Short-chain antibacterial peptide with anti-candida albicans activity and application thereof

Abstract

The application provides a short-chain antibacterial peptide with anti-candida albicans activity and application thereof, and belongs to the application field of peptide antibiotics. The antibacterial peptide is a derivative peptide Pm-2W obtained by modification of an antibacterial peptide from Hainan Rana spinosa; the derivative peptide Pm-2W is obtained by modification of the antibacterial peptide; the Pm-2W has high anti-candida albicans activity; the Pm-2W has anti-candida albicans biofilm activity, is not prone to inducing drug resistance, has high stability and low hemolytic toxicity, and can be applied to preparation of drugs or bacteriostatic agents for treating candida albicans infection, and has good application prospect.

Description

Technical Field

[0001] This invention relates to the field of peptide antibiotic application technology, specifically to a short-chain antimicrobial peptide with anti-Candida albicans activity and its application. Background Technology

[0002] Due to the overuse of antifungal drugs, hospital transmission, and the large-scale use of fungicides in agriculture, pathogenic fungi are rapidly developing resistance to antifungal drugs. Currently available drugs for treating fungal infections include azoles (fluconazole), echinocandins (caspofungin), and polyenes (amphotericidal B). However, their high cytotoxicity necessitates low-dose administration during treatment, leading not only to reduced antifungal efficacy but also to increased fungal resistance. Drug-resistant fungi have been isolated from many patients with fungal infections (Blackman LD, Sutherland TD, De Barro PJ, et al. Addressing a future pandemic: how can non-biological complex drugs prepare us for antimicrobial resistance threats?). Mater Horiz . 2022; 9(8):2076-2096).

[0003] Candida albicans ( Candida albicans Candida albicans can cause both superficial and systemic infections, colonizing various mucosal sites, including the oral cavity, gastrointestinal tract, and genitourinary tract, in symptomatic immunocompromised patients. It can transform its morphology from a symbiotic budding yeast into a pathogenic filamentous form, capable of triggering cell surface adhesion and invasion to damage host cells. If local infections are not treated promptly or adequately, they can spread via the bloodstream, leading to systemic infection and a high mortality rate (Riera FO, Caeiro JP, Angiolini SC, et al. Invasive Candidiasis: Update and Current Challenges in the Management of This Mycosis in South America). Antibiotics (Basel) . 2022; 11(7): 877).

[0004] Antimicrobial peptides (AMPs) are promising antifungal drug candidates with broad-spectrum antimicrobial activity and low resistance. Numerous studies have demonstrated their significant advantages over traditional antibiotics, and their clinical potential is rapidly attracting attention. Most antimicrobial peptides lack specific molecular targets; they act on the cell membrane, causing cell lysis and death through membrane rupture or alteration of membrane permeability. Therefore, they are considered novel alternatives to synthetic antibiotics (Li R, Wang X, Yin K, et al. Fatty acid modification of antimicrobial peptide CGA-N9 and the combats against Candida albicans infection). Biochem Pharmacol .2023; 211: 115535). Summary of the Invention

[0005] To address the aforementioned technical shortcomings, the purpose of this invention is to provide a short-chain antimicrobial peptide and its application. The Pm-2W of this invention exhibits highly efficient anti-Candida albicans activity. The anti-Candida albicans biofilm activity, high stability, low susceptibility to inducing drug resistance, and low hemolytic toxicity of Pm-2W make it a promising candidate for treating Candida albicans infections.

[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0007] A short-chain antimicrobial peptide Pm-2W, characterized in that the amino acid sequence of the antimicrobial peptide is as shown in SEQ ID NO: 1, specifically H-Arg-Lys-Ile-Leu-Arg-Pro-Trp-Trp-Trp-NH2.

[0008] Preferably, the antimicrobial peptide has broad-spectrum antimicrobial activity.

[0009] Preferably, the antimicrobial peptide has the activity of inhibiting the growth of Candida albicans hyphae and resisting biofilm formation.

[0010] Preferably, the antimicrobial peptide has high stability, is not prone to inducing drug resistance, and has low hemolytic toxicity.

[0011] Preferably, the antimicrobial peptide is used in the treatment of Candida albicans infection.

