Antimicrobial agents and antimicrobial methods

Abstract

To provide simple and easy means and methods capable of imparting antimicrobial activity to materials such as fibers.SOLUTION: An antimicrobial agent includes a self-assembling peptide with a full length of 4-30 amino acids and metal ions binding to the peptide. The self-assembling peptide includes one or more amino acids bindable to the metal ions, one or more basic amino acids, and two or more nonpolar amino acids or neutral amino acids. Positions of the amino acids bindable to the metal ions from a C terminus of the self-assembling peptide are all expressed as 2 m+a-th; and positions of basic amino acids from the C terminus of the self-assembling peptide are all expressed as 2n+1+a-th, where m is an integer of 0-15, n is an integer of 0-14, and a is 0 or 1. The metal ions include a metal ion having antimicrobial activity.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a self-assembling peptide capable of attaching metal ions to the surface of a material, and also to an antimicrobial agent and material, and a method for making a material antimicrobial, using the self-assembling peptide and metal ions. [Background technology]

[0002] Many metal elements are known as substances having antimicrobial activity against microorganisms such as viruses and bacteria. As described in Patent Document 1, among them, metal elements such as silver, copper, and zinc have long been known as metal elements that are relatively less harmful to the human body. For this reason, many antimicrobial compositions containing these metal elements in the form of atoms or ions as active ingredients have been developed.

[0003] On the other hand, it is known that heavy metal ions are oxidized by irradiation with light, exposure to heat, or reaction with chemical substances such as dyes, and mainly become heavy metal atoms, which then aggregate and precipitate, causing discoloration and dullness. This discoloration and dullness are not only undesirable because they impair the appearance of the product, but also because the antimicrobial activity caused by the heavy metal ions is localized to the precipitated area. In particular, discoloration of textile products is difficult to eliminate, and in the development of antimicrobial compositions containing heavy metals for textile products, it has been considered particularly important to devise ways to suppress discoloration caused by heavy metals.

[0004] To date, several methods have been developed to achieve this. The most well-known method is to use special complexes such as zeolite (for example, Patent Document 2). Although this method significantly improves discoloration caused by light irradiation, it is difficult to prepare and obtain the complexes, and there is a problem in that the production costs are high. In addition, a method is known in which the fibers themselves are processed to impart antimicrobial activity. However, in addition to the production cost problem, these antimicrobial fibers have the problem that once the antimicrobial activity of the fibers is lost, it is difficult to impart antimicrobial activity again. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] JP 2017-133137 A [Patent Document 2] JP 2007-223995 A Summary of the Invention [Problem to be solved by the invention]

[0006] There has been a demand for simple means and methods for stably dispersing heavy metal ions uniformly in response to stimuli such as light irradiation and imparting antimicrobial activity to materials such as fibers. In particular, there has been a demand for the development of a stable, inexpensive antimicrobial agent that can be repeatedly applied to existing materials. [Means for solving the problem]

[0007] The present inventor attempted to solve the above problems using his own technology related to self-assembling peptides. This self-assembling peptide technology has been used as a dispersant for metal particles in liquids and as a base with excellent dielectric properties for making metal particle aggregates take a specific shape. It was found that when this self-assembled peptide and metal ions are applied to a material, the metal ions are uniformly held on the surface of the material via the self-assembling peptide. It was also found that the metal ions are stable and do not precipitate even after irradiation with light, discoloration is suppressed, and the metal ions held on the surface of the material exhibit strong antimicrobial activity. Based on the above findings, the present invention has been completed.

[0008] For example, the present invention encompasses the following embodiments: [1] An antimicrobial agent comprising a self-assembling peptide having a total length of 4 to 30 amino acids and a metal ion bound to the peptide, the self-assembling peptide comprises at least one amino acid capable of binding to a metal ion, at least one basic amino acid, and at least two nonpolar or neutral amino acids; the positions of the amino acids capable of binding to the metal ion from the C-terminus of the self-assembling peptide are all expressed as 2m+a; the positions of the basic amino acids from the C-terminus of the self-assembling peptide are all expressed as 2n+1+a; The m is an integer of 0 to 15, The n is an integer of 0 to 14, The a is 0 or 1, The antimicrobial agent, wherein the metal ion comprises a metal ion having antimicrobial activity. [2] The antimicrobial agent described in [1], wherein the self-assembling peptide forms a β-sheet structure. [3] The antimicrobial agent according to [1] or [2], wherein the metal ions are applied at a concentration that inhibits aggregation of the metal ions compared to when only the metal ions are applied. [4] The antimicrobial agent according to any one of [1] to [3], wherein the metal ion having antimicrobial activity is applied at a concentration capable of exerting antimicrobial activity. [5] The antimicrobial agent according to any one of [1] to [4], wherein the molar concentration of the amino acid capable of binding to the metal ion is applied at a molar concentration equivalent to that of the metal ion having antimicrobial activity. [6] The antimicrobial agent according to any one of [1] to [5], wherein the total length of the self-assembling peptide is 4 to 15 amino acids. [7] The antimicrobial agent according to any one of [1] to [6], wherein the self-assembling peptide has a protecting group at its terminal. [8] The antimicrobial agent according to [7], wherein the protecting group is a carbamate-based protecting group. [9] The antimicrobial agent according to [8], wherein the carbamate-based protecting group is an Fmoc group or a Boc group.

[0009]

[10] The antimicrobial agent according to any one of [1] to [9], wherein all of the amino acids capable of binding to metal ions are located at odd-numbered positions from the C-terminus of the self-assembling peptide.

[11] The antimicrobial agent according to any one of [1] to

[10] , wherein the amino acid capable of binding to the metal ion includes at least one type of amino acid selected from the group consisting of cysteine, histidine, lysine, aspartic acid, and glutamic acid.

[12] The antimicrobial agent according to any one of [1] to

[11] , wherein the basic amino acid includes lysine. [12-2] The antimicrobial agent according to any one of [1] to

[11] , wherein the nonpolar amino acid or neutral amino acid includes at least one amino acid selected from the group consisting of valine, isoleucine, leucine, tyrosine, tryptophan, glutamine and serine. [12-3] The antimicrobial agent according to any one of [1] to

[12] , wherein the self-assembling peptide contains at least two nonpolar amino acids or neutral amino acids between the amino acid capable of binding to the metal ion and the basic amino acid.

[13] The antimicrobial agent according to any one of [1] to

[12] , wherein the self-assembling peptide comprises a sequence of at least two consecutive valines.

[14] The antimicrobial agent according to any one of [1] to

[13] , wherein the self-assembling peptide comprises the sequence set forth in Val-Lys-Val-Val-Cys (SEQ ID NO: 9).

[15] The antimicrobial agent according to any one of [1] to

[14] , wherein the metal ion comprises at least one type of metal ion selected from the group consisting of silver ions, copper ions, zinc ions, cobalt ions, nickel ions, lead ions, aluminum ions, mercury ions, bismuth ions, cadmium ions, and chromium ions.

[16] The antimicrobial agent according to any one of [1] to

[15] , which is applied to a surface of a material.

[17] The antimicrobial agent according to any one of [1] to

[16] , wherein the material is an organic material.

[18] The antimicrobial agent according to

[17] , wherein the organic material is a fibrous material.

[19] The antimicrobial agent according to any one of [1] to

[18] , wherein the microorganism is a virus, a bacterium or a fungus.

[20] An antimicrobial composition comprising the antimicrobial agent according to any one of [1] to

[19] as an active ingredient.

[0010]

[21] An antimicrobial kit comprising a self-assembling peptide having a total length of 4 to 30 amino acids and a metal ion having antimicrobial activity, the self-assembling peptide comprises at least one amino acid capable of binding to a metal ion, at least one basic amino acid, and at least two nonpolar or neutral amino acids; the positions of the amino acids capable of binding to the metal ion from the C-terminus of the self-assembling peptide are all expressed as 2m+a; the positions of the basic amino acids from the C-terminus of the self-assembling peptide are all expressed as 2n+1+a; The m is an integer of 0 to 15, The n is an integer of 0 to 14, An antimicrobial kit, wherein a is 0 or 1. [21-2] The antimicrobial kit described in

[21] , wherein the metal ion includes at least one type of metal ion selected from the group consisting of silver ions, copper ions, zinc ions, cobalt ions, nickel ions, lead ions, aluminum ions, mercury ions, bismuth ions, cadmium ions and chromium ions. [21-3] An antimicrobial kit according to

[21] , which is applied to a surface of a material.

