阅读说明：本技术 用于快速合成抗菌化妆品粉扑的生物相容性组合物及其制备方法 (Biocompatible composition for rapid synthesis of antibacterial cosmetic puff and method for preparing the same ) 是由 金容泰 李相载 曹世熙 于 2018-07-04 设计创作，主要内容包括：本发明提供了生物相容性组合物,以使得能够用抗菌粘附蛋白快速涂覆化妆品粉扑。牢固附着的抗菌蛋白可赋予化妆品粉扑抗菌功能,其抗菌活性持续6个月以上。本发明还描述了用生物相容性组合物中的抗菌粘附蛋白快速涂覆化妆品粉扑的方法。(The present invention provides a biocompatible composition to enable the rapid coating of a cosmetic puff with an antimicrobial adhesive protein. The firmly attached antibacterial protein can endow the cosmetic puff with an antibacterial function, and the antibacterial activity of the cosmetic puff lasts for more than 6 months. The present invention also describes a method for the rapid coating of cosmetic puffs with antimicrobial adhesion proteins in biocompatible compositions.)

1. A coating composition comprising a buffer solution and a naturally occurring adhesion protein recombinantly functionalized with an antimicrobial peptide.

2. The coating composition of claim 1, wherein the buffer solution comprises water.

3. The coating composition of claim 1, wherein the buffer solution comprises an alcohol.

4. The coating composition of claim 3, wherein the alcohol is ethanol.

5. The coating composition of claim 1, wherein the buffer solution comprises ethanol and water.

6. The coating composition of claim 5, wherein the mass ratio of ethanol to water in the buffer solution ranges from 1:9 to 9: 1.

7. The coating composition of claim 2, wherein the buffer solution further comprises a salt at a concentration of 10 to 100 mM.

8. The coating composition of claim 7, wherein the salt is an inorganic salt.

9. The coating composition of claim 8, wherein the inorganic salt is selected from the group consisting of: sodium acetate, sodium carbonate, sodium bicarbonate, sodium phosphate, sodium hydroxide, potassium acetate, potassium carbonate, potassium bicarbonate, potassium phosphate, and potassium hydroxide.

10. The coating composition of claim 7, wherein the salt is an organic salt.

11. The coating composition of claim 10, wherein the organic salt is selected from the group consisting of: tris, Bis-Tris, MES, PIPES, MOPS and HEPES.

12. The coating composition of claim 1, wherein the naturally occurring adhesion protein is selected from the group consisting of: barnacle adhesion protein, collagen, fibrin, and mussel adhesion protein.

13. The coating composition of claim 12, wherein the mussel adhesive protein has the sequence ID number: 13 or 14, pre-treated with tyrosinase to convert tyrosine residues to DOPA residues.

14. The coating composition of claim 1, wherein the antimicrobial peptide is selected from the group consisting of: KLWKKWAKKWLKLWKA (SEQ ID NO: 16), FALALKALKKL (SEQ ID NO: 17), ILRWPWPWRRK (SEQ ID NO: 18), AKRHHGYKRKFH (SEQ ID NO: 19), KWKLFKKIGAVLKVL (SEQ ID NO: 20), LVKLVAGIKKFLKWK (SEQ ID NO: 21), IWSILAPLGTTLVKLVAGIGQQKRK (SEQ ID NO: 22), GIGAVLKVLTTGLPALISWI (SEQ ID NO: 23), SWLSKTAKKGAVLKVL (SEQ ID NO: 24), KKLFKKILKYL (SEQ ID NO: 25), GLKKLISWIKRAAQQG (SEQ ID NO: 26), and GWLKKIGKKIERVGQHTRDATIQGLGIAQQAANVAATAR (SEQ ID NO: 27).

15. A cosmetic puff comprising the coating composition of any one of claims 1-14.

16. The cosmetic puff of claim 15, wherein the cosmetic puff is made from a material selected from the group consisting of: polyurethane (PUR), acrylonitrile butadiene rubber (NBR), Styrene Butadiene Rubber (SBR), Natural Rubber (NR), polyvinyl chloride, polyethylene, ethylene vinyl acetate butyl rubber (EVA), latex, silicone, styrene-isoprene-styrene (SIS), styrene-ethylene-butylene-styrene (SEBS), polyvinyl alcohol (PVA), nitrile rubber, butyl rubber, chloroprene rubber, and combinations thereof.

17. A method of coating a cosmetic puff with the coating composition of claims 1-14, comprising: a) providing the coating composition comprising an antimicrobial peptide according to any one of claims 1-14, and wherein the concentration of the antimicrobial peptide in the coating composition ranges from 0.02 to 0.1 mg/ml, b) applying the coating composition to the surface of a cosmetic puff, and c) drying the antimicrobial peptide coated cosmetic puff.

18. The method of claim 17, wherein the antimicrobial peptide is present in the coating composition at a concentration ranging from 0.02 to 0.1 mg/ml.

