the solid-phase synthesis tube, bubble in N2for 1 h-8 h for coupling reaction, and then release the liquid. Wash the resin with appropriate amounts of DMF, DCM and methanol in sequence. Add 50 mL of 10%-50% v / v piperidine in DMF solution, bubble in N2for 2 h. Wash the resin with appropriate amounts of DMF, DCM and methanol in sequence to obtain Lys(Boc)-OH resin.

[0026] B. Take 2 g Fmoc-Lys(Boc)-Wang resin and place it in a solid-phase synthesis tube, add 50 mL of 10%-50% v / v piperidine in DMF solution, bubble in N2to remove the Fmoc protecting group. Wash the resin with appropriate amounts of DMF, DCM and methanol in sequence to obtain Lys(Boc)-OH resin.

[0027] (2) Lys(Boc)-Lys(Boc)-OH resin Take 2 g Fmoc-Lys(Boc)-Wang resin and place it in a solid-phase synthesis tube, add 50 mL of 10%-50% piperidine / DMF (v / v) deprotection solution, bubble in N2to remove the Fmoc protecting group. Wash the resin with appropriate amounts of DMF, DCM and methanol in sequence. Take 3 g Fmoc-Lys(Boc)-OH and dissolve it in 30 mL DMF, add 1 g HoBt and 1.5 mL DIC for pre-activation. Add the pre-activated amino acid to the solid-phase synthesis tube containing the amino acid with all Fmoc removed, bubble in N2for 0.5 h-6 h for coupling reaction, and then release the liquid. Wash the resin with appropriate amounts of DMF, DCM and methanol in sequence. Add 50 mL of 10%-50% piperidine / DMF (v / v) deprotection solution, bubble in N2to remove the Fmoc protecting group. Wash the resin with appropriate amounts of DMF, DCM and methanol in sequence to obtain Lys(Boc)-Lys(Boc)-OH resin.

[0028] (3) Arg(Pbf)-OH resin Take 2 g Wang resin and place it in a solid-phase synthesis tube, add 20 mL DCM, and swell for 30 min. Filter by suction. Take 8 g Fmoc-Arg(Pbf)-OH and dissolve it in 20 mL DMA, add 1.5 g HoBt and 2 mL DIC, and place it in a shaking bed at 20°C, 140 rpm, and react for 0.5-4 h. Add the above mixture to the solid-phase synthesis tube, bubble in N2for 1-8 h of coupling reaction, release the liquid after the reaction is complete, and wash the resin with appropriate amounts of DMF, DCM, and methanol in sequence. Dry the product at 40°C under vacuum for 3 h to obtain Fmoc-Arg(Pbf)-Wang resin.

[0029] Take 2 g Fmoc-Arg(Pbf)-Wang resin and place it in a solid-phase synthesis tube, add 50 mL of 10%-50% piperidine / DMF (v / v) deprotection solution, bubble in N2, and remove the Fmoc protecting group. Wash the resin with appropriate amounts of DMF, DCM, and methanol in sequence to obtain Arg(Pbf)-OH resin.

[0030] (4) Preparation method of Arg(Pbf)-Arg(Pbf)-OH resin Take 2 g Fmoc-Arg(Pbf)-Wang resin and place it in a solid-phase synthesis tube, add 50 mL of 10%-50% piperidine / DMF (v / v) deprotection solution, bubble in N2, and remove the Fmoc protecting group. Wash the resin with appropriate amounts of DMF, DCM, and methanol in sequence. Take 3 g Fmoc-Arg(Pbf)-OH, dissolve it in 30 mL DMF, add 1 g HoBt and 1.5 mL DIC for pre-activation. Add the pre-activated amino acid to the solid-phase synthesis tube containing the amino acid from which the Fmoc has been completely removed, bubble in N2for 0.5-6 h of coupling reaction, release the liquid after the reaction is complete, and wash the resin with appropriate amounts of DMF, DCM, and methanol in sequence. Add 50 mL of 10%-50% piperidine / DMF (v / v) deprotection solution, bubble in N2, and remove the Fmoc protecting group. Wash the resin with appropriate amounts of DMF, DCM, and methanol in sequence to obtain Arg(Pbf)-Arg(Pbf)-OH resin.