[22] The antimicrobial kit described in

[21] , wherein the self-assembling peptide forms a β-sheet structure.

[23] The antimicrobial kit according to

[21] or

[22] , wherein the metal ion is applied at a concentration that inhibits aggregation of the metal ion compared to application of the metal ion alone.

[24] The antimicrobial kit according to any one of

[21] to

[23] , wherein the metal ion having antimicrobial activity is applied at a concentration capable of exerting antimicrobial activity.

[0011]

[25] An antimicrobial material comprising a self-assembling peptide having a total length of 4 to 30 amino acids and a metal ion bound to the peptide, the self-assembling peptide comprises at least one amino acid capable of binding to a metal ion, at least one basic amino acid, and at least two nonpolar or neutral amino acids; the positions of the amino acids capable of binding to the metal ion from the C-terminus of the self-assembling peptide are all expressed as 2m+a; the positions of the basic amino acids from the C-terminus of the self-assembling peptide are all expressed as 2n+1+a; The m is an integer of 0 to 15, The n is an integer of 0 to 14, The a is 0 or 1, the self-assembling peptide forms a β-sheet structure; the metal ions include metal ions having antimicrobial activity; The metal ions are arranged on the β-sheet structure. [25-1] The antimicrobial material described in

[25] , wherein the material is a material made of fiber.

[26] The antimicrobial material according to

[25] , in which aggregation of the metal ions is suppressed compared to the case where only the metal ions are applied.

[0012]

[27] A method for making a material antimicrobial, comprising the steps of: adding a self-assembling peptide solution to the material; adding metal ions having antimicrobial activity to the material; and Drying the material Including, the total length of the self-assembling peptide is 4 to 30 amino acids; the self-assembling peptide comprises at least one amino acid capable of binding to a metal ion, at least one basic amino acid, and at least two nonpolar or neutral amino acids; the positions of the amino acids capable of binding to the metal ion from the C-terminus of the self-assembling peptide are all expressed as 2m+a; the positions of the basic amino acids from the C-terminus of the self-assembling peptide are all expressed as 2n+1+a; The m is an integer of 0 to 15, The n is an integer of 0 to 14, The method, wherein a is 0 or 1.

[28] The antimicrobial method described in

[27] , wherein the self-assembling peptide forms a β-sheet structure.

[29] The antimicrobial method described in

[27] or

[28] , wherein the metal ions are applied at a concentration that inhibits aggregation of the metal ions compared to when only the metal ions are applied.

[30] The antimicrobial method according to any one of

[27] to

[29] , wherein the step of adding metal ions to the material is carried out simultaneously with or after the step of adding a self-assembling peptide to the material.

[31] The antimicrobial method according to any one of

[27] to

[30] , wherein the step of adding metal ions to the material is carried out while the material is in a wet state. [31-1] The antimicrobial method described in any of

[27] to

[30] , wherein the step of adding a self-assembling peptide solution to the material and the step of adding a metal ion to the material are carried out after mixing the self-assembling peptide solution with the metal ion.

[32] The antimicrobial method according to any one of

[27] to

[30] , wherein the step of adding metal ions to the material is carried out after the step of adding a self-assembling peptide solution to the material and before the self-assembling peptide solution dries.

[0013]

[33] A method for producing an antimicrobial material, comprising the steps of: adding a self-assembling peptide solution to the material; adding metal ions having antimicrobial activity to the material; and Drying the material Including, the total length of the self-assembling peptide is 4 to 30 amino acids; the self-assembling peptide comprises at least one amino acid capable of binding to a metal ion, at least one basic amino acid, and at least two nonpolar or neutral amino acids; the positions of the amino acids capable of binding to the metal ion from the C-terminus of the self-assembling peptide are all expressed as 2m+a; the positions of the basic amino acids from the C-terminus of the self-assembling peptide are all expressed as 2n+1+a; The m is an integer of 0 to 15, The n is an integer of 0 to 14, The method, wherein a is 0 or 1.

[0014]

[34] A composition for attaching metal ions, comprising a self-assembling peptide having a total length of 4 to 30 amino acids, the self-assembling peptide comprises at least one amino acid capable of binding to a metal ion, at least one basic amino acid, and at least two nonpolar or neutral amino acids; the positions of the amino acids capable of binding to the metal ion from the C-terminus of the self-assembling peptide are all expressed as 2m+a; the positions of the basic amino acids from the C-terminus of the self-assembling peptide are all expressed as 2n+1+a; The m is an integer of 0 to 15, The n is an integer of 0 to 14, The a is 0 or 1, A composition applied to organic materials.

[35] The composition for attaching metal ions according to

[34] , wherein the self-assembling peptide has the ability to form a β-sheet structure.

[36] A composition for attaching metal ions described in

[34] or

[35] , wherein the self-assembling peptide has a structure such that, when applied to the organic material, amino acids capable of binding to the metal ions are present in a dispersed state.

[37] The composition for adsorbing metal ions according to any one of

[34] to

[36] , which is applied to a surface of the organic material.

[38] The composition for attaching metal ions according to any one of

[34] to

[37] , wherein the metal ions include metal ions having antimicrobial activity.

[0015]

[39] A self-assembling peptide having a metal ion bound thereto, the total length of the self-assembling peptide is 4 to 30 amino acids; the self-assembling peptide comprises at least one amino acid capable of binding to a metal ion, at least one basic amino acid, and at least two nonpolar or neutral amino acids; the positions of the amino acids capable of binding to the metal ion from the C-terminus of the self-assembling peptide are all expressed as 2m+a; the positions of the basic amino acids from the C-terminus of the self-assembling peptide are all expressed as 2n+1+a; The m is an integer of 0 to 15, The n is an integer of 0 to 14, The self-assembling peptide, wherein a is 0 or 1. Effect of the Invention

[0016] The antimicrobial agent, antimicrobial kit, and antimicrobial method according to the present invention can easily render a material antimicrobial in a state in which it is less likely to discolor due to stimuli such as light, etc. Furthermore, the antimicrobial material according to the present invention can inhibit the growth of microorganisms on the material. [Brief description of the drawings]

[0017] [Figure 1] The infrared transmission spectrum of the synthesized self-assembling peptide by Fourier transform infrared spectroscopy (FT-IR) is shown. The dashed rectangle indicates the wave number region showing the formation of an α-helix structure, and the solid rectangle indicates the wave number region showing the formation of a β-sheet structure. [Diagram 2] Transmission electron microscope (TEM) images of the self-assembling peptide-silver complex. A: Low magnification image, B: High magnification image. [Diagram 3]Scanning electron microscope (SEM) images of the surface of silver-doped silk fabric, A: without self-assembling peptide, B: with self-assembling peptide. [Figure 4] The images are taken by an energy dispersive X-ray (EDX) analyzer of the surface of silver-added silk fabric. A: elemental sulfur (S) without the self-assembling peptide, B: elemental sulfur (S) with the self-assembling peptide, C: elemental silver (Ag) without the self-assembling peptide, D: elemental silver (Ag) with the self-assembling peptide. [Diagram 5] Photographs of silver-doped silk fabric are shown: A: without self-assembling peptide, B: with self-assembling peptide. [Figure 6] Photographs of bacterial culture media obtained in the evaluation of the bactericidal activity of silver-added silk fabric are shown in Fig. 1. A: without the self-assembling peptide, B: with the self-assembling peptide. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] The present invention will be described in detail below. The present invention particularly relates to an antimicrobial agent, an antimicrobial kit, an antimicrobial material, and an antimicrobial method that utilize the antimicrobial activity of a metal ion. In the present invention, when a metal ion is applied to a material, a self-assembling peptide is used, whereby the metal ion can be dispersed on the surface of the material and stably held thereon, and as a result, the properties of the metal ion, such as antimicrobial activity, can be imparted to the material in a state in which it is less likely to discolor due to stimuli such as light.

