JP7630787B2 - Antimicrobial agents and antimicrobial methods - Google Patents

Description

The present invention relates to a self-assembling peptide that can attach metal ions to the surface of a material. The present invention also relates to an antimicrobial agent and an antimicrobial material that use such a self-assembling peptide and a metal ion, as well as a method for making a material antimicrobial.

Many metal elements are known to have antimicrobial activity against microorganisms such as viruses and bacteria. As described in Patent Document 1, among these, metal elements such as silver, copper, and zinc have long been known as metal elements that are relatively harmless to the human body. For this reason, many antimicrobial compositions have been developed that contain these metal elements in the atomic or ionic state as active ingredients.

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 is not only undesirable because it impairs the appearance of the product, but also because the antimicrobial activity caused by the heavy metal ions is localized to the precipitated areas. In particular, discoloration of textile products is difficult to eliminate, and in the development of antimicrobial compositions containing heavy metals for use in textile products, it has been considered particularly important to find ways to suppress discoloration caused by heavy metals.

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 produce and obtain the complexes, and there is a problem in that the production costs are high. There is also a known method of imparting antimicrobial activity by processing the fiber itself. However, in addition to the production cost problem, these antimicrobial fibers have the problem that once the antimicrobial activity of the fiber is lost, it is difficult to impart antimicrobial activity again.

JP 2017-133137 A JP 2007-223995 A

There has been a demand for simple means and methods for stably dispersing heavy metal ions uniformly in response to stimuli such as exposure to light, 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.

The inventor attempted to solve the above problem 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. The inventor discovered 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. The inventor also discovered 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.

For example, the present invention includes 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 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 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.

[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 a 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 comprises 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 ion, copper ion, zinc ion, cobalt ion, nickel ion, lead ion, aluminum ion, mercury ion, bismuth ion, cadmium ion, and chromium ion.
[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.

[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 from 0 to 14,
An antimicrobial kit, wherein a is 0 or 1.
[21-2] The antimicrobial kit according to [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 according to [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.

[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 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.
[25-1] The antimicrobial material according to [25], wherein the material is a material made of fibers.
[26] The antimicrobial material according to [25], in which aggregation of the metal ions is suppressed compared to when only the metal ions are applied.

[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,
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 from 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 according to [27] or [28], wherein the metal ions are applied at a concentration that inhibits aggregation of the metal ions compared to the case where 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 according to 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 is dried.

[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,
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 from 0 to 14,
The method, wherein a is 0 or 1.

[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 from 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] A composition for attaching 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.

[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 from 0 to 14,
The self-assembling peptide, wherein a is 0 or 1.

The antimicrobial agent, antimicrobial kit, and antimicrobial method of 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. Furthermore, the antimicrobial material of the present invention can inhibit the growth of microorganisms on the material.

The infrared transmission spectrum of the synthesized self-assembling peptide by Fourier transform infrared spectroscopy (FT-IR). The dashed rectangle indicates the wave number region showing the formation of an α-helical structure, and the solid rectangle indicates the wave number region showing the formation of a β-sheet structure. Transmission electron microscope (TEM) images of the self-assembling peptide-silver complex. A: Low magnification image, B: High magnification image. Scanning electron microscope (SEM) images of the surface of silver-doped silk fabric, A: without self-assembling peptide, B: with self-assembling peptide. 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. Photographs of silver-doped silk fabric are shown: A: without self-assembling peptide, B: with self-assembling peptide. Photographs of bacterial culture media obtained in the evaluation of the bactericidal activity of silver-added silk fabric are shown, A: without the self-assembling peptide, B: with the self-assembling peptide.

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.

In the present invention, "antimicrobial" or "antimicrobial activity" means to inhibit the growth, activity, growth or proliferation of microorganisms, and in some cases to inhibit the adhesion of microorganisms and to inhibit phenomena resulting from the growth of microorganisms. Therefore, antimicrobial activity includes antiviral activity, antibacterial activity, sterilizing activity, bactericidal activity, disinfecting activity, bacteriostatic activity, disinfecting activity, antifungal activity and antifungal activity. In the present invention, it is not necessary for microorganisms to be completely killed by antimicrobial activity. Furthermore, 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, it does not matter whether the object is intended to exhibit antimicrobial activity or not, as long as the object exhibits antimicrobial activity when exposed to microorganisms.

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.).