[0031] (5) Preparation method of Lys(Boc)-Arg(Pbf)-OH resin Take 2 g Fmoc-Lys(Boc)-Wang resin into a solid-phase synthesis tube, add 50 mL of 10%-50% piperidine / DMF (v / v) deprotection solution, and bubble N2to remove the Fmoc protecting group. Wash the resin with appropriate amounts of DMF, DCM, and methanol in turn. Take 3 g Fmoc-Arg(Pbf)-OH, dissolve in 30 mL DMF, add 1 g HoBt and 1.5 mL DIC for pre-activation. Add the pre-activated amino acid to the solid-phase synthesis tube containing the completely Fmoc-removed amino acid, bubble N2for 0.5-5 h for coupling reaction, release the liquid after the end of the reaction, and wash the resin with appropriate amounts of DMF, DCM, and methanol in turn. Add 50 mL of 10%-50% piperidine / DMF (v / v) deprotection solution, and bubble N2to remove the Fmoc protecting group. Wash the resin with appropriate amounts of DMF, DCM, and methanol in turn to obtain the Arg(Pbf)-Arg(Pbf)-OH resin.

[0032] (6) Preparation method of Arg(Pbf)-Lys(Boc)-OH resin Take 2 g Fmoc-Arg(Pbf)-Wang resin into a solid-phase synthesis tube, add 50 mL of 10%-50% piperidine / DMF (v / v) deprotection solution, and bubble N2to remove the Fmoc protecting group. Wash the resin with appropriate amounts of DMF, DCM, and methanol in turn. Take 2.5 g Fmoc-Lys(Boc)-OH, dissolve in 30 mL DMF, add 1 g HoBt and 1.5 mL DIC for pre-activation. Add the pre-activated amino acid to the solid-phase synthesis tube containing the completely Fmoc-removed amino acid, bubble N2for 0.5-5 h for coupling reaction, release the liquid after the end of the reaction, and wash the resin with appropriate amounts of DMF, DCM, and methanol in turn. Add 550 mL of 10%-50% piperidine / DMF (v / v) deprotection solution, and bubble N2to remove the Fmoc protecting group. Wash the resin with appropriate amounts of DMF, DCM, and methanol in turn to obtain the Lys(Boc)-Lys(Boc)-OH resin.

[0033] 4. Synthesis of antibacterial peptide All activated peptide solutions were added to a solid-phase synthesis tube containing Lys(Boc)-OH resin. The tube was bubbled with N2 for 0.5–5 h for coupling reaction. After the reaction, the liquid was released, and the resin was washed sequentially with appropriate amounts of DMF, DCM, and methanol. A suitable amount of lysis buffer (TFA: TIS: H2O = 95:2.5:2.5 v / v / v) was added to the solid-phase synthesis tube at a ratio of 1 mL lysis buffer per 150 mg of resin. The tube was bubbled with N2 for 1–8 h for lysis reaction. The resin was removed by filtration, and the lysis buffer was concentrated under N2. The concentrated lysis buffer was added to frozen anhydrous diethyl ether at approximately -30 °C at a ratio of 1:10 v / v to precipitate the peptides. The sample was centrifuged at 7000 r / min for 20 min, and the anhydrous diethyl ether was discarded. This process was repeated five times to obtain a sample named CP-K.

[0034] Following the above method, the remaining amino acid resins or dipeptide resins were coupled together. Based on the different types and numbers of amino acids linked, the samples were named CP-2K, CP-R, CP-2R, CP-KR, and CP-RK, respectively. After being dissolved in deionized water, they were lyophilized and stored.

[0035] The antimicrobial peptides obtained by the above method were analyzed using a fully automated amino acid analyzer (L-8900, Hitachi). The amino acid analysis results for CP-K and CP-2K are as follows: Figure 1 As shown, the amino acid analysis results of CP-R and CP-2R are as follows: Figure 2 As shown, the amino acid analysis results of CP-KR and CP-RK are as follows: Figure 3 As shown.

[0036] The amino acid analysis results of antimicrobial peptides CP-K and CP-2K are as follows: Figure 1 As shown. Based on the characteristic amino acid composition of collagen—its hydroxyproline content accounts for approximately 14% of its dry weight—this amino acid was selected as an endogenous reference indicator to establish an amino acid content analysis system. The lysine / hydroxyproline ratio in the CP-K group increased to 1.24, and in the CP-2K group, this ratio further increased to 2.97, reaching 2.4 times that of the CP-K group, precisely corresponding to the molecular design of the double lysine modification.