[0019] In the present invention, "antimicrobial" or "antimicrobial activity" means inhibiting the growth, activity, growth or proliferation of microorganisms, and in some cases inhibiting the adhesion of microorganisms and inhibiting phenomena resulting from the growth of microorganisms. Therefore, antimicrobial activity includes antiviral activity, antibacterial activity, sterilization activity, bacteriostatic activity, disinfection activity, antifungal activity and antifungal activity. In the present invention, it is not necessary for microorganisms to be completely killed by antimicrobial activity. In addition, phenomena resulting from the growth of microorganisms include, for example, the generation of harmful substances, the generation of odors, deterioration of materials, decay, etc. Therefore, antimicrobial activity also includes deodorizing activity, preservative activity, and deterioration resistance activity. In the present invention, when an object "has antimicrobial activity", it means that the object has antimicrobial activity. Therefore, regardless of whether the object is intended to exhibit antimicrobial activity or not, it is sufficient that the object exhibits antimicrobial activity when exposed to microorganisms.

[0020] Microorganisms include, for example, viruses (e.g., pathogenic viruses, phages, etc.), prokaryotes (e.g., bacteria and archaea, etc.), unicellular eukaryotes (e.g., fungi and protozoa, etc.), cell colonies (e.g., hyphae-forming fungi and lichens, etc.), and parts thereof (e.g., spores, etc.).

[0021] Preferred examples of bacteria include gram-positive bacteria such as Staphylococcus bacteria including Staphylococcus aureus, and gram-negative bacteria such as Escherichia coli. Preferred examples of fungi include yeast fungi and mold fungi.

[0022] In the present invention, a self-assembling peptide is used. Here, "self-assembly" refers to autonomously forming a three-dimensional structure over time, and "self-assembling peptide" refers to a peptide that self-assembles under specific conditions. The specific conditions include the environment in which self-assembly occurs, the reaction time, and, in some cases, the stimulus required for self-assembly.

[0023] The environment may be in a liquid, and the properties of the liquid, such as physical properties (viscosity, melting point, boiling point, etc.) and chemical properties (pH, composition, polarity, etc.), are not particularly limited as long as they do not adversely affect the self-organization and the stability of the formed structure. Specific examples of preferred liquids include water, lower alcohols (e.g., having 1 to 4 carbon atoms) such as methanol, ethanol, propanol, and butanol, lower alkyl esters such as ethyl acetate, glycols such as ethylene glycol, butylene glycol, propylene glycol, and glycerin, other polar solvents such as acetone and acetic acid, mixtures of two or more solvents selected from these, and solutions containing other solutes.

[0024] The reaction time is not particularly limited. It is preferably a time sufficient for the peptide to self-assemble. Self-assembly only needs to occur before the self-assembling peptide is applied to a material, and does not need to be completed before contact with a metal ion having antimicrobial activity as described below. The formation of a three-dimensional structure can be analyzed precisely by observation with a transmission electron microscope or acquisition of an infrared transmission spectrum, but may also be judged with the naked eye by an increase in the viscosity of the liquid. The specific reaction time may be, for example, 5 days or more, 6 days or more, 7 days or more, 8 days or more, 9 days or more, or 10 days or more.

[0025] Optionally, the progress of self-assembly may be regulated by applying a specific stimulus, including, but not limited to, physical stimuli, such as thermodynamic stimuli such as temperature, and mechanical stimuli, such as mixing, chemical stimuli, such as changes in pH and addition of specific substances, and combinations thereof.

[0026] The three-dimensional structure formed by the self-assembling peptide is not limited as long as it is a higher-order structure, but is preferably a nearly planar structure, and more preferably a structure including a β-sheet structure. A β-sheet structure is formed by forming hydrogen bonds between two peptide chains that are parallel and close to each other mainly due to hydrophobic interactions, between the NH part of the main chain of one peptide chain and the C=O part of the main chain of the other peptide chain. The above-mentioned two peptide chains may belong to the same molecule of peptides linked by a turn structure or a loop structure, but the β-sheet structure is preferably formed between molecules. The β-sheet structure formed by the self-assembling peptide may include either a parallel β-sheet structure or an antiparallel β-sheet structure, or may be a combination thereof. The formed β-sheet structure may have folds as a whole, or a plurality of β-sheet structures may be stacked in layers.

[0027] The three-dimensional structure formed by the self-assembling peptide does not need to be a β-sheet structure in its entirety, and may be, for example, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more of the structure is occupied by a β-sheet structure. In addition, it is not necessary for all the self-assembling peptides to form a single three-dimensional structure, and they may be an aggregate of several three-dimensional structures, or there may be a peptide that exists alone. For example, it is sufficient that 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more of the self-assembling peptide contributes to the formation of a three-dimensional structure.

[0028] A self-assembling peptide is characterized in that when it forms a three-dimensional structure, metal ions are dispersed on its surface and can bind thereto. That is, the metal ions are arranged on the β-sheet structure of the self-assembling peptide. Therefore, when the self-assembling peptide is applied to a material, amino acids capable of binding to metal ions are dispersed on the β-sheet structure of the self-assembling peptide in the material.

[0029] The self-assembling peptide is a peptide with a total length of 4 to 30 amino acids. For example, the upper limit is 30 amino acids or less, 25 amino acids or less, 20 amino acids or less, 19 amino acids or less, 18 amino acids or less, 17 amino acids or less, 16 amino acids or less, 15 amino acids or less, 14 amino acids or less, 13 amino acids or less, 12 amino acids or less, 11 amino acids or less, 10 amino acids or less, 9 amino acids or less, 8 amino acids or less, or 7 amino acids or less, and the lower limit is 4 amino acids or more, or 5 amino acids or more. In a preferred embodiment, the self-assembling peptide is a peptide with a total length of 4 to 15 amino acids.

[0030] As the amino acids constituting the self-assembling peptide, 20 kinds of standard amino acids and their stereoisomers can be used, but modified amino acids in which any functional group has been modified to the extent that self-assembly is not inhibited may also be included. The modified amino acids may be natural or artificial. Modifications of the peptide include, but are not limited to, acetylation of the amino group at the N-terminus and amidation of the carboxyl group at the C-terminus. In order not to inhibit the formation of a β-sheet structure, it is preferable that the NH and C=O parts of the main chain are not modified or have the same charge at the same position after modification.

[0031] The termini of the self-assembling peptide may be bound to a label, modification or protecting group, sugar compound, lipid compound, or other compound that is useful in processes such as detection, synthesis, or purification.

[0032] The self-assembling peptides contain at least one amino acid capable of binding to a metal ion, at least one basic amino acid, and at least two non-polar or neutral amino acids in a specific arrangement.

[0033] An amino acid capable of binding to a metal ion refers to an amino acid capable of binding to a metal (preferably a metal having antimicrobial activity), its ion and / or metal salt, and examples thereof include cysteine ​​(Cys / C), histidine (His / H), aspartic acid (Asp / D), glutamic acid (Glu / E), and lysine (Lys / K), etc. Whether or not an amino acid is capable of binding to a metal ion is known in the art, and a person skilled in the art can confirm this using a conventional method.

[0034] Basic amino acids include, for example, lysine (Lys / K), arginine (Arg / R) and histidine (His / H). In a preferred embodiment, the basic amino acid comprises lysine. It is preferable that the basic amino acid is different from the amino acid capable of binding to a metal ion.

[0035] Examples of non-polar or neutral amino acids include asparagine (Asn / N), serine (Ser / S), glutamine (Gln / Q), threonine (Thr / T), glycine (Gly / G), tyrosine (Tyr / Y), tryptophan (Trp / W), methionine (Met / M), proline (Pro / P), phenylalanine (Phe / F), alanine (Ala / A), valine (Val / V), leucine (Leu / L) and isoleucine (Ile / I). In a preferred embodiment, the non-polar or neutral amino acid includes at least one amino acid selected from the group consisting of valine, isoleucine, leucine, tyrosine, tryptophan, glutamine and serine.

[0036] The particular configuration is determined by optional conditions that favor conformation, particularly including β-sheet conformation, and conditions for binding both to the substrate and to the metal ion. In order to facilitate the formation of a structure including a β-sheet structure, it is preferable to contain a nonpolar amino acid or a neutral amino acid between the amino acid capable of binding to a metal ion and the basic amino acid. The number of nonpolar amino acids or neutral amino acids contained is not particularly limited, but is preferably at least 2, for example, 2 to 28, 2 to 26, 2 to 24, 2 to 22, 2 to 20, 2 to 18, 2 to 16, 2 to 14, 2 to 12, 2 to 10, 2 to 8, 2 to 6, or 2 to 4. The number of amino acids capable of binding to a metal ion and basic amino acids contained in one molecule of the self-assembling peptide is not limited.