Preferred bacteria include, for example, gram-positive bacteria such as Staphylococcus bacteria including Staphylococcus aureus, and gram-negative bacteria such as Escherichia coli. Preferred fungi include, for example, yeast fungi and mold fungi.

In the present invention, a self-assembling peptide is used. Here, "self-assembly" refers to the autonomous formation of a three-dimensional structure over time, and a "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.

The environment may be 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.

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 the material, and does not need to be completed before contact with metal ions 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 by obtaining an infrared transmission spectrum, but it 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.

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

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 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.

The three-dimensional structure formed by the self-assembling peptide does not need to be entirely β-sheet structure, but may be, for example, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more of the structure being β-sheet structure. In addition, it is not necessary for all the self-assembling peptides to form a single three-dimensional structure, but may be an aggregate of several three-dimensional structures, or some peptides may exist 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 peptides contribute to the formation of the three-dimensional structure.

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 to it. In other words, 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, the amino acids capable of binding to metal ions are dispersed on the β-sheet structure of the self-assembling peptide in the material.

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.

As the amino acids constituting the self-assembling peptide, 20 types 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 N-H and C=O parts of the main chain are not modified or have similar charges at the same positions even after modification.

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.

The self-assembling peptide contains 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 in a specific arrangement.

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 a metal salt, and examples thereof include cysteine (Cys/C), histidine (His/H), aspartic acid (Asp/D), glutamic acid (Glu/E), and lysine (Lys/K). 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 conventional methods.

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.

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 acids include at least one amino acid selected from the group consisting of valine, isoleucine, leucine, tyrosine, tryptophan, glutamine, and serine.

The particular configuration is determined by optional conditions that favor conformation, particularly including β-sheet structure, 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 nonpolar amino acids or neutral amino acids between the amino acids capable of binding to metal ions and the basic amino acids. 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 metal ions and basic amino acids contained in one molecule of the self-assembling peptide is not limited.

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 from 0 to 15 (provided that when a is 0, m is an integer from 1 to 15, and when a is 1, m is an integer from 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.

By adopting this arrangement, the side chains of amino acids capable of binding to metal ions are exposed on one side of the formed β-sheet structure, and the side chains of basic amino acids are exposed on the other side. With this configuration, the β-sheet structure formed by the self-assembling peptide is capable of binding to metal ions on one side, and is capable of binding to a negatively charged material on the other side.

Whether or not a certain peptide is a self-assembling peptide can be routinely determined by a person skilled in the art. For example, a prepared peptide can be determined to have a structure including a β-sheet structure by observing it with a transmission electron microscope or obtaining an infrared transmission spectrum.

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 the opposite positions:
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).

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.)

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 the opposite positions:
Zaa-Val-Val-Xaa (SEQ ID NO: 4)
[Wherein,
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.

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).

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

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)
[Wherein,
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)
[Wherein,
Xaa is Lys, Arg or His;
Zaa is Cys, His, Asp, Glu or Lys.

Specific sequences that can be used include, for example, Val-Lys-Val-Val-Cys (SEQ ID NO: 9).

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

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 are prepared in which functional groups other than the amino and carboxyl groups to be bonded are protected with protecting groups, 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 with the carboxyl group of the next amino acid. By carrying out this reaction in succession, a polypeptide with protected functional groups in the side chain is synthesized. Finally, all protecting groups are removed to complete the peptide synthesis.

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 for the protecting group to be completely removed when the peptide is used as a self-assembling peptide.

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

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

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.

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.

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. Examples include nitrate ions, tetrafluoroborate ions, trifluoromethanesulfonate ions, behenate ions, sulfate ions, acetate ions, perchlorate ions, chlorate ions, and chloride ions.

In the present invention, when applied to a material, the metal ions are preferably in solution, but may also 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 preferred antimicrobial activity for use 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 sulfate (I), copper sulfate (II), copper chloride (II), copper perchlorate, copper acetate, potassium tetracyanocuprate, zinc nitrate, zinc sulfate, zinc acetate, zinc chloride, zinc perchlorate, zinc thiocyanate, cobalt (II) nitrate, 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. Here, 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.

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, the two may be brought into contact so that they bind, as described below.

In one aspect, the present invention provides a self-assembling peptide having a metal ion bound thereto.

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.

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.

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, a light stimulus (e.g., mixing by inversion, shaking, stirring, etc.) may be applied after contact.