[0037] The amino acid analysis results of antimicrobial peptides CP-R and CP-2R are as follows: Figure 2 As shown in the figure, after modification, the arginine / hydroxyproline ratio of the CP-R group was 2.858, and the arginine / hydroxyproline ratio of the CP-2R group increased to 5.62, which is 1.97 times that of the CP-K group, precisely corresponding to the design target of diarginine modification.

[0038] Example 2: Inhibition of pathogenic bacteria by antimicrobial peptides Single colonies of E. coli (BNCC 357959) and S. aureus (ATCC 6538) after picking and activating were inoculated in LB broth medium and expanded at 37°C for 16-18 h, and the bacterial liquid concentration was adjusted to 1×10 6 CFU / mL.

[0039] Each well of a sterile 96-well plate was added with 100 μL of diluted bacterial liquid, and then 100 μL of each of the test samples diluted with normal saline at a gradient was added to each well, which was blown and stirred with a pipette to mix, and then placed in a 37°C constant temperature incubator for overnight culture. The test samples were the antibacterial peptides CP-K, CP-2K, CP-R, CP-2R, CP-KR and CP-RK prepared in Example 1. The concentration of the diluted test samples was 0-16 mg / mL. Each well of the 96-well plate was respectively injected with a test group (50 μL of sample liquid, 50 μL of bacterial liquid), a positive control group (50 μL of ciprofloxacin, 50 μL of bacterial liquid), a negative control group (50 μL of sample liquid, 50 μL of culture medium), and a blank control group (50 μL of culture medium, 50 μL of bacterial liquid). The 96-well plate was placed in a 37°C constant temperature incubator for culture. The OD value of each well at 600 nm was determined every hour by using an enzyme marker, and the bacterial growth was evaluated by the change of the OD value.

[0040] The results of the influence of the antibacterial peptides CP-K and CP-2K on the growth curve of E. coli are shown in Figure 4 A, and the antibacterial peptides CP-K and CP-2K can dose-dependently inhibit the growth of E. coli.

[0041] The results of the influence of the antibacterial peptides CP-R and CP-2R on the growth curve of S. aureus are shown in Figure 4 B, and the antibacterial peptides CP-R and CP-2R can dose-dependently inhibit the growth of S. aureus.

[0042] The results of the influence of the antibacterial peptides CP-K and CP-2K on the growth curve of E. coli are shown in Figure 5 A, and the antibacterial peptides CP-K and CP-2K can dose-dependently inhibit the growth of E. coli.

[0043] The results of the influence of the antibacterial peptides CP-R and CP-2R on the growth curve of S. aureus are shown in Figure 5 B, and the antibacterial peptides CP-R and CP-2R can dose-dependently inhibit the growth of S. aureus.

[0044] The results of the influence of the antibacterial peptides CP-KR and CP-RK on the growth curve of E. coli are shown in Figure 6 A, and the antibacterial peptides CP-KR and CP-RK can dose-dependently inhibit the growth of E. coli.

[0045] The results of the influence of antibacterial peptides CP-KR and CP-RK on the growth curve of S. aureus are shown in Figure 6 As shown in Figure B, the antibacterial peptides CP-KR and CP-RK can dose-dependently inhibit the growth of S. aureus.

[0046] The above results show that the prepared antibacterial peptides CP-K, CP-2K, CP-R, CP-2R, CP-KR and CP-RK have inhibitory effects on both gram-negative bacteria and gram-positive bacteria. The antibacterial effect of the antibacterial peptide with double amino acid modification is better than that of the antibacterial peptide with single amino acid modification. Among them, the antibacterial peptide CP-2R has the most obvious inhibitory effect on the two bacteria.

[0047] Preparation of antibacterial peptides 1. Preparation of antibacterial peptides The cationic component of the collagen peptide sample in Example 1 was separated by ion exchange chromatography: the ion exchange separation pattern of the hydrolyzed polypeptide using 732 cationic resin is shown in Figure 7 , wherein the first elution peak is the anionic and non-polar component eluted with distilled water, and the second elution peak is the cationic component eluted with 0.5 mol / L NaCl. The second component collected by ion exchange chromatography was freeze-dried and resampled, and eluted with distilled water. The obtained pattern is shown in Figure 7 . The collected components were scanned by visible-ultraviolet spectrophotometer, and the ultraviolet spectrum of the eluted components is shown in Figure 8 . There is a strong absorption peak of peptide bond between 200-240 nm, and the absorption at 280 nm is weak, which is the typical spectrum of collagen protein. It can be shown that there is collagen polypeptide in the sample, which is named CP as an antibacterial peptide.