[0037] Conditions for binding both the material and the metal ion are, for example: The positions of the amino acids capable of binding to metal ions from the C-terminus of the self-assembling peptide are all expressed as 2m+a. The positions of basic amino acids from the C-terminus of the self-assembling peptide are all expressed as 2n+1+a, m is an integer of 0 to 15 (provided that when a is 0, m is an integer of 1 to 15, and when a is 1, m is an integer of 0 to 14); n is an integer from 0 to 14; a is 0 or 1. By satisfying the above conditions, when the positions of the amino acids capable of binding to metal ions from the C-terminus of the self-assembling peptide are all even-numbered, the positions of the basic amino acids from the C-terminus of the self-assembling peptide are odd-numbered. Conversely, when the positions of the amino acids capable of binding to metal ions are all odd-numbered, the positions of the basic amino acids are all even-numbered.

[0038] This arrangement exposes the side chains of amino acids capable of binding to metal ions on one side of the formed β-sheet structure, and the side chains of basic amino acids on the other side, making the β-sheet structure formed by the self-assembling peptide capable of binding to metal ions on one side and to a negatively charged material on the other side.

[0039] Those skilled in the art can routinely determine whether a peptide is a self-assembling peptide, for example, by observing the prepared peptide with a transmission electron microscope or acquiring an infrared transmission spectrum to determine whether the peptide forms a structure including a β-sheet structure.

[0040] In a specific embodiment, the self-assembling peptide is, for example, composed of a sequence in which an even number of consecutive nonpolar or neutral amino acids are sandwiched between basic amino acids and amino acids capable of binding to metal ions. Thus, the minimum peptide unit preferred as a self-assembling peptide is, for example, the following sequence: Xaa-Yaa-Zaa (SEQ ID NO: 1), or The following sequence with the basic amino acids and the amino acids capable of binding to metal ions in reverse position: Zaa-Yaa-Xaa (SEQ ID NO: 2) and In the above formula, Xaa is Lys, Arg or His (basic amino acid); Yaa is independently at least two (but an even number of) amino acids selected from the group consisting of Asn, Ser, Gln, Thr, Gly, Tyr, Trp, Met, Pro, Phe, Ala, Val, Leu, and Ile (non-polar or neutral amino acids); Zaa is Cys, His, Asp, Glu or Lys (an amino acid capable of binding to a metal ion).

[0041] The number of nonpolar or neutral amino acids sandwiched between the basic amino acids and the amino acids capable of binding to metal ions is not limited as long as it is an even number. Therefore, the amino acid sequence of the self-assembling peptide used in the present invention contains one or more of the following sequences consecutively, partially consecutively, or separated: Yaa1-Yaa2 (In the formula, Yaa1 and Yaa2 are independently Asn, Ser, Gln, Thr, Gly, Tyr, Trp, Met, Pro, Phe, Ala, Val, Leu, or Ile.)

[0042] Preferably, the self-assembling peptide contains at least one amino acid sequence of Val-Val. A preferred minimum peptide unit containing this amino acid sequence is, for example, the following sequence: Xaa-Val-Val-Zaa (SEQ ID NO: 3), or The following sequence with the basic amino acids and the amino acids capable of binding to metal ions in reverse position: Zaa-Val-Val-Xaa (SEQ ID NO: 4) [During the ceremony, Xaa is Lys, Arg or His (basic amino acid); Zaa is Cys, His, Asp, Glu or Lys (an amino acid capable of binding to a metal ion). It is.

[0043] More specifically, the self-assembling peptide includes a sequence such as Lys-Val-Val-Cys (SEQ ID NO: 5) or Cys-Val-Val-Lys (SEQ ID NO: 6).

[0044] The self-assembling peptide may also comprise one or more optional further amino acids at its N-terminus and / or C-terminus. Preferably, the further amino acids at the termini of the peptide are non-polar or neutral amino acids.

[0045] Preferably, the additional amino acid at the end of the peptide is a valine, and the self-assembling peptide may have, for example, the following sequence: Val-Xaa-Yaa-Yaa-Zaa (SEQ ID NO: 7) [During the ceremony, Xaa is Lys, Arg or His; Yaa is independently Asn, Ser, Gln, Thr, Gly, Tyr, Trp, Met, Pro, Phe, Ala, Val, Leu, or Ile; Zaa is Cys, His, Asp, Glu or Lys. The self-assembling peptide also includes, for example, the following sequence, in which the nonpolar or basic amino acid within the peptide is Val: Val-Xaa-Val-Val-Zaa (SEQ ID NO: 8) [During the ceremony, Xaa is Lys, Arg or His; Zaa is Cys, His, Asp, Glu or Lys.

[0046] A specific sequence that can be used is, for example, Val-Lys-Val-Val-Cys (SEQ ID NO: 9).

[0047] Self-assembling peptides can be synthesized using peptide synthesis methods well known in the art, including, but not limited to, chemical synthesis and genetic engineering synthesis.

[0048] Chemical synthesis methods that can be used in the present invention are well known in the art, but the following is an example of a general method outlined below. First, amino acids in which functional groups other than the amino and carboxyl groups to be bonded are protected by protective groups are prepared, and a peptide bond formation reaction is carried out between the amino and carboxyl groups of each amino acid. Next, the N-terminus of the resulting peptide is deprotected, and a peptide bond is formed between the carboxyl group of the next amino acid. By carrying out this reaction in succession, a polypeptide in which the functional groups of the side chains are protected is synthesized. Finally, all protecting groups are removed to complete the peptide synthesis.

[0049] Any known protecting group can be used as the protecting group. In addition, since the protecting group can have a positive effect on self-assembly, it is not necessary to completely remove the protecting group when the peptide is used as a self-assembling peptide.

[0050] Examples of the protecting group for the amino group include carbamate-based protecting groups such as Boc group, Fmoc group, methoxycarbonyl group, ethoxycarbonyl group, benzyloxycarbonyl (Z or Cbz) group, 2,2,2-trichloroethoxycarbonyl (Troc) group, or allyloxycarbonyl (Alloc) group, sulfonyl-based protecting groups such as methanesulfonyl group, ethanesulfonyl group, benzenesulfonyl group, tosyl group, or nitrobenzenesulfonyl group, alkylcarbonyl-based protecting groups such as acetyl group, ethylcarbonyl group, trifluoroacetyl group, or benzoyl group, arylcarbonyl-based protecting groups, or imide-based protecting groups such as phthaloyl group, etc. A carbamate-based protecting group is preferably used, and more specifically, a Boc group or Fmoc group is used.

[0051] Furthermore, when protection of a carboxyl group is necessary (for example, when carrying out liquid phase peptide synthesis), the carboxyl group can be protected by, for example, alkyl esterification (using, for example, methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl, or the like), aralkyl esterification (using, for example, benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl ester, or the like), phenacyl esterification, benzyloxycarbonyl hydrazide formation, t-butoxycarbonyl hydrazide formation, trityl hydrazide formation, or the like.

[0052] The self-assembling peptide can also be synthesized by genetic engineering. Examples of the genetic engineering synthesis method include synthesis by gene recombination. Synthesis by recombinant gene synthesis methods that can be used in the present invention are well known in the art, but the following is an example of a general method outline. First, a nucleic acid sequence encoding the peptide of interest is designed, and a DNA fragment containing the sequence is inserted into an appropriate expression vector. This is then introduced into an appropriate host cell, and the cell is cultured to express the peptide of interest. The peptide of interest can then be recovered from the inside of the host cell or from the extracellular fluid. Such expression of the peptide of interest in a host cell and purification of the peptide of interest from the cell are routinely performed in the art, and can be performed appropriately by a person skilled in the art.

[0053] The above-mentioned self-assembling peptides have the property of being able to disperse metal ions and stably retain them on the surface of a material. Such metal ions are not particularly limited as long as the amino acid capable of binding to the metal ion contained in the self-assembling peptide can bind thereto. The metal ion to be used can be selected according to the properties to be imparted to the material. For example, when antimicrobial activity is to be imparted to the material, a metal ion having antimicrobial activity is used.