[0012] Preferably, the present invention provides a pharmaceutical composition, characterized in that the active ingredient contains the above-mentioned antimicrobial peptide.

[0013] Preferably, the present invention provides an antibacterial additive, characterized in that the active ingredient contains the above-mentioned antimicrobial peptide.

[0014] The beneficial effects of this invention are as follows: Pm-2W of this invention has highly efficient anti-Candida albicans activity; the anti-Candida albicans biofilm activity, high stability, low susceptibility to inducing drug resistance and low hemolytic toxicity of Pm-2W make it a promising new antibacterial candidate drug for the treatment of Candida albicans infection. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 The growth inhibition curve of the antimicrobial peptide Pm-2W against Candida albicans ( Figure 1 A) Bactericidal kinetics ( Figure 1 B) In vitro induced drug resistance ( Figure 1 C) and cell membrane integrity ( Figure 1 D).

[0017] Figure 2 To observe the effect of antimicrobial peptide Pm-2W on the morphology of Candida albicans using scanning electron microscopy, A: negative control without antimicrobial peptide treatment, B: antimicrobial peptide Pm-2W treatment group.

[0018] Figure 3 The ability of the antimicrobial peptide Pm-2W to inhibit mycelial growth.

[0019] Figure 4 This assay demonstrates the antimicrobial peptide Pm-2W's ability to inhibit Candida albicans biofilm formation. A: Inhibition of biofilm formation; B: Disruption of mature biofilm. * represents the difference compared to the negative control without antimicrobial peptide treatment. P <0.05,** P <0.01, *** P <0.001.

[0020] Figure 5 * indicates the cytotoxicity of the antimicrobial peptide Pm-2W, representing the difference compared to the negative control without antimicrobial peptide treatment. P <0.05,** P <0.01, *** P <0.001.

[0021] Figure 6* indicates the hemolytic toxicity of the antimicrobial peptide Pm-2W, representing the difference compared to the negative control without antimicrobial peptide treatment. P <0.05,** P <0.01, *** P <0.001. Detailed Implementation

[0022] To more clearly illustrate the present invention, the invention will be further described in detail below with reference to embodiments and accompanying drawings. Those skilled in the art should understand that the specific description below is illustrative rather than restrictive and should not be construed as limiting the scope of protection of the present invention.

[0023] The implementation of this invention mainly includes the following steps:

[0024] 1. Derivative peptides were designed using antimicrobial peptides extracted from the spotted-legged tree frog as templates. The physicochemical properties of the peptides were analyzed using the DBAASP and APD3 databases to assist in the design of a derivative peptide, Pm-2W, with enhanced antimicrobial activity. Its amino acid sequence is shown in SEQ ID NO: 1.

[0025] 2. By determining the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), the results showed that the antimicrobial peptide Pm-2W has broad-spectrum antimicrobial activity.

[0026] 3. Fluorescence spectroscopy and scanning electron microscopy showed that the antimicrobial peptide Pm-2W could disrupt the cell membrane integrity of Candida albicans, thereby achieving a bactericidal effect.

[0027] 4. Through 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetraazolium bromide (MTT) experiment and microscopic observation, the results showed that the antimicrobial peptide Pm-2W has inhibitory activity on Candida albicans hyphae growth and anti-biofilm activity.

[0028] 5. Stability experiments, drug resistance induction experiments, and hemolytic toxicity experiments showed that the antimicrobial peptide Pm-2W has high stability, is not prone to inducing drug resistance, and has low hemolytic toxicity, indicating its potential application in the treatment of Candida albicans infections.

[0029] Example 1: Design, synthesis and physicochemical property analysis of antimicrobial peptides

[0030] Previously, we extracted antimicrobial peptides from the spotted-legged tree frog using cDNA library construction and designed derived peptides based on these peptides. The physicochemical properties of the peptides were analyzed using the DBAASP and APD3 databases to aid in the design of a derived peptide with stronger antimicrobial activity. The amino acid sequence of the derived peptide Pm-2W is shown in SEQ ID NO: 1, specifically H-Arg-Lys-Ile-Leu-Arg-Pro-Trp-Trp-Trp-NH2. The peptides were synthesized using an N-9-fluorenylmethoxycarbonyl (Fmoc) solid-phase synthesis method, with C-terminal amidation modification, followed by purification by reversed-phase high-performance liquid chromatography (RP-HPLC) to a purity ≥95%. The antimicrobial peptides were identified by electrospray mass spectrometry (ESMMS). Their physicochemical properties are shown in Table 1 below.