[0048] 2. Antibacterial effect The antibacterial ability of each component was tested by using P. phocinidus T3 serotype. At room temperature of 20-25℃, 0.7 mL of stable luminous bacteria liquid was taken in a 1.5 mL centrifuge tube, the sample to be tested was added, and the fluorescence visible spectrophotometer was used to measure the luminosity after precise reaction for 15 min in vortex mixing instrument. The blank sample was reacted with pure water and the luminosity was measured.

[0049] The antibacterial ability of CP and CP-K, CP-2K is shown in Figure 9 . The antibacterial peptide CP has antibacterial activity, but the antibacterial ability is weak, much lower than that of CP-K and CP-2K with increased amino acid suffix.

[0050] Preparation of antibacterial peptides 1. Preparation of antibacterial peptides Take 8 g of the collagen peptide prepared in Example 1 and 1 g of sodium bicarbonate, dissolve in 50 mL of deionized water to prepare a polypeptide solution; Take 3 g of Boc, dissolve in 50 mL of THF, mix the Boc solution with the polypeptide solution, and react at room temperature for 12 h; after the reaction is completed, remove the THF by rotary evaporation, dissolve with water, and freeze-dry to obtain Boc-polypeptide; Take 5 g of Boc-polypeptide, dissolve in 30 mL of DMF, add 1 g of HoBt and 1.5 mL of DIC for pre-activation to obtain an activated polypeptide solution.

[0051] Take 2 g of Fmoc-Arg(Pbf)-Wang resin and place it in a solid-phase synthesis tube, add 50 mL of 10%-50% piperidine / DMF (v / v) deprotection solution, and bubble N2 to remove the Fmoc protecting group. Wash the resin with appropriate amounts of DMF, DCM, and methanol in turn. Take 3 g of Fmoc-Arg(Pbf)-OH, dissolve in 30 mL of DMF, add 1 g of HoBt and 1.5 mL of DIC for pre-activation. Add the pre-activated amino acid to the solid-phase synthesis tube with all the Fmoc-removed amino acids, bubble N2 for 1-3 h for coupling reaction, release the liquid after the reaction is completed, and wash the resin with appropriate amounts of DMF, DCM, and methanol in turn. Add 50 mL of 10%-50% piperidine / DMF (v / v) deprotection solution, and bubble N2 to remove the Fmoc protecting group. Wash the resin with appropriate amounts of DMF, DCM, and methanol in turn to obtain Arg(Pbf)-Arg(Pbf)-OH resin. Take 3 g of Fmoc-Arg(Pbf)-OH again, dissolve in 30 mL of DMF, add 1 g of HoBt and 1.5 mL of DIC for pre-activation. Add the pre-activated amino acid to the solid-phase synthesis tube with all the Fmoc-removed amino acids, bubble N2 for 1-3 h for coupling reaction, release the liquid after the reaction is completed, and wash the resin with appropriate amounts of DMF, DCM, and methanol in turn. Add 50 mL of 10%-50% v / v piperidine in DMF solution, bubble N2 to remove the Fmoc protecting group. Wash the resin with appropriate amounts of DMF, DCM, and methanol in turn to obtain Arg(Pbf)-Arg(Pbf)-Arg(Pbf)-OH resin.

[0052] The whole activated polypeptide solution was added to the Fmoc-removed tripeptide solid-phase synthesis tube, and N2 was bubbled for 1-5 h for coupling reaction. After the reaction, the liquid was released, and the resin was washed with appropriate amounts of DMF, DCM and methanol in turn. According to the proportion of 1 mL of lysis solution per 150 mg of resin, an appropriate amount of lysis solution (TFA: TIS: H2O = 95:2.5:2.5 v / v / v) was added to the solid-phase synthesis tube, and N2 was bubbled for 3-5 h for lysis reaction. The resin was filtered and the lysis solution was concentrated under N2. The concentrated lysis solution was added to about -30℃ frozen anhydrous ether at a ratio of 1:10 v / v to precipitate the polypeptide, centrifuged at 7000 r / min for 20 min, and the anhydrous ether was discarded. Repeat five times to obtain the CP-3R antibacterial peptide sample, which was dissolved in deionized water and freeze-dried for storage.