[0054] Metal ions having antimicrobial activity are well known in the art. The metal ions having antimicrobial activity used in the present invention are not particularly limited, and include, for example, one or more metal ions selected from the group consisting of heavy metal ions such as silver ions, copper ions, zinc ions, cobalt ions, nickel ions, lead ions, mercury ions, bismuth ions, cadmium ions, chromium ions, thallium ions, molybdenum ions, zirconium ions, palladium ions, gold ions, platinum ions, manganese ions, and tungsten ions, and other metal ions such as aluminum ions. Preferably, one or more metal ions are selected from the group consisting of silver ions, copper ions, zinc ions, cobalt ions, nickel ions, lead ions, aluminum ions, mercury ions, bismuth ions, cadmium ions, and chromium ions. The anion paired with the metal ion is not limited as long as it is soluble in the liquid. For example, nitrate ions, tetrafluoroborate ions, trifluoromethanesulfonate ions, behenate ions, sulfate ions, acetate ions, perchlorate ions, chlorate ions, and chloride ions can be mentioned.

[0055] In the present invention, when applied to a material, the metal ion is preferably in the form of a solution, but may be provided in the form of a salt or in the form of a solution in which the salt is dissolved. Examples of salts having antimicrobial activity that are preferably used in the present invention include silver nitrate, silver sulfate, silver acetate, silver perchlorate, silver chlorate, diammine silver nitrate, diammine silver sulfate, silver tetrafluoroborate, silver trifluoromethanesulfonate, silver behenate, copper (II) nitrate, copper (I) sulfate, copper (II) sulfate, copper (II) chloride, copper perchlorate, copper acetate, potassium tetracyanocuprate, zinc nitrate, zinc sulfate, zinc acetate, zinc chloride, zinc perchlorate, zinc thiocyanate, copper nitr ... Examples of the salt include mercury (II), cobalt (II) sulfate, cobalt (II) chloride, nickel (II) nitrate, nickel (II) sulfate, nickel (II) chloride, lead (II) nitrate, lead (II) acetate, lead (II) chloride, aluminum nitrate, aluminum sulfate, aluminum chloride, mercury perchlorate, cadmium perchlorate, cadmium sulfate, cadmium nitrate, cadmium acetate, chromium perchlorate, chromium sulfate, ammonium chromium sulfate, chromium nitrate, and hydrates thereof. The liquid used to dissolve the salt is not limited as long as it can dissolve metal ions having antimicrobial activity. Preferred liquids are the same as those described above for the self-assembling peptide.

[0056] The self-assembling peptide may be provided in a state bound to the metal ion, or may be provided separately. For example, the self-assembling peptide and the metal ion may be provided separately, and when applied to a material as described below, the two may be brought into contact with each other so as to bind to each other.

[0057] In one aspect, the present invention provides self-assembling peptides having metal ions bound thereto.

[0058] In another aspect, the present invention provides an antimicrobial agent comprising a self-assembling peptide and a metal ion having antimicrobial activity bound to the self-assembling peptide.

[0059] In the present invention, the self-assembling peptide and the metal ion are bound to each other. In this specification, bound and bound to each other include a state in which the self-assembling peptide and the metal ion coexist under conditions that allow them to bind to each other. By contacting the self-assembling peptide with the metal ion, the two become capable of binding to each other.

[0060] The method of contact is not limited. For example, contact can be achieved by adding metal ions to the self-assembling peptide, or vice versa, or by adding both to the same container or the like. The form of contact is not limited. For example, a liquid containing metal ions may be added to a liquid containing the self-assembling peptide, or a solid salt containing metal ions may be added. Optionally, mild stimulation or the like (for example, mixing by inversion, shaking, stirring, etc.) may be applied after contact.

[0061] The concentrations of the self-assembling peptide and metal ions are not limited as long as they are concentrations at which they can exert their intended functions and activities.

[0062] The preferred concentration of the self-assembling peptide is a concentration capable of self-assembly. The self-assembling concentration may be, for example, 0.1 mM or more, 0.2 mM or more, 0.3 mM or more, 0.4 mM or more, 0.5 mM or more, 0.6 mM or more, 0.8 mM or more, 1 mM or more, 1.5 mM or more, 2.5 mM or more, 5 mM or more, 7.5 mM or more, 8 mM or more, 8.5 mM or more, 9 mM or more, 9.5 mM or more, or 10 mM or more during self-assembly. In particular, the concentration after self-assembly is not limited. Therefore, after self-assembly at a concentration capable of self-assembly, it can be diluted with an appropriate liquid, adjusted to an arbitrary concentration, and then contacted with metal ions.

[0063] In addition, the preferred concentration of the metal ion is not particularly limited as long as it is a concentration that can exhibit the desired characteristics. For example, the preferred concentration of the metal ion having antimicrobial activity is a concentration that can exhibit antimicrobial activity. The desired characteristics, such as the concentration that can exhibit antimicrobial activity, may be, for example, 0.01 mM or more, 0.02 mM or more, 0.05 mM or more, 0.10 mM or more, 0.15 mM or more, 0.25 mM or more, 0.5 mM or more, 1 mM or more, 1.5 mM or more, 2 mM or more, 2.5 mM or more, 5 mM or more, 7.5 mM or more, 8 mM or more, 8.5 mM or more, 9 mM or more, 9.5 mM or more, or 10 mM or more at the time of application.

[0064] The ratio or concentration at the time of contact is not limited, but is preferably a ratio or concentration at which aggregation of metal ions is suppressed compared to the case where only metal ions are applied. Therefore, it is sufficient that aggregation or precipitation of metal ions is suppressed when a stimulus such as light irradiation is applied. Aggregation or precipitation of metal ions can be easily determined, for example, by whether or not a precipitate of metal particles occurs when a stimulus is applied to the liquid after contact. Specifically, the ratio at the time of contact may be a ratio at which the concentration of amino acids capable of binding to metal ions in the self-assembling peptide is equal to or greater than the concentration of metal ions after contact. Equal to or greater means, for example, 80% or more, 85% or more, 90% or more, 92.5% or more, 95% or more, 96% or more, 97% or more, 97.5% or more, 98% or more, 98.5% or more, 99% or more, 99.5% or more, or 100% or more. In one embodiment, the molar concentration of amino acids capable of binding to metal ions in the self-assembling peptide is applied at a molar concentration equivalent to that of the metal ions.

[0065] The form of the self-assembling peptide and antimicrobial agent of the present invention is not particularly limited. They are preferably provided in liquid form, but may also be provided in solid form such as crystals, which may be diluted in a liquid for use.

[0066] The self-assembling peptide and antimicrobial agent of the present invention can be used as a composition. For example, the antimicrobial agent of the present invention can be mixed with a suitable carrier or diluted or suspended in a carrier to form a composition. The composition may contain other active ingredients, such as antimicrobial ingredients, in addition to commonly used excipients, surfactants, dispersants, buffers, preservatives, solubilizers, stabilizers, or isotonicity agents. The dosage form of the composition is not particularly limited. For example, it may be any of solutions, wettable powders, emulsions, powders, suspensions, granules, capsules, coatings, and sprays.

[0067] The self-assembling peptide and the antimicrobial agent are applied to a material, for example, the surface of a material. The method of application is not limited. It may be appropriately selected depending on the dosage form and the material to which it is applied, and may be applied, for example, by coating, spraying, immersion, or the like. The material to which it is applied is described in detail elsewhere in this specification. It is not necessary to apply the agent directly to the material or part to which it is desired to impart a desired property, for example, antimicrobial activity, but it may be applied to a material or part that comes into contact with the material or part to which it is desired.

[0068] In particular, the following embodiment is directed to imparting antimicrobial activity to a material using a metal ion having antimicrobial activity. However, in the following embodiment, by using a metal ion having a desired activity instead of the metal ion having antimicrobial activity, the metal ion can be stably retained in the material, and the desired activity of the metal ion can be imparted to the material. The present invention also provides such a metal ion addition kit and addition method, as well as a material to which any metal ion has been added.

[0069] In another aspect, the present invention provides an antimicrobial kit comprising a self-assembling peptide and a metal ion having antimicrobial activity. In the kit of the present invention, the self-assembling peptide and the metal ion may be provided in the same container or in separate containers, but are preferably provided in separate containers. The form of the self-assembling peptide and the metal ion contained in the kit is not particularly limited. For example, each may be in liquid form or solid form, but is preferably in liquid form when applied.

[0070] The kit of the present invention may further include additional elements, such as a diluent and instructions for use, as necessary. The diluent is not particularly limited, but the liquids described above for the self-assembling peptide and the antimicrobial agent may be used. The liquid used to dissolve the self-assembling peptide or the liquid used to dissolve the metal ions may be used, or another liquid may be used.