The concentrations of the self-assembling peptide and metal ions are not limited as long as they are capable of exerting the intended functions and activities.

The preferred concentration of the self-assembling peptide is a concentration at which self-assembly is possible. The self-assembly 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 self-assembly concentration, it can be diluted in an appropriate liquid, adjusted to an arbitrary concentration, and then contacted with metal ions.

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, a preferred concentration of a metal ion having antimicrobial activity is a concentration that can exhibit antimicrobial activity. A concentration that can exhibit the desired characteristics, such as antimicrobial activity, may be, for example, a concentration of 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 when applied.

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 the 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.

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 they may also be provided in solid form such as crystals, which may be diluted in a liquid for use.

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 made into a composition by mixing with a suitable carrier or diluting or suspending in a carrier. 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 a liquid, wettable powder, emulsion, powder, suspension, granules, capsule, coating agent, or spray.

The self-assembling peptide and the antimicrobial agent are applied to a material, for example, the surface of a material. There are no limitations on the method of application. The method may be appropriately selected depending on the dosage form and the material to which it is to be applied, and may be applied, for example, by painting, spraying, immersion, or the like. The material to which it is to be 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.

In particular, the following describes an embodiment that aims to impart 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.

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 preferably, both are 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 it is preferable that they are in liquid form when applied.

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 may be the liquid described above for the self-assembling peptide and the antimicrobial agent. 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.

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 methods described below, when using the kit, the self-assembling peptide and metal ion may be combined and then applied, or may be applied separately.

The antimicrobial agent and antimicrobial kit of the present invention can disperse and stably retain metal ions having antimicrobial activity on the surface of a material, so that by simply applying the antimicrobial agent and antimicrobial kit of the present invention to the material, the material can be made antimicrobial easily and quickly in a state in which it is resistant to discoloration due to stimuli such as light.

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 steps of:
adding a self-assembling peptide solution to the material;
adding metal ions to the material; and drying the material.

In this method, 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 in a manner similar to the application of an antimicrobial agent to the material as described above.

In addition, the step of adding the self-assembling peptide solution to the material and the step of adding the metal ions to the material can be carried out simultaneously or separately. Therefore, the self-assembling peptide and the metal ions may be added after contacting each other, or may be added separately.

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.

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, 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 in which the slurry is left exposed to the outside air (including drying in the sun), or a combination thereof can be mentioned. 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.

For example, the material may be dried once after the step of adding the self-assembling peptide solution, and then the material may be re-moistened 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.

In the process 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 ions may be used, or a different liquid may be used.

For example, the antimicrobial method of the present invention can easily and quickly make materials antimicrobial. The antimicrobial method of the present invention can make materials antimicrobial in a state where they are less likely to discolor due to stimuli such as light, and is therefore particularly useful for making antimicrobial materials whose appearance is adversely affected by discoloration or materials that are used for long periods of time.

By using the antimicrobial method of the present invention, an antimicrobial material can be produced in which the material is antimicrobialized. Thus, 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.

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. Being able 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 part of the material to which the self-assembling peptide is applied.

There are no limitations on the part of the material to which the self-assembling peptide is applied, but it is preferable that it 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.

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, surfaces of biological materials, resins such as plastics, etc.

Examples of fibers include natural fibers including plant 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.

Plant fibers are fibers that 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.

Animal fibers include sheep's wool, silk, cashmere, and angora fibers.

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 their copolymers, polyvinyl alcohol and its copolymers, etc. Negatively charged functional groups, such as carboxyl groups, sulfo groups, sulfonyl groups, and hydroxyl groups, may be introduced into synthetic fibers that do not have a negative charge, such as polyester fibers and polyolefin fibers, by post-processing such as graft polymerization and corona discharge treatment.

Properties other than the charge and composition, such as the length of the fiber, are not particularly limited. Furthermore, 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 items such as duvet covers, blankets, and sheets, interior decoration textile products such as curtains, wallpaper, and tablecloths, clothing items such as hats, shirts, and underwear, automobile parts such as car seats and car mats, toys such as stuffed animals, and filter products such as air purifier filters, air conditioner filters, and masks.

Biological materials include materials of biological origin, including wood, leather, hide, and fur materials, as well as the surfaces of living organisms such as humans and pet animals. There are no particular limitations on the organisms from which the materials are derived. Thus, for example, application to the epidermis of humans or the surfaces of pet animals for the purpose of deodorization is also included.