[0053] 2. Bacteriostatic effect The bacteriostatic effect of CP-3R on E. coli and S. aureus was determined by the method in Example 2, and the 96-well plate was placed in a 37℃ constant temperature incubator for 12 h. The OD value of each well at 600 nm was determined by using a microplate reader, and the bacterial growth was evaluated by the change of OD value. The results are shown in Figure 10 As the concentration of antibacterial peptide increased from 0.5 mg / mL to 16 mg / mL, the OD 600 value decreased significantly, especially at high concentrations, which was significantly different from the blank control, indicating that CP-3R could effectively inhibit the growth of E. coli and S. aureus, and the inhibition was concentration-dependent. However, compared with CP-2K, CP-2R, CP-KR and CP-RK, there was no obvious advantage in bacteriostatic effect, and the preparation was more difficult.

[0054] Example 4 Broad-spectrum bacteriostatic ability of antibacterial peptide The antibacterial ability of CP-K, CP-2K, CP-R and CP-2R was determined by using P. fluorescens (BNCC 336632); B. licheniformis (ATCC 11946); P. aeruginosa (BNCC 125486); S. typhi (CMCC 50071) as test bacteria: Activated single colonies were picked and inoculated in LB broth medium and cultured at 37℃ for 16-18 h. The bacterial solution was adjusted to 1×10 6CFU / mL. 100 μL of the diluted bacterial solution was added to each well of a sterile 96-well plate, followed by 100 μL of the test sample diluted in physiological saline in gradient, and then mixed by pipetting. The plate was incubated in a 37°C incubator overnight. The test sample was added (0-16 mg / mL). The plate was incubated in a 37°C incubator for 12 h. The OD value of each well at 600 nm was measured every hour using a microplate reader, and the bacterial growth was evaluated by the change in OD value.

[0055] The test results are shown in Figure 11 , 12 The antibacterial peptides CP-K, CP-2K, CP-R and CP-2R all had antibacterial activity against several test bacteria, and the activity was basically concentration-dependent.

[0056] Example 5. Antibacterial stability 1. Salt ion concentration stability The antibacterial peptides CP-K, CP-2K, CP-R and CP-2R obtained in Example 1 were dissolved in solutions containing 50, 100, 150, 200 and 250 mM NaCl, respectively, to prepare a series of concentration gradient samples. The inhibitory effect of the antibacterial peptides on E. coli was determined by the microbroth dilution method under each NaCl concentration. To exclude the potential influence of NaCl itself on the growth of E. coli, a series of NaCl solutions (50-250 mM) without antibacterial peptides were set as a control group. The results are shown in Figure 13 and Figure 14 The antibacterial peptides CP-K, CP-2K, CP-R and CP-2R had good salt ion concentration stability in the range of 0-200 mM NaCl, and CP-R and CP-2R showed more excellent salt ion concentration stability.

[0057] 2. pH stability The antibacterial peptides CP-K, CP-2K, CP-R and CP-2R obtained in Example 1 were dissolved in a series of PBS buffer solutions with different pH values (3.0, 5.0, 7.0, 9.0, 11.0), and the inhibitory effect of the antibacterial peptides on E. coli was determined by the microbroth dilution method under different pH conditions. To exclude the potential influence of pH change on the growth of E. coli, a series of PBS buffer solutions (pH 3.0-11.0, with an interval of 2 pH units) without antibacterial peptides were set as a control group. The results are shown in Figure 15 and Figure 16 The antibacterial peptides CP-K, CP-2K, CP-R and CP-2R had good pH stability in a wide pH range, and the antibacterial activity was relatively stronger under acidic conditions.

[0058] Example 6. Biological safety 1. Cytotoxicity HepG2 hepatoma cells were cultured in a 5% CO2, 37℃ constant temperature incubator. The antibacterial peptides CP-K, CP-2K, CP-R and CP-2R obtained from Example 1 and Example 2 were weighed, respectively, and added to complete culture medium at a proportion of 16 mg / mL, mixed and dissolved, and then filtered with a 0.22 μm filter to remove bacteria for standby HepG2 cells in the logarithmic growth phase were selected and inoculated in a 96-well culture plate at a density of 6×10³ cells per well. Then, 100 μL of the sample solution to be tested was added to each well of the experimental group, and an equal volume (100 μL / well) of complete culture medium was added to the control group. The culture plate was placed in a 37℃, 5% CO2 constant temperature incubator for incubation for 24 h. After incubation, the liquid in the wells was aspirated and washed 3 times with 0.01 M PBS buffer. 100 μL of MTT-containing medium (0.5 mg / mL) was added to each well, and incubation was continued under the same conditions for 4 h. After the reaction was completed, the supernatant was discarded, and 100 μL of DMSO was added to each well. After 10 min of reaction, the absorbance value at 570 nm was determined using an enzyme marker.