[0071] The kit of the present invention is applied to a material, for example, a surface of a material. The application method and the material to which it is applied are described in detail elsewhere in this specification. As with the antimicrobial method described below, when using the kit, the self-assembling peptide and the metal ion may be combined and then applied, or may be applied separately.

[0072] The antimicrobial agent and antimicrobial kit of the present invention enable metal ions having antimicrobial activity to be dispersed on the surface of a material and stably retained thereon. Therefore, by simply applying the antimicrobial agent and antimicrobial kit of the present invention to a material, the material can be made antimicrobial easily and quickly in a state that is less likely to discolor due to stimuli such as light.

[0073] The self-assembling peptide and the metal ion having antimicrobial activity can be used to make a material antimicrobial. Therefore, a further aspect of the present invention relates to a method for making a material antimicrobial. The method for making a material antimicrobial of the present invention comprises the following steps: adding a self-assembling peptide solution to the material; adding metal ions to the material; and The process of drying materials.

[0074] In this method, the step of adding a self-assembling peptide solution to a material and the step of adding metal ions to a material are carried out in a manner similar to the application of an antimicrobial agent to a material as described above.

[0075] Furthermore, the step of adding the self-assembling peptide solution to the material and the step of adding the metal ion to the material can be carried out simultaneously or separately, and therefore the self-assembling peptide and the metal ion may be added after contacting each other, or may be added separately.

[0076] When both steps are carried out simultaneously, the self-assembling peptide solution and the metal ions are mixed together before carrying out both steps. When both steps are performed separately, either step may be performed first, but preferably, the step of adding a self-assembling peptide solution to the material is performed before the step of adding metal ions to the material. The step of adding metal ions to the material is desirably performed while the material is in a wet state. Therefore, the step of adding metal ions to the material is preferably performed after the step of adding a self-assembling peptide solution and before the self-assembling peptide solution on the material dries.

[0077] The step of drying the material reduces the moisture in the material, particularly at least in the portion of the material to which the self-assembling peptide has been applied. The drying method is not particularly limited as long as it can reduce the moisture content. For example, there are a ventilation drying method in which hot or cold air is blown using a blower or the like, a windless drying method in which moisture is evaporated by heating, a spray drying method in which the slurry is suspended in an appropriate buffer and then the suspension is sprayed into gas to rapidly dry it, a freeze-drying method, a vacuum drying method in which degassing is performed using a vacuum pump or the like in a sealed container, a natural drying method (including drying in the sun) in which the mixture is exposed to the outside air, or a combination thereof. In the actual process, various drying devices that apply the above methods may be used. For example, a drum dryer, a far-infrared band dryer, a continuous vacuum drying device, a spray dryer, a freeze dryer, etc. can be used.

[0078] Also, for example, the material may be dried once after the step of adding the self-assembling peptide solution, and then the material may be moistened again by any method before the step of adding metal ions to the material. Thus, the antimicrobial method of the present invention may further include a step of drying the material between the step of adding the self-assembling peptide solution to the material and the step of adding metal ions to the material, and may further include a step of moistening the material thereafter.

[0079] In the step of wetting the material, a liquid is added to the material. The liquid used here is not particularly limited, but the liquids described above for the antimicrobial agent can be used. The liquid used to dissolve the self-assembling peptide or the liquid used to dissolve the metal ion may be used, or another liquid may be used.

[0080] For example, the antimicrobial method of the present invention can easily and quickly render a material antimicrobial. The antimicrobial method of the present invention can render a material antimicrobial in a state in which it is unlikely to discolor due to stimuli such as light, and is therefore particularly useful for rendering antimicrobial a material whose appearance is adversely affected by discoloration or a material that is to be used for a long period of time.

[0081] By using the method for providing an antimicrobial agent of the present invention, an antimicrobial material can be produced in which the material is provided with an antimicrobial agent. Accordingly, a further aspect of the present invention relates to an antimicrobial material to which a self-assembling peptide and a metal ion bound to the peptide are applied.

[0082] The antimicrobial material of the present invention is not particularly limited as long as it is a material to which a self-assembling peptide can be bound. The ability to bind a self-assembling peptide means that the applied self-assembling peptide does not easily fall off, and for example, it is sufficient that a negatively charged functional group is present in the portion of the material to which the self-assembling peptide is applied.

[0083] The portion of the material to which the self-assembling peptide is applied is not limited, but it is preferable that the self-assembling peptide is applied to the surface of the material. The surface of the material does not have to be the outermost surface, and includes all surfaces that may be contaminated with microorganisms. In addition, the surface may be made of one type of material or multiple types of materials.

[0084] The material of the present invention is preferably an organic material, for example an organic material having a negative charge, and includes, for example, materials made of fibers such as paper, knitted fabrics, woven fabrics and nonwoven fabrics, biological material surfaces, resins such as plastics, and the like.

[0085] Examples of fibers include natural fibers including plant fibers and animal fibers, regenerated fibers, semi-synthetic fibers, and synthetic fibers. The material made of fibers may be composed of blended fibers using two or more types of fibers including the above-mentioned fibers. A preferred blended fiber to be used is, for example, one of the fibers that accounts for the largest weight of the fibers that make up the fiber. The material made of fibers preferably contains 20% by weight or more of a material containing the above-mentioned fibers, preferably at least one type of fiber having a negative charge.

[0086] Vegetable fibers are fibers that mainly contain cellulose as a raw material, and specific examples include natural fibers such as cotton, linen, jute, pulp, and hemp, refined cellulose fibers such as viscose rayon, Tencel, lyocell, and cupra, and semi-synthetic fibers such as acetate.

[0087] Examples of animal fibers include sheep's wool, silk, cashmere, and angora.

[0088] Examples of synthetic fibers having a negative charge include fibers that can be obtained by melt spinning resins containing hydroxyl groups, such as nylon, and copolymers thereof, polyvinyl alcohol and copolymers thereof, etc. Negatively charged functional groups, such as carboxyl groups, sulfo groups, sulfonyl groups, and hydroxyl groups, may be introduced into synthetic fibers not having a negative charge, such as polyester fibers and polyolefin fibers, by post-processing, such as graft polymerization and corona discharge treatment.

[0089] Properties other than the charge and composition, such as the length of the fiber, are not particularly limited. In addition, the material made of fiber may be a part of a textile product, and the type of textile product is not limited. For example, textile products include bedding products such as duvet covers, blankets, and sheets, interior decoration textile products such as curtains, wallpapers, and tablecloths, clothing products such as hats, shirts, and underwear, automobile parts such as car seats and car mats, toys such as stuffed toys, and filter products such as air purifier filters, air conditioner filters, and masks.

[0090] Biological materials include materials of biological origin, including wood, leather, hide, and fur materials, as well as living biological surfaces such as humans and pet animals. There is no particular limitation on the organism from which the material originates. Thus, for example, application to the epidermis of humans or the surface of pet animals for the purpose of deodorization is also included.

[0091] For example, the antimicrobial material of the present invention can inhibit the growth of microorganisms on a material. The antimicrobial material of the present invention is made antimicrobial in a state where it is not easily discolored by stimuli such as light, and is therefore advantageously applicable to materials whose appearance is adversely affected by discoloration or materials that are used for a long period of time. EXAMPLES

[0092] The present invention will be described in more detail below with reference to examples and drawings, although the present invention is not limited to the following examples.

[0093] [Example 1] Synthesis of self-assembling peptides In this example, a self-assembling peptide was synthesized by solid-phase peptide synthesis using an Fmoc group.

[0094] (1) Solid-phase peptide synthesis The peptide solid-phase synthesis was carried out using the Fmoc method. The Fmoc method was carried out according to a standard method widely known in the art. During peptide synthesis, the N-terminus was protected by substituting the N-terminus with an Fmoc group, and a peptide having the sequence Fmoc-VKVVC (SEQ ID NO: 10) was synthesized. Subsequently, mass analysis of the peptide obtained by synthesis was carried out by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOFMS).

[0095] Mass spectrometry by MALDI-TOFMS revealed that the accurate mass of the synthesized Fmoc-VKVVC peptide was 767.40.

[0096] (2) Confirmation of the ability of peptides to form β-sheet structures The synthesized Fmoc-VKVVC was dissolved and dispersed in a methanol solution (99.5%, Wako Pure Chemical Industries, Ltd.) at a concentration of 1% (w / w) (equivalent to approximately 0.01 M) and allowed to stand at room temperature (approximately 20°C) for approximately one week to promote molecular accumulation. After standing, the solution was measured using a Fourier transform infrared spectrometer (FT-IR) (JASCO Corporation) in the wavenumber region where amide I absorption is observed (1600-1700 cm -1 ) was detected.