[0031] Table 1. Sequence and physicochemical properties of the antimicrobial peptide Pm-2W

[0032] Antimicrobial peptides amino acid sequence MW AI Charge pI BI GRAVY Pm-2W RKILRPWWW 1339.64 86.67 +3 12.01 2.06 -0.989

[0033] Note: MW: molecular weight; AI: aliphatic index; Charge: charge number; pI: isoelectric point; BI: Boman index, indicating the binding ability of a protein; GRAVY: hydrophilicity / hydrophobicity.

[0034] Example 2: Determination of minimum inhibitory concentration and minimum bactericidal concentration

[0035] Antimicrobial peptide solutions with concentration gradients of 0.4–800 μM were prepared using the two-fold dilution method. 50 μL of each solution was placed in a 96-well cell culture plate, and an equal volume of bacterial culture was added to each well to achieve a final bacterial concentration of 1 × 10⁻⁶. 6 CFU / mL. A positive control containing bacterial culture but no antimicrobial peptide was used, and a negative control containing neither bacterial culture nor antimicrobial peptide was used. After incubation at 37 ℃ for 16-24 hours, the lowest concentration of peptide at which no turbidity appeared at the bottom of the well was visually observed, and this was defined as the minimum inhibitory concentration (MIC). Cultures with MIC or higher concentrations were plated and incubated overnight; the lowest concentration of peptide at which no colonies grew was defined as the minimum bactericidal concentration (MBC). The MICs and MBCs of the antimicrobial peptide Pm-2W against methicillin-resistant Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Candida albicans are shown in Table 2. This peptide exhibits good antimicrobial activity against Gram-positive bacteria, Gram-negative bacteria, and fungi.

[0036] Table 2. MIC and MBC (μM) of the antimicrobial peptide Pm-2W against the tested strains.

[0037] Antimicrobial peptides amino acid sequence MRSA CA-1 CA-2 Pm-2W RKILRPWWW 50 / 100 50 / 50 50 / 100 200 / 200 200 / 200 200 / 200

[0038] Note: MRSA: Methicillin-resistant Staphylococcus aureus (ATCC 43300); E. coli Escherichia coli (ATCC25922); P. aeruginosa Pseudomonas aeruginosa (ATCC 27853); C. albicans Candida albicans (ATCC10231); CA-1 and CA-2: clinically isolated Candida albicans.

[0039] Example 3 Stability Study

[0040] 1. The effect of temperature on the activity of antimicrobial peptides

[0041] The MICs of the antimicrobial peptide against Candida albicans were determined after incubating the samples at 40℃, 70℃, and 100℃ for 30 minutes. Untreated samples were used as negative controls. As shown in Table 3, the MIC values ​​of the antimicrobial peptide Pm-2W remained unchanged after treatment at different temperatures, demonstrating its good thermal stability.

[0042] Table 3. Effect of temperature on the activity of antimicrobial peptides (MIC (μM))

[0043] Antimicrobial peptides room temperature 40℃ 70℃ 100℃ Pm -2W 200 200 200 200

[0044] 2. Effect of pH on antimicrobial peptide activity

[0045] SDB liquid culture media with pH values ​​of 5, 7, and 9 were prepared. The MIC of the samples against Candida albicans was determined using these media. Wells treated with SDB liquid culture medium at pH 5.6 served as negative controls. As shown in Table 4, the antimicrobial peptide Pm-2W maintained its original antimicrobial activity under different pH conditions, indicating its good acid-base tolerance.