[0059] Cell relative survival rate (RGR, %) = (OD 600 value of the sample group - OD 600 value of the blank well) / (OD 600 value of the control group - OD 600 value of the blank well) × 100%.

[0060] The results are shown in Figure 17 and Figure 18 Within the tested concentration range, the antibacterial peptides CP-K, CP-2K, CP-R and CP-2R had no obvious cytotoxicity on HepG2 hepatoma cells, laying a foundation for further development of antibacterial peptides.

[0061] 2. Hemolytic toxicity The anticoagulated rabbit blood sample was placed in a centrifuge tube and centrifuged at 1500 rpm for 10 min. After centrifugation, the supernatant plasma layer was carefully removed, and then the red blood cells were repeatedly washed with pre-cooled 0.01 M PBS buffer until the supernatant was completely clear. The washed red blood cells were prepared into a 12% cell suspension with the same buffer, ready for use. During the experiment, an appropriate amount of red blood cell suspension was dispensed into 1.5 mL centrifuge tubes, and different concentrations of antibacterial peptide solution prepared with PBS were added. At the same time, the control group was set up: the positive control used 0.25% Triton-X 100 solution, and the negative control used the same volume of PBS buffer. Each group of experiments was set up with 3 repeated samples to ensure data reliability. The centrifuge tubes were placed in an incubator for 1 h, and the culture conditions were set at 37°C and 60 rpm. After incubation, the experimental samples were centrifuged at 1000 rpm for 10 min for precipitation separation. The degree of hemolysis was evaluated by visual method: when the contents of the centrifuge tube presented uniform transparent red color and no obvious red blood cell sedimentation at the bottom of the tube, it was determined as positive hemolytic reaction; if the complete red blood cell sedimentation was observed and the supernatant remained colorless and transparent, it was determined that no hemolysis occurred. Carefully pipette 100 μL of supernatant into a 96-well plate and measure the absorbance at 540 nm.

[0062] Hemolysis rate (%) = (positive control OD 540 value-negative control OD 540 value) / (sample group OD 540 value-negative control OD 540 value) x 100%.

[0063] The results are shown in Figure 19 and Figure 20 , within the test concentration range, antibacterial peptides CP-K, CP-2K, CP-R and CP-2R had no obvious hemolytic effect on red blood cells, and CP-R and CP-2R showed better hemolytic safety.

[0064] Example 7 Antibacterial effect of antibacterial peptides prepared by different molecular weight collagen peptides Weigh 100 g of gelatin in a beaker, add 400 mL of deionized water, and place it in a 60°C constant temperature water bath to heat until completely dissolved, adjust the pH to 7.0; Add Alcalase enzyme 0.5 mL to the gelatin solution, and hydrolyze at 55°C with magnetic stirring for 2-10 h; after the reaction is completed, completely inactivate the enzyme at 100°C water bath for 5 min, and obtain the hydrolysate; The gelatin hydrolysate was separated and purified according to the molecular weight of the hydrolyzed collagen polypeptide by using a tangential flow ultrafiltration system. First, the components less than 1 kDa were separated by using a 1 kDa MWCO ultrafiltration membrane; then, the components of 1-3 kDa, 3-5 kDa and 5-10 kDa were separated by using 3 kDa, 5 kDa and 10 kDa MWCO ultrafiltration membranes, respectively, and the polypeptides greater than 10 kDa were retained in the final retentate.

[0065] The hydrolyzed polypeptide solutions less than 1 kDa, 1-3 kDa, 3-5 kDa and 5-10 kDa were freeze-dried, respectively, and then the C-terminal lysine-modified collagen polypeptides were synthesized according to the method of Example 1, and were named as CP(1000)-K, CP(3000)-K, CP(5000)-K and CP(10000)-K, respectively.

[0066] The bacteriostatic effect was tested by using P. luminescens T3 serotype according to the method in Comparative Example 1, and the results are shown in Table 1. Figure 21 As shown in Table 1, CP(1000)-K and CP(3000)-K have stronger bacteriostatic ability than other molecular weights.