[0097] The infrared transmission spectrum by FT-IR is shown in Figure 1. The peak at 1640–1660 cm indicates the formation of an α-helical structure. -1 No clear drop was observed in the wavenumber region of 1615–1640 cm , indicating the formation of a β-sheet structure. -1 A large drop was observed in the wavenumber region of 1615–1640 cm for the synthesized peptide. -1 The selective absorption of light in the wavenumber region of 100 nm indicated that this peptide has the ability to self-assemble into a β-sheet structure.

[0098] Example 2: Preparation of peptide-metal complexes In this example, a complex between the peptide synthesized in Example 1 and a metal ion was prepared and observed using a transmission electron microscope.

[0099] For observation by a transmission electron microscope, silver was used as the metal. The Fmoc-VKVVC peptide methanol solution (0.01 M) prepared in Example 1(2) after standing was mixed with an aqueous silver nitrate solution (0.01 M, Sigma-Aldrich Japan LLC). Specifically, equal amounts of each solution were added to a sample tube using a micropipette, and the tubes were mixed by shaking by hand. The operation was carried out in a dark place.

[0100] The peptide-silver complex solution was dropped onto a TEM grid (Oken Shoji Co., Ltd., #10-9999 Elastic Carbon ELS-C075) to prepare a specimen for observation. The prepared specimen for observation was observed using a transmission electron microscope (TEM, JEOL Ltd.).

[0101] The appearance of the peptide-silver complex observed by TEM is shown in Figure 2. As shown in Figure 2, A and B, the β-sheet structure of the peptide was observed to be mesh-like or misty, and black silver particles were found to be dispersed on top of it. Many silver particles smaller than 100 nm were observed.

[0102] [Example 3] Application of peptide-metal complex to fabric In this example, peptide-silver-doped silk fabric was prepared by using a peptide-metal complex.

[0103] Onto a piece of silk fabric cut into a 2 cm square, 200 μl of the peptide-silver complex solution obtained in Example 2 was dropped using a micropipette. Then, the silk fabric on which the solution had been dropped was rapidly dried using a vacuum pump.

[0104] Comparative Example In this comparative example, silver nitrate-loaded silk fabric was prepared using the same procedure as in Example 3, but using a silver nitrate aqueous solution instead of the peptide-silver complex solution.

[0105] [Example 4] Evaluation of the state of peptide-metal complexes on fabric In this example, the surface of the silver-added silk fabric produced was observed, and discoloration due to light exposure was also observed.

[0106] (1) Observation of the silk fabric surface The surfaces of the peptide-silver-added silk cloth obtained in Example 3 and the silver nitrate-added silk cloth obtained in the comparative example were observed using a scanning electron microscope (SEM, Hitachi) and an energy dispersive X-ray (EDX) analyzer (Oxford Instruments).

[0107] The results of observation by SEM are shown in Figure 3, and the results of observation by EDX are shown in Figure 4. As shown in Figures 3A and 3B, no clear difference was observed in the appearance of the silk fabric surface between the silk fabric of Comparative Example (Figure 3A) and Example 3 (Figure 3B).

[0108] On the other hand, as shown in Figures 4A and 4B, more sulfur atom signals were observed on the surface of the silk fabric of Example 3 (Figure 4B) compared to the silk fabric of the comparative example (Figure 4A). This indicates that the Fmoc-VKVVC peptide containing a cysteine ​​(C) residue with a thiol group in the side chain is abundantly present on the silk fabric surface.

[0109] In addition, as shown in C and D of FIG. 4, many silver atom signals were observed on the surface of the silk fabric of Example 3 (D of FIG. 4) compared to the silk fabric of the comparative example (C of FIG. 4). This indicates that in the comparative example, many of the silver atoms have penetrated into the silk fabric or formed large aggregates, and there are few fine silver particles on the surface of the silk fabric (C of FIG. 4). On the other hand, in the silk fabric of Example 3, it was revealed that many fine silver particles remained on the surface of the silk fabric due to the formation of a peptide-silver complex (D of FIG. 4). In addition, some of the sulfur signals were colocalized with some of the silver signals (B and D of FIG. 4). This is consistent with the conventional knowledge that cysteine ​​can bind to metals via the thiol group in the side chain.

[0110] (2) Observation of the discoloration reaction of fabric exposed to light The precipitation of silver aggregates due to light exposure can be observed by the fact that the cloth containing silver ions turns brown after light exposure. Therefore, to investigate the effect of peptides on the aggregation of silver due to light exposure, light was irradiated onto silver-added silk cloth and the effect was examined.

[0111] Using a solar simulator (Minakata Electric Manufacturing Co., Ltd.) that emits light simulating sunlight, 500 W white light was irradiated onto the silk fabrics of Comparative Example and Example 3 for 1 hour. After that, the silk fabrics after the light irradiation were visually observed.

[0112] The appearance of the silk fabric after light irradiation is shown in Figure 5. As shown in Figures 5A and 5B, the silk fabric of the comparative example changed color to brownish-red after light irradiation (Figure 5A). On the other hand, the silk fabric of Example 3 did not change color even after light irradiation (Figure 5B). Therefore, it was found that the peptide-silver complex formed by the self-assembling peptide was stable even when exposed to sunlight, and that the formation of silver aggregates was greatly suppressed by the formation of the peptide-silver complex.

[0113] [Example 5] Evaluation of the antibacterial effect of peptide-silver-added silk fabric In this example, the antibacterial effect of peptide-silver-added silk fabric was tested according to the JIS L1902 standard.

[0114] The test was carried out according to the bacterial liquid absorption method. The test was carried out using 0.4 g of peptide-silver-added silk fabric onto which a total amount (2.8 mL) of peptide-silver complex, which was a mixture of 1.4 mL each of self-assembling peptide and silver nitrate, was dropped, and 0.4 g of silk fabric (control) to which nothing was added. The outline of the operation is as follows.

[0115] First, a standard cloth (silk 14 mesh) was used as the silk cloth. The sample sterilized by autoclaving (120°C, 20 min) was packed into a sterilized tube, and 0.2 ml of the test inoculum was inoculated onto the silk cloth using a micropipette. The test inoculum was a gram-positive bacterium, Staphylococcus aureus (provided by the National Institute of Technology and Evaluation, Biotechnology Center: NBRC; NBRC No. 12732), at 1.2 x 10 5 A bacterial solution containing bacteria at a concentration of 1000 / ml was used. The inoculated silk fabric was cultured at 37°C for 18 hours. After culture, 20 ml of physiological saline was added to a sterilized tube and washed out by stirring. To obtain the initial value of the viable bacterial count, a similar washout was performed immediately after inoculation on a portion of the control silk fabric. After washing out, the viable bacterial count was quantified by the pour culture method. The pour culture method is outlined below. 1 ml of the washout liquid was collected and diluted 10 times with physiological saline. This operation was repeated to prepare a 10-fold dilution series of the washout liquid, such as 1-fold dilution, 10-fold dilution, 100-fold dilution, and 1000-fold dilution. 1 ml of each dilution was cultured on two agar plates at 37°C for 24 hours. The number of colonies was counted in the dilution series with a colony count of 30 to 300 after culture, and the average of the two plates was taken. When the number of colonies was 1 or less, the average was taken as 1.

[0116] Viable bacterial count (C B ), growth value (F) and bactericidal activity value (L) were calculated as follows. Viable bacteria count: C B = Z × R (Z: average number of colonies, R: dilution factor) Growth value: F=M b -M a (M a: Common logarithm of the number of viable bacteria counted from the control silk fabric immediately after inoculation, M b : Common logarithm of the number of viable bacteria counted from the control silk fabric after 18 hours of incubation) Bactericidal activity value: L=M a -M c (M c (Decimal logarithm of the number of viable bacteria counted on peptide-silver-added silk fabric after 18 hours of incubation)

[0117] The appearance of the bacterial culture medium after the pour plate culture method is shown in Figure 6. As shown in Figure 6, A and B, bacterial growth was observed when the control silk fabric was used (Figure 6, A), but no colony formation was observed when the peptide-silver-added silk fabric was used (Figure 6, B).