19. The method of claim 17, wherein the drying is performed at 60 ℃.

Technical Field

The present invention relates to a cosmetic puff having antibacterial properties to prevent the growth of microorganisms in the puff during use and storage after use in a humid environment.

Background

Cosmetic puffs made of various materials are known, preferably those that are soft and comfortable on the skin. Examples of such cosmetic puffs include those made of polyester velour and polyester warp-knit pile, polytrimethylene terephthalate (JP 2004-033432, US 2015/0173485) as described in japanese patent (JP 11-000224). Puffs are usually made of velvet, sponge, etc., and are particularly made for application of cosmetic cakes, powders and foundations.

Cosmetic puffs are used to scrape cosmetic products (mainly powdered cosmetic products) from a cosmetic container and transfer the scraped cosmetic products to the skin surface when the puff is applied to the skin. When this occurs, it is necessary to transfer the cosmetic scraped off and adhered to the powder puff surface to the skin surface as much as possible.

Cosmetic powders are sometimes contaminated with microorganisms (such as staphylococcus aureus, pseudomonas aeruginosa, clostridium tetani, yeasts and molds) which may be from raw materials, or from the presence of dust during manufacture, processing, and breakage or damage of cosmetic powder containers, in retail markets, and also from the periods of use of the products (see michael macvren Dashen et al, Microbiological quality assessment of soil fibers of cosmetics products soil with soil joints, pateaustate.j.

In fact, every time we come into contact with our cosmetics, all dirt and micro-organisms on our skin are transferred to the product. The same is true of any brush or sponge applicator, all of which inevitably enter our cosmetics. Furthermore, we get bacteria-laden cosmetics into our mouth, eyes and any damaged skin.

Silver-based antibacterial agents, including silver nanoparticles, are widely used in commercial products for their antibacterial properties in order to protect the products and our bodies from microbial contamination. For example, silver nanoparticles are added to medical products such as bandages, as well as textiles and household items. For the cosmetic applicator, some korean patents disclose silver-based cosmetic powder puff (see KR 10-1750832, KR 10-2015-.

However, silver compounds such as silver nanoparticles are known to cause Toxicity in many different species, and long term exposure to silver is known to cause psoriasis and/or silver deposition in humans (see ChristianeBeer et al, perception of silver nanoparticles-nanoparticie or silver ion.

Coating compositions based on antibacterial adhesion proteins are disclosed in korean patent (KR 10-1652263). However, DOPA-mediated adhesion requires long contact times, typically at least 6 hours, without the use of oxidizing agents toxic to our tissues. In order to rapidly solidify the DOPA-containing compound, the pH of the buffer or solvent must be strongly basic, which is also disadvantageous to our tissues.

For this reason, there is a need for novel, less or non-toxic, fast curing antimicrobial agents for cosmetic puffs. We have solved the toxicity problem by providing naturally occurring antimicrobial peptides for recombinant addition to naturally occurring adhesion proteins. The self-adhesive of the antimicrobial adhesive protein can be coated or adhered quickly without any chemicals (e.g., cross-linking agents).

Disclosure of Invention

Technical scheme for solving technical problem

In one aspect of the invention, a coating composition is provided comprising a buffer solution and a naturally occurring adhesion protein recombinantly functionalized with an antimicrobial peptide.

In one embodiment of the present invention, the buffer solution may comprise water, alcohol, or a mixture thereof. In a preferred embodiment of the present invention, the alcohol may be a lower alcohol such as methanol, ethanol, propanol and butanol. Among them, ethanol is more preferable. In another preferred embodiment of the present invention, the mass ratio of the alcohol to the water in the buffer solution may be 1:9 to 9: 1.

In another embodiment of the present invention, the buffer solution in the coating composition may further contain a salt at a concentration of 10 to 100 mM. In a preferred embodiment of the invention, the salt may be an inorganic salt or an organic salt. In a more preferred embodiment, the inorganic salt may be sodium acetate, sodium carbonate, sodium bicarbonate, sodium phosphate, sodium hydroxide, potassium acetate, potassium carbonate, potassium bicarbonate, potassium phosphate, or potassium hydroxide, and the organic salt may be Tris, Bis-Tris, MES, PIPES, MOPS, or HEPES.

In another embodiment of the invention, the naturally occurring adhesion protein in the coating composition may be a barnacle adhesion protein, collagen, fibrin, or mussel adhesion protein. In a preferred embodiment of the invention, the mussel adhesive protein may have the sequence ID: 13 or 14. In another example, the mussel adhesive protein may be pre-treated with tyrosinase to convert tyrosine residues to DOPA residues.

In another embodiment of the present invention, the antimicrobial peptide in the coating composition may be KLWKKWAKKWLKLWKA (SEQ ID NO: 16), FALALKALKKL (SEQ ID NO: 17), ILRWPWPWRRK (SEQ ID NO: 18), AKRHHGYKRKFH (SEQ ID NO: 19), KWKLFKKIGAVLKVL (SEQ ID NO: 20), LVKLVAGIKKFLKWK (SEQ ID NO: 21), IWSILAPLGTTLVKLVAGIGQQKRK (SEQ ID NO: 22), GIGAVLKVLTTGLPALISWI (SEQ ID NO: 23), SWLSKTAKKGAVLKVL (SEQ ID NO: 24), KKLFKKILKYL (SEQ ID NO: 25), GLKKLISWIKRAAQQG (SEQ ID NO: 26), or GWLKKIGKKIERVGQHTRDATIQGLGIAQQAANVAATAR (SEQ ID NO: 27).