[0067] Example 8 Bacteriostatic effect of antibacterial peptides prepared from collagen peptides obtained by different degradation methods 100 g of gelatin was weighed into a beaker, 400 mL of deionized water was added, and the beaker was placed in a 60°C constant temperature water bath to heat until completely dissolved; Trypsin was added to the gelatin solution, the pH was adjusted to 8.0, and the solution was hydrolyzed at 37°C under magnetic stirring for 2-10 h; after the reaction was completed, the enzyme was completely inactivated by water bath at 100°C for 5 min to obtain a hydrolysate; Pepsin was added to the gelatin solution, the pH was adjusted to 3.0, and the solution was hydrolyzed at 37°C under magnetic stirring for 2-10 h; after the reaction was completed, the enzyme was completely inactivated by water bath at 100°C for 5 min to obtain a hydrolysate; Papain was added to the gelatin solution, the pH was adjusted to 6.0, and the solution was hydrolyzed at 55°C under magnetic stirring for 2-10 h; after the reaction was completed, the enzyme was completely inactivated by water bath at 100°C for 5 min to obtain a hydrolysate; Bromelain was added to the gelatin solution, the pH was adjusted to 6.0, and the solution was hydrolyzed at 45°C under magnetic stirring for 2-10 h; after the reaction was completed, the enzyme was completely inactivated by water bath at 100°C for 5 min to obtain a hydrolysate; The hydrolysates obtained by using different enzymes were used to prepare CP-K according to the method in Example 1, and the effects on E. coli and S. aureus were determined according to the method in Example 2, and the results are shown in Table 2. Figure 22 As shown in Table 2, the antibacterial peptides prepared from collagen peptides obtained by using different enzymes have the effect of inhibiting the growth of both pathogenic bacteria.

[0068] Example 9: Inhibition of vaginal pathogens by antimicrobial peptides 1. Preparation of the antimicrobial peptide CP(1000)-2K Weigh 100 g of gelatin into a beaker, add 400 mL of deionized water, and heat in a 60 ℃ constant temperature water bath until completely dissolved. Adjust the pH to 7.0. Add 0.5 mL of Subtilisin enzyme to the gelatin solution and hydrolyze it with magnetic stirring at 55℃ for 2-10 h; after the reaction is complete, completely inactivate the enzyme by water bath at 100℃ for 5 min to obtain the hydrolysate. The gelatin hydrolysate was purified by tangential flow ultrafiltration to separate and purify the hydrolyzed collagen peptides according to their molecular weight. First, collagen peptides smaller than 1 kDa were separated using a 1 kDa MWCO ultrafiltration membrane, then freeze-dried and named CP(1000).

[0069] Weigh 8 g of collagen peptide CP(1000) and 1 g of sodium bicarbonate, dissolve them in 50 mL of deionized water to prepare a peptide solution; Weigh 3 g of ditert-butyl dicarbonate (Boc), dissolve it in 50 mL of tetrahydrofuran (THF), mix the Boc solution with the peptide solution, and react at room temperature for 12 h; after the reaction is complete, remove THF by rotary evaporation, dissolve in water, freeze dry to obtain Boc-peptide. Weigh 5 g of Boc-peptide, dissolve it in 30 mL of N,N-dimethylformamide (DMF), and add 1 g of 1-hydroxybenzotriazole (HoBt) and 1.5 mL of N,N'-diisopropylcarbodiimide (DIC) for pre-activation.

[0070] All activated peptide solutions were added to a solid-phase synthesis tube containing Lys(Boc)-Lys(Boc)-OH resin. The tube was bubbled with N2 for 1 h to initiate the coupling reaction. After the reaction, the liquid was released, and the resin was washed sequentially with appropriate amounts of DMF, dichloromethane (DCM), and methanol. A suitable amount of lysis buffer (TFA:TIS:H2O = 95:2.5:2.5v / v / v) was added to the solid-phase synthesis tube at a ratio of 1 mL lysis buffer per 150 mg of resin. The tube was bubbled with N2 for 3 h to initiate the lysis reaction. The resin was removed by filtration, and the lysis buffer was concentrated under N2. The concentrated lysis buffer was added to frozen anhydrous diethyl ether at approximately -30 °C at a ratio of 1:10 v / v to precipitate the peptides. The sample was centrifuged at 7000 r / min for 20 min, and the anhydrous diethyl ether was discarded. This process was repeated five times to obtain a sample named CP(1000)-2K.