[0046] Table 4. Effect of pH on the activity of antimicrobial peptides (MIC (μM))

[0047] Antimicrobial peptides negative control pH 5 pH 7 pH 9 Pm-2W 200 200 200 200

[0048] 3. Effects of serum on antimicrobial peptide activity

[0049] SDB liquid culture media containing 2%, 5%, and 10% fetal bovine serum were prepared. The MIC (micron activity) of the samples against Candida albicans was determined using these media. Untreated wells were used as negative controls. As shown in Table 5, the MIC value of the antimicrobial peptide Pm-2W remained unchanged in the presence of serum, indicating that the peptide exhibited good stability in the presence of serum.

[0050] Table 5. Effect of different serum concentrations on the activity of antimicrobial peptides (MIC (μM))

[0051] Antimicrobial peptides 0 2% serum 5% serum 10% serum Pm-2W 200 200 200 200

[0052] Example 4 Growth Inhibition Curve

[0053] Antimicrobial peptide solutions with concentration gradients of 1 / 8–1×MIC were prepared using a two-fold dilution method. 50 μL of the above solutions were placed in a 96-well cell culture plate, and equal volumes of bacterial culture were added to each well to achieve a final bacterial concentration of 1×10⁻⁶. 6 CFU / mL. A positive control containing bacterial culture but no antimicrobial peptides was used, and a negative control containing neither bacterial culture nor antimicrobial peptides was used. The culture was incubated at 37 ℃ for 24 hours, and the OD600 was measured every hour using a microplate reader to plot a growth inhibition curve. Figure 1 As shown in Figure A, the antimicrobial peptide Pm-2W completely inhibited the growth of Candida albicans within 24 hours at a concentration of 200 μM, and delayed the growth of Candida albicans by 12 hours at a concentration of 100 μM, demonstrating that the peptide has good antibacterial activity.

[0054] Example 5: Sterilization Kinetics

[0055] Candida albicans was cultured to the logarithmic growth phase, and the bacterial culture was adjusted to a concentration of 2 × 10⁻⁶. 6 Prepare CFU / mL for later use. Dilute the antimicrobial peptide using the two-fold dilution method in 5 mL centrifuge tubes. Add 1 mL of diluted bacterial suspension to each tube to achieve a final peptide concentration of 1 / 2–4 × MIC. Incubate at 37°C, collecting samples at 0, 15, 30, 60, 90, 120, and 180 minutes. Dilute to the appropriate concentration with PBS, then plate 50 μL onto agar plates and incubate at 37°C for 24 hours. Count the colonies. SDB without the peptide serves as a negative control. Construct a bactericidal curve by plotting 1g CFU / mL versus time. Figure 1 As shown in B, the antimicrobial peptide Pm-2W can completely kill Candida albicans within 60 minutes at a concentration of 400 μM, and can completely kill Candida albicans within 120 minutes at a concentration of 1×MIC, i.e., 200 μM, proving that the peptide has good bactericidal activity.

[0056] Example 6 In vitro drug resistance induction test

[0057] Candida albicans in its logarithmic growth phase was inoculated into SDB medium containing 1 / 2×MIC antimicrobial peptide and cultured at 37°C with shaking for 24 hours. An appropriate amount of the bacterial culture was then inoculated into fresh SDB medium containing 1 / 2×MIC antimicrobial peptide, and the culture was repeated under the same conditions for 30 days. The MIC of the antimicrobial peptide against Candida albicans was determined every 5 days. Amphotericin B and ciclopirox olamine were used as positive controls, and medium without antimicrobial peptide was used as a negative control. Figure 1As shown in Table C, on day 10, the MIC value of amphotericin B against Candida albicans increased eightfold, from 0.4 μM to 3.2 μM. On day 20, the MIC value of ciclopirox olamine against Candida albicans increased fourfold, from 25 μM to 100 μM, indicating that the sensitivity of Candida albicans to amphotericin B and ciclopirox olamine decreased rapidly, leading to resistance. The MIC value of the antimicrobial peptide Pm-2W against Candida albicans remained unchanged over 30 days, indicating that Pm-2W is unlikely to induce resistance in Candida albicans. On day 30, strains resistant to amphotericin B and ciclopirox olamine were isolated, and the MIC values ​​of Pm-2W against them were determined. The results are shown in Table 6. The antimicrobial activity of Pm-2W against resistant strains was no different from that against sensitive strains, with a MIC value still of 200 μM, indicating that Pm-2W can effectively kill resistant Candida albicans.