In another aspect of the present invention, there is provided a cosmetic puff comprising the coating composition of the present invention. In a preferred embodiment of the present invention, the cosmetic puff may be prepared by: polyurethane (PUR), acrylonitrile butadiene rubber (NBR), Styrene Butadiene Rubber (SBR), Natural Rubber (NR), polyvinyl chloride, polyethylene, ethylene vinyl acetate butyl rubber (EVA), latex, silicone, styrene-isoprene-styrene (SIS), styrene-ethylene-butylene-styrene (SEBS), polyvinyl alcohol (PVA), nitrile rubber, butyl rubber, chloroprene rubber, or a combination thereof.

In another aspect of the present invention, there is provided a method of coating a cosmetic puff with the coating composition of the present invention. In a preferred embodiment of the invention, the method may comprise the steps of: a) providing a coating composition of the present invention comprising an antimicrobial peptide; b) applying a coating composition to a surface of a cosmetic puff; c) drying the antimicrobial peptide coated puff. In one embodiment of the present invention, the concentration of the antimicrobial peptide in the coating composition ranges from 0.01 to 0.2 mg/ml, more preferably from 0.02 to 0.1 mg/ml. In a more preferred embodiment, the drying may be carried out at 60 ℃.

In one aspect of the present invention, a novel antibacterial coating agent for a cosmetic puff and a method of preparing a cosmetic puff coated with the antibacterial coating agent, wherein the antibacterial peptide may be derived from human blood or frog skin.

The prior art of DOPA-containing adhesives, such as mussel adhesive protein or polydopamine, requires a high pH (at least 8 or higher) and the addition of a curing agent, such as peroxide, to promote curing, otherwise at least 10 hours or more are required for coating or attachment.

The present invention provides an adhesive composition and method for antimicrobial coating of cosmetic puffs without the use of any pH-adjusting or curing-promoting agent.

In a first aspect, there is provided an antimicrobial coating composition comprising an antimicrobial adhesive protein, wherein the antimicrobial adhesive protein may comprise at least 1 mol% of catechol groups in the adhesive protein backbone.

In a second aspect, there is provided a method of applying an antimicrobial adhesive coating to a cosmetic puff surface, the method comprising the steps of: a) providing a composition comprising a solution of an antimicrobial adhesive protein, b) applying the composition to a surface to be coated to obtain an antimicrobial adhesive coating on the surface, and c) drying the adhesive coating. In one embodiment of the invention, the concentration of the polymer in the composition is 0.01 to 0.2 mg/ml, more preferably 0.02 to 0.1 mg/ml.

Drawings

FIG. 1 shows a schematic representation of the rapid antimicrobial coating of antimicrobial adhesion proteins with L-dopamine. L-dopamine rapidly adsorbs to the substrate surface and reacts with DOPA derived from the antimicrobial mussel adhesive protein to form a solid and durable antimicrobial coating.

FIG. 2 shows antimicrobial activity from antimicrobial coated surfaces of alcohol-containing buffer solutions, where the antimicrobial adhesion proteins are dissolved at different concentrations. The growth of escherichia coli (2A) and staphylococcus aureus (2B) on the antimicrobial-coated surface was completely inhibited.

Fig. 3 shows an antibacterial coated cosmetic puff. No change in appearance was observed for the antimicrobial coated surface and the control sample.

FIG. 4 shows the antimicrobial efficacy of antimicrobial adhesion protein coated surfaces. The addition of dopamine is beneficial to the coating, and the antibacterial effect is not influenced by L-DOPA.

Fig. 5 shows the durability test results. When both samples were subjected to 3 to 10 washing cycles, the antibacterial adhesive protein-coated puff (5B) exhibited superior long-lasting antibacterial activity compared to the commercially available antibacterial cosmetic puff (5C).

Detailed Description

The present invention is directed to an antimicrobial adhesive protein coating composition for antimicrobial cosmetic puffs, wherein the antimicrobial adhesive protein comprises a naturally occurring adhesive protein recombinantly functionalized with a naturally occurring or synthetic antimicrobial peptide. The natural adhesive protein in the present invention may be derived from mussel adhesive protein, barnacle adhesive protein, fibrin or collagen. Due to its self-adhesive properties, the invention makes it possible, unlike the prior art, to apply cosmetic puffs without any chemical substance.

Any suitable adhesion protein may be used in the present invention, including but not limited to extracted or recombinant barnacle adhesion protein, collagen, fibrin, or mussel adhesion protein. Preferably, the protein is a recombinant mussel adhesive protein.

Any suitable recombinant mussel