[0071] 2. Inhibition of vaginal pathogens by CP(1000)-2K Escherichia coli (E. coli Klebsiella pneumoniae ( ) K. oxytoca ), Klebsiella pneumoniae ( K. pneumoniae ), Enterococcus faecalis ( E. faecalis ) and agalactococcus ( S. agalactiae All samples were obtained from Jinan Maternal and Child Health Hospital. The prepared antimicrobial peptide CP(1000)-2K was dissolved in sterile physiological saline, filtered through a 0.22 μm microporous membrane for sterilization, and test solutions with three concentration gradients of 8, 16, and 32 mg / mL were prepared.

[0072] Single colonies were picked from the -80 ℃ glycerol-preserved bacterial strain and inoculated into LB liquid medium. The culture was incubated at 37 ℃ and 180 rpm for 12–16 h until the logarithmic growth phase. The culture was then serially diluted with sterile physiological saline to prepare 1×10⁻⁶ colonies. 6 CFU / mL standard bacterial suspension. Take a sterile EP tube, add 1 mL of the prepared bacterial suspension, then add 1 mL of antimicrobial peptide solution of different concentrations and 1 mL of physiological saline (control group), respectively. Mix thoroughly and incubate at 37 ℃ for 1 h. Transfer 100 μL of the mixture and spread it evenly on LB solid medium. Incubate upside down at 37°C for 16 h, then count the colonies. The experiment was set up in triplicate. The antimicrobial effect of the antimicrobial peptide was calculated based on the change in colony count on the plates.

[0073] The test results are as follows Figure 23 As shown, the antimicrobial peptide CP(1000)-2K exhibits significant inhibitory effects against *Escherichia coli*, *Klebsiella pneumoniae*, *Enterococcus faecalis*, and *Streptococcus agalactiae*, showing a concentration-dependent effect. At higher concentrations, it almost completely inhibits the growth of these bacteria. These results indicate that the antimicrobial peptide CP(1000)-2K has potential applications in the biomedical field.

[0074] The above description is merely an embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principle of this application should be included within the scope of the claims of this application.

Claims

1. A method for preparing an antibacterial peptide, characterized by, comprising the following steps: connecting the carboxyl of collagen peptide with molecular weight of 800-20000 to amino acid, dipeptide or tripeptide; the amino acid is lysine or arginine; the dipeptide is any one of lysyl lysine, lysyl arginine, arginyl arginine, arginyl lysine; the tripeptide is any one of lysyl lysyl lysine, arginyl lysyl lysine, lysyl lysyl arginine, lysyl arginyl lysine, lysyl arginyl arginine, arginyl lysyl arginine, arginyl arginyl lysine, arginyl arginyl arginine.

2. The production method according to claim 1, characterized by, comprising the following steps: (1) degrading collagen to obtain collagen peptide with molecular weight of 800-20000 Da; (2) reacting the collagen peptide with Boc to obtain Boc-collagen peptide, and then activating with HoBt and DIC; (3) coupling Fomc-modified amino acid protected by amino acid with Wang resin by Fmoc solid-phase synthesis method, and then removing Fmoc protecting group; (4) adding the activated Boc-collagen peptide of step (2) to the product of step (3) to perform coupling reaction under N2, washing the resin, and then adding cleavage solution to react, and removing the resin by filtration; obtaining the antibacterial peptide by separating and purifying the filtrate.

3. The preparation method according to claim 2, characterized in that, The method for degrading collagen is selected from enzyme method or physical method; the molecular weight of the collagen peptide is 1000-10000 Da.

4. The production method according to claim 2, characterized by, The enzyme is selected from alkaline protease, trypsin, pepsin, papain, bromelain; the molecular weight of the collagen peptide is 1000-5000 Da.

5. An antibacterial peptide obtained by the preparation method according to any one of claims 1-4.

6. Use of the antibacterial peptide according to claim 5 in the preparation of antibacterial products and antibacterial products prepared thereby.

7. A medicament, a drug, a cosmetic and an additive prepared from the antibacterial peptide according to claim 5.

8. The pharmaceutical, medical, cosmetic and additive according to claim 7, characterized in that, The medicament is selected from dressing and surgical implant material; the drug is selected from adhesive plaster, suppository and gel.

9. The pharmaceutical, medical, cosmetic and additive according to claim 7, characterized in that, The drug is medical or veterinary drug; the additive is food additive, cosmetic additive or feed additive.

Images