[0118] This was also confirmed by the growth and bactericidal activity values. a =log(9.4×10 4 )=4.97, M b =log(1.39×10 7 ) = 7.14, the growth value was 2.17, which exceeded the test validity condition of 1.5. In addition, the number of viable bacteria on the peptide-silver-added silk fabric after 18 hours of incubation was 0, so M c From the above, the bactericidal activity value was 4.97, which was much higher than the condition for bactericidal activity, 0. This demonstrated that the peptide-silver-added silk fabric obtained in Example 3 has strong antibacterial activity.

[0119] [Example 6] Comparison of peptide and metal ion addition methods In this example, the method of adding the self-assembling peptide and metal ions was changed to explore the addition conditions under which the metal ions can be dispersed and stably present on the material surface.

[0120] The experimental method was carried out according to Examples 2 to 4. Whether metal ions can be dispersed and stably present on the surface of the material was judged by whether the silk cloth changed color after light irradiation when silver ions were applied to the silk cloth. Under some conditions, instead of mixing the self-assembling peptide solution and the silver nitrate aqueous solution as in Example 2, the self-assembling peptide was added to the silk cloth and then the silver nitrate aqueous solution was added. The experimental conditions are shown in Table 1.

[0121] [Table 1]

[0122] The presence or absence of discoloration after light irradiation under each condition was as shown in Table 1. When only methanol, which is a solvent for the self-assembling peptide, was mixed with silver nitrate, discoloration due to light irradiation was observed, so it was confirmed that the suppression of discoloration due to light irradiation was the effect of the self-assembling peptide. In addition, the only cases in which the addition of an aqueous silver nitrate solution did not result in discoloration after light irradiation were when the same treatment as in Example 2 was performed, and when the aqueous silver nitrate solution was added to the silk cloth before the silk cloth dried after the self-assembling peptide was added. From this, it was found that in order to disperse silver ions on the surface of the material using the self-assembling peptide and have them exist stably, it is not necessarily necessary to mix the self-assembling peptide and the silver ions in advance, and they may be added separately if the silk cloth is wet when the silver ions are added. [Sequence List Free Text]

[0123] SEQ ID NOs: 1 to 10: Artificial (synthetic peptides)

Claims

1. An antimicrobial agent comprising a self-assembling peptide having a total length of 4 to 30 amino acids and a metal ion bound to the peptide, the self-assembling peptide comprises at least one amino acid capable of binding to a metal ion, at least one basic amino acid, and at least two nonpolar or neutral amino acids; the self-assembling peptide comprises a sequence as set forth in Lys-Val-Val-Cys (SEQ ID NO: 5) or Cys-Val-Val-Lys (SEQ ID NO: 6); the positions of the amino acids capable of binding to the metal ion from the C-terminus of the self-assembling peptide are all expressed as 2m+a; the positions of the basic amino acids from the C-terminus of the self-assembling peptide are all expressed as 2n+1+a; The m is an integer of 0 to 15, The n is an integer from 0 to 14, The a is 0 or 1, The antimicrobial agent, wherein the metal ion comprises a metal ion having antimicrobial activity.

2. The antimicrobial agent of claim 1 , wherein the self-assembling peptide forms a beta-sheet structure.

3. The antimicrobial agent according to claim 1 or 2, wherein the total length of the self-assembling peptide is 4 to 15 amino acids.

4. The antimicrobial agent according to any one of claims 1 to 3, wherein the self-assembling peptide has a protecting group at its terminus.

5. The antimicrobial agent according to any one of claims 1 to 4, wherein the amino acid capable of binding to a metal ion comprises at least one type of amino acid selected from the group consisting of cysteine, histidine, lysine, aspartic acid, and glutamic acid.

6. The antimicrobial agent according to any one of claims 1 to 5, wherein the nonpolar or neutral amino acid comprises at least one amino acid selected from the group consisting of valine, isoleucine, leucine, tyrosine, tryptophan, glutamine and serine.

7. The antimicrobial agent according to any one of claims 1 to 6, wherein the self-assembling peptide comprises the sequence set forth in Val-Lys-Val-Val-Cys (SEQ ID NO: 9).

8. The antimicrobial agent according to any one of claims 1 to 7, wherein the metal ions include at least one type of metal ion selected from the group consisting of silver ions, copper ions, zinc ions, cobalt ions, nickel ions, lead ions, aluminum ions, mercury ions, bismuth ions, cadmium ions, and chromium ions.

9. The antimicrobial agent according to any one of claims 1 to 8, which is applied to a surface of a material.

10. The antimicrobial agent according to claim 9 , wherein the material is a fibrous material.

11. The antimicrobial agent according to any one of claims 1 to 10, wherein the microorganism is a virus, a bacterium or a fungus.

12. An antimicrobial composition comprising the antimicrobial agent according to any one of claims 1 to 11 as an active ingredient.

13. An antimicrobial kit comprising a self-assembling peptide having a total length of 4 to 30 amino acids and a metal ion having antimicrobial activity, the self-assembling peptide comprises at least one amino acid capable of binding to a metal ion, at least one basic amino acid, and at least two nonpolar or neutral amino acids; the self-assembling peptide comprises a sequence as set forth in Lys-Val-Val-Cys (SEQ ID NO: 5) or Cys-Val-Val-Lys (SEQ ID NO: 6); the positions of the amino acids capable of binding to the metal ion from the C-terminus of the self-assembling peptide are all expressed as 2m+a; the positions of the basic amino acids from the C-terminus of the self-assembling peptide are all expressed as 2n+1+a; The m is an integer of 0 to 15, The n is an integer from 0 to 14, An antimicrobial kit, wherein a is 0 or 1.

14. 14. The antimicrobial kit of claim 13, wherein the metal ions include at least one metal ion selected from the group consisting of silver ions, copper ions, zinc ions, cobalt ions, nickel ions, lead ions, aluminum ions, mercury ions, bismuth ions, cadmium ions, and chromium ions.

15. The antimicrobial kit according to claim 13 or 14, which is applied to a surface of a material.

16. An antimicrobial material comprising a self-assembling peptide having a total length of 4 to 30 amino acids and a metal ion bound to the peptide, the self-assembling peptide comprises at least one amino acid capable of binding to a metal ion, at least one basic amino acid, and at least two nonpolar or neutral amino acids; the self-assembling peptide comprises a sequence as set forth in Lys-Val-Val-Cys (SEQ ID NO: 5) or Cys-Val-Val-Lys (SEQ ID NO: 6); the positions of the amino acids capable of binding to the metal ion from the C-terminus of the self-assembling peptide are all expressed as 2m+a; the positions of the basic amino acids from the C-terminus of the self-assembling peptide are all expressed as 2n+1+a; The m is an integer of 0 to 15, The n is an integer from 0 to 14, The a is 0 or 1, the self-assembling peptide forms a β-sheet structure; the metal ions include metal ions having antimicrobial activity; The metal ions are arranged on the β-sheet structure.

17. 17. The antimicrobial material of claim 16, wherein the material is a fibrous material.

18. A method for making a material antimicrobial, comprising the steps of: adding a self-assembling peptide solution to the material; adding metal ions having antimicrobial activity to the material; and Drying the material Including, the total length of the self-assembling peptide is 4 to 30 amino acids; the self-assembling peptide comprises at least one amino acid capable of binding to a metal ion, at least one basic amino acid, and at least two nonpolar or neutral amino acids; the self-assembling peptide comprises a sequence as set forth in Lys-Val-Val-Cys (SEQ ID NO: 5) or Cys-Val-Val-Lys (SEQ ID NO: 6); the positions of the amino acids capable of binding to the metal ion from the C-terminus of the self-assembling peptide are all expressed as 2m+a; the positions of the basic amino acids from the C-terminus of the self-assembling peptide are all expressed as 2n+1+a; The m is an integer of 0 to 15, The n is an integer from 0 to 14, The method, wherein a is 0 or 1.

19. The antimicrobial method according to claim 18, wherein the step of adding metal ions to the material is carried out while the material is wet.

20. The antimicrobial method according to claim 18 or 19, wherein the step of adding a self-assembling peptide solution to the material and the step of adding metal ions to the material are carried out after mixing the self-assembling peptide solution with the metal ions.

21. The antimicrobial method according to any one of claims 18 to 20, wherein the step of adding metal ions to the material is carried out after the step of adding a self-assembling peptide solution to the material and before the self-assembling peptide solution is dried.

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