[0058] Table 6. Antimicrobial peptide Pm-2W against drug-resistant bacteria C. albicans MIC (μM) for strain type

[0059] Antimicrobial peptides ATCC 10231 Amphotericin B resistant strains Ciclopirox-resistant strains Pm-2W 200 200 200

[0060] Example 7: Detection of Candida albicans cell integrity by fluorescence spectroscopy

[0061] Prepare peptide solutions with final concentrations of 1 / 2–4 × MIC in 96-well plates. Add propidium iodide (PI) to each well with a final concentration of 50 μM and a final concentration of 1 × 10⁻⁶ μM. 8 CFU / mL Candida albicans. Untreated bacterial culture served as a negative control. The culture was continuously incubated at 37°C for 2 hours using a multi-mode microplate reader, with fluorescence intensity measured every 5 minutes. Excitation and emission wavelengths were 584 nm and 620 nm, respectively. Figure 1 As shown in Figure D, the PI fluorescence in the Pm-2W treatment group increased in a concentration- and time-dependent manner at high concentrations. The results indicate that the antimicrobial peptide Pm-2W can disrupt the integrity of the Candida albicans cell membrane.

[0062] Example 8: Scanning electron microscopy observation of the effect of antimicrobial peptide Pm-2W on the morphology of Candida albicans.

[0063] Resuspend the Candida albicans culture in the logarithmic growth phase to a concentration of 2 × 10⁻⁶. 8 CFU / mL, bacterial suspension was mixed with antimicrobial peptide (final concentration 4×MIC) at a 1:1 volume ratio and incubated for 4 hours. Untreated bacterial suspension served as a negative control. Bacteria were collected by centrifugation and washed three times, then fixed with 2.5% glutaraldehyde at 4°C for 4 hours. Bacteria were collected by centrifugation and washed three times. The samples were then dehydrated sequentially with 30%, 45%, 60%, 75%, 90%, and 100% ethanol for 10 minutes each time. Bacteria were then dropped onto aluminum foil, freeze-dried overnight, and vacuum-autoclaved with gold. Bacterial morphology was observed using a scanning electron microscope. Figure 2 As shown, the surface of *Candida albicans* in the blank control group was smooth. The cell membrane integrity of the Pm-2W-treated group was severely disrupted, with contents leaking out, cells shrinking, and losing their intact morphology. These results indicate that the antimicrobial peptide Pm-2W exerts its antimicrobial effect by disrupting the cell membrane integrity of *Candida albicans*.

[0064] Example 9: Observation of the effect of antimicrobial peptide Pm-2W on morphological transformation of Candida albicans using an inverted microscope

[0065] Dilute Candida albicans in the logarithmic growth phase to a concentration of 1×10⁻⁶. 6 CFU / mL was added to peptide solutions with a final concentration of 1 / 8–1×MIC, using SDB medium without antimicrobial peptides as a negative control. The cultures were incubated at 37°C for 2 or 8 hours, and the morphological changes of the bacteria in each group were observed under an inverted microscope. Figure 3 As shown, the antimicrobial peptide Pm-2W can inhibit the transformation of Candida albicans from yeast morphology to hyphal morphology at a concentration of 100-200 μM, and the effect increases in a concentration-dependent manner.

[0066] Example 10: Determination of anti-biofilm activity using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazoleonium bromide (MTT) method

[0067] 1. Inhibits biofilm formation

[0068] Dilute Candida albicans in the logarithmic growth phase to a concentration of 1×10⁻⁶. 6 CFU / mL was added to peptide solutions with a final concentration of 1 / 8–1×MIC, using SDB medium without antimicrobial peptides as a negative control. The mixture was incubated at 37°C for 24 hours, and biomass of the biofilm was measured using the MTT assay. Figure 4 As shown in Figure A, Pm-2W inhibits the formation of Candida albicans biofilm in a dose-dependent manner, with an inhibition rate as high as 73.1% at a concentration of 1 / 8×MIC (25 μM) and almost completely inhibits biofilm formation at a concentration of 200 μM.