For example, the antimicrobial material of the present invention can inhibit the growth of microorganisms on materials. The antimicrobial material of the present invention is antimicrobial in a state in which it is not easily discolored by stimuli such as light, and is therefore advantageously applicable to materials in which discoloration can have a detrimental effect on the appearance or materials that are used for long periods of time.

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

Example 1 Synthesis of Self-Assembling Peptide In this example, a self-assembling peptide was synthesized by solid-phase peptide synthesis using an Fmoc group.

(1) Peptide solid-phase 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 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).

Mass spectrometry using the MALDI-TOFMS method revealed that the exact mass of the synthesized Fmoc-VKVVC peptide was 767.40.

(2) Confirmation of the peptide's ability to form β-sheet structure 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 aggregation. After standing, the solution was subjected to Fourier transform infrared (FT-IR) spectrometer (JASCO Corporation) to detect transmitted light in the wavenumber region (1600-1700 cm ^-1 ) where amide I absorption is observed.

The infrared transmission spectrum by FT-IR is shown in Figure 1. No clear drop was observed in the wavenumber region of 1640-1660 cm ^-1 , which indicates the formation of an α-helical structure, while a large drop was observed in the wavenumber region of 1615-1640 cm ^-1 , which indicates the formation of a β-sheet structure. The selective absorption of light in the wavenumber region of 1615-1640 cm ^-1 by the synthesized peptide indicated that this peptide has the ability to self-assemble into a β-sheet structure.

Example 2 Preparation of Peptide-Metal Complex In this example, a complex of the peptide synthesized in Example 1 and a metal ion was prepared and observed using a transmission electron microscope.

For observation with 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 mixed by shaking by hand. The work was carried out in a dark place.

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.).

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

Example 3 Application of Peptide-Metal Complex to Fabric In this example, peptide-silver-added silk fabric was produced by using a peptide-metal complex.

200 μl of the peptide-silver complex solution obtained in Example 2 was dripped onto a piece of silk fabric cut into a 2 cm square using a micropipette. After that, the silk fabric with the dripped solution was quickly dried using a vacuum pump.

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

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

(1) Observation of the silk cloth 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).

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 the Comparative Example (Figure 3A) and Example 3 (Figure 3B).

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 Fmoc-VKVVC peptides containing cysteine (C) residues with thiol groups in their side chains are abundant on the silk fabric surface.

As shown in Figures 4C and 4D, more silver atom signals were observed on the surface of the silk fabric of Example 3 (Figure 4D) than on the silk fabric of the comparative example (Figure 4C). This indicates that in the comparative example, most of the silver atoms have penetrated into the silk fabric or formed large aggregates, and there are fewer tiny silver particles on the surface of the silk fabric (Figure 4C). On the other hand, in the silk fabric of Example 3, it was revealed that many tiny silver particles remain on the surface of the silk fabric due to the formation of a peptide-silver complex (Figure 4D). In addition, some of the sulfur signals were colocalized with some of the silver signals (Figures 4B and 4D). This is consistent with the conventional knowledge that cysteine can bind to metals via the thiol group in the side chain.

(2) Observation of discoloration of cloth exposed to light The precipitation of silver aggregates due to light exposure can be observed by the cloth containing silver ions turning brown after light exposure. Therefore, to investigate the effect of peptides on the aggregation of silver due to light exposure, light was irradiated to a silver-added silk cloth and the effect was examined.

Using a solar simulator (Minakata Electric Manufacturing Co., Ltd.) that emits light simulating sunlight, the silk fabrics of the comparative example and example 3 were irradiated with 500 W white light for 1 hour. After that, the silk fabrics after irradiation were visually observed.

Figure 5 shows the appearance of the silk fabric after light irradiation. As shown in Figures 5A and 5B, the silk fabric of the comparative example turned 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.

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

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 the entire amount (2.8 mL) of peptide-silver complex, a mixture of 1.4 mL each of self-assembling peptide and silver nitrate, was dripped, and 0.4 g of silk fabric (control) to which nothing was added. The procedure is outlined below.

First, a standard silk cloth (silk 14 mesh) was used as the silk cloth. The samples sterilized by autoclaving (120°C, 20 min) were 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 contained a gram-positive bacterium, Staphylococcus aureus (Provided by National Institute of Technology and Evaluation, Biotechnology Center: NBRC No. 12732), at a concentration of 1.2 × ¹⁰⁵ /ml. The inoculated silk cloth was cultured at 37°C for 18 hours. After culture, 20 ml of physiological saline was added to the sterilized tube and washed out by stirring. To obtain the initial value of the viable cell count, a similar washout was performed on a part of the control silk cloth immediately after inoculation. After washing, the viable cell 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 procedure was repeated to prepare a 10-fold dilution series of the washout solution, i.e., 1-fold, 10-fold, 100-fold, 1000-fold, etc. 1 ml of each dilution solution was cultured on two agar plates at 37°C for 24 hours. After culture, the number of colonies was counted for the dilution series with colony counts of 30 to 300, and the average of the two plates was taken. If the number of colonies was 1 or less, the average was taken as 1.

The viable cell count (C _B ), growth value (F) and bactericidal activity value (L) were calculated as follows.
Viable cell count: C _B = Z × R (Z: average colony count, 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 : common logarithm of the number of viable bacteria counted from the peptide-silver-added silk fabric after 18 hours of incubation)

Figure 6 shows the appearance of the bacterial culture medium after the pour plate culture method. As shown in Figure 6, A and B, bacterial growth was observed when the control silk fabric was used (Figure 6, A), but no colonies were observed when the peptide-silver-added silk fabric was used (Figure 6, B).

This was also confirmed by the growth value and bactericidal activity value. From _Ma = log (9.4 x ¹⁰⁴ ) = 4.97 and _Mb = log (1.39 x ¹⁰⁷ ) = 7.14, the growth value was 2.17, which exceeded the test validity condition of 1.5. In addition, since the viable bacteria count on the peptide-silver-added silk fabric after 18 hours of culture was 0, from _Mc = log (1) = 0, the bactericidal activity value was 4.97, which far exceeded the bactericidal activity condition of 0. From this, it was found that the peptide-silver-added silk fabric obtained in Example 3 has strong antibacterial activity.

[Example 6] Comparison of methods for adding peptides and metal ions In this example, the methods for adding the self-assembling peptides and metal ions were varied to explore the addition conditions under which the metal ions would be dispersed and stably present on the material surface.

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. In 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.

The presence or absence of discoloration after light irradiation under each condition is as shown in Table 1. When only methanol, the solvent for the self-assembling peptide, was mixed with silver nitrate, discoloration due to light irradiation was observed, confirming that the suppression of discoloration due to light irradiation is 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 after the self-assembling peptide was added and before the silk cloth dried. From this, it was found that in order to use the self-assembling peptide to disperse and stably distribute silver ions on the surface of the material, it is not necessarily necessary to mix the self-assembling peptide and silver ions in advance, and they can be added separately as long as the silk cloth is wet when the silver ions are added.

Sequence numbers 1 to 10: Artificial (synthetic peptides)

Claims (21)

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. The antimicrobial agent according to claim 1, wherein the self-assembling peptide forms a β-sheet structure. The antimicrobial agent according to claim 1 or 2, wherein the total length of the self-assembling peptide is 4 to 15 amino acids. The antimicrobial agent according to any one of claims 1 to 3, wherein the self-assembling peptide has a protecting group at its terminus. The antimicrobial agent according to any one of claims 1 to 4, wherein the amino acid capable of binding to the metal ion includes at least one amino acid selected from the group consisting of cysteine, histidine, lysine, aspartic acid, and glutamic acid. The antimicrobial agent according to any one of claims 1 to 5, wherein the nonpolar or neutral amino acid includes at least one amino acid selected from the group consisting of valine, isoleucine, leucine, tyrosine, tryptophan, glutamine, and serine. 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). 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. The antimicrobial agent according to any one of claims 1 to 8 , which is applied to a surface of a material. The antimicrobial agent according to claim 9 , wherein the material is a fibrous material. The antimicrobial agent according to any one of claims 1 to 10 , wherein the microorganism is a virus, a bacterium or a fungus. An antimicrobial composition comprising the antimicrobial agent according to any one of claims 1 to 11 as an active ingredient. 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. 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. The antimicrobial kit according to claim 13 or 14 , which is applied to a surface of a material. 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. The antimicrobial material of claim 16 , wherein the material is a fibrous material. 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,
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. 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. 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. 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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