[0069] 2. Disruption of mature biofilms

[0070] Dilute Candida albicans in the logarithmic growth phase to a concentration of 1×10⁻⁶. 7 After incubating at CFU / mL at 37°C for 24 hours, a peptide solution with a final concentration of 1 / 4-4×MIC was added, using SDB medium without antimicrobial peptides as a negative control. After incubation at 37°C for 4 hours, the ability of the peptide to disrupt biofilms was detected using the MTT assay. Figure 4As shown in B, Pm-2W disrupts the mature biofilm of Candida albicans in a dose-dependent manner, achieving a clearance rate of up to 88.5% at a concentration of 4×MIC.

[0071] Example 11 Cytotoxicity

[0072] Human immortalized keratinocytes (HaCaT) were cultured in DMEM medium containing 10% fetal bovine serum (FBS), and human umbilical vein endothelial cells (HUVECs) were cultured in DMEM / F12 medium containing 10% FBS. Both cell lines were cultured in a 5% CO2 incubator at 37°C. When the cells reached 80% confluence, they were digested with trypsin and diluted to 5 × 10⁶ cells / mL with culture medium. 4 Cells / mL, add 100 μL to each well of a 96-well plate, and incubate overnight at 37°C with 5% CO2. When cells reach 70-80% confluence, remove the supernatant and add 100 μL of a 2-fold diluted 100 μM sample. Use culture medium without the sample as a blank control. Continue incubation for 4 hours, then restore culture overnight with complete culture medium. Add 10 μL of CCK-8 solution to each well and incubate for 2-4 hours. Measure the absorbance at 450 nm using a microplate reader. The cell viability is calculated as: Cell viability = [(OD2 / 2) / mL] 实验组 -OD 空白组 ) / (OD 对照组 -OD 空白组 )]×100%. For example... Figure 5 As shown, Pm-2W did not exhibit cytotoxicity against HaCaT and HUVEC at concentrations ranging from 3.1 to 100 μM. At a concentration of 1×MIC (200 μM), Pm-2W maintained a cell viability of 96.6% against HaCaT and 90.2% against HUVEC, indicating slight cytotoxicity against HUVEC at this concentration. The half-maximal inhibitory concentration (IC50) of Pm-2W against both cell types was greater than 400 μM.

[0073] Example 12 Hemolytic Toxicity

[0074] Mouse red blood cells were washed three times with PBS until the supernatant was clear, and then diluted to a final concentration of 2 × 10⁻⁶. 8 cells / mL (4%). 300 μL of red blood cell suspension and 300 μL of two-fold diluted sample were mixed and incubated at 37°C for 45 min. 100 μL of the supernatant was collected and transferred to a new 96-well plate. Each sample was tested in triplicate, and absorbance was measured at 570 nm. PBS treatment of cells served as a negative control, and 0.1% Triton X-100 treatment served as a positive control. Figure 6As shown, the hemolysis rate of mouse erythrocytes increased with increasing concentration of the antimicrobial peptide Pm-2W, but the hemolysis rate was only 3.23% at a concentration of 1×MIC and only 7.62% at a high concentration of 800μM, indicating that the antimicrobial peptide Pm-2W has no obvious hemolytic toxicity.

[0075] Obviously, the above embodiments of the present invention are merely examples to illustrate the present invention more clearly, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is impossible to exhaustively list all implementation methods here. Any obvious variations or modifications derived from the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. A short chain antibacterial peptide, characterized in that, The antibacterial peptide is H-Arg-Lys-Ile-Leu-Arg-Pro-Trp-Trp-Trp-NH2.

2. The antimicrobial peptide of claim 1, wherein, The antibacterial peptide has broad-spectrum antimicrobial activity.

3. The antimicrobial peptide of claim 1, wherein, The antibacterial peptide has the activities of inhibiting Candida albicans hypha growth and resisting biofilm.

4. The antimicrobial peptide of claim 1, wherein, The antibacterial peptide has high stability and low hemolytic toxicity.

5. Use of the antibacterial peptide of claim 1 in the preparation of a drug for treating Candida albicans infection diseases.

6. A pharmaceutical composition, characterized by, The effective component contains the antibacterial peptide of claim 1.

7. A bacteriostatic additive characterized in that, The effective component contains the antibacterial peptide of claim 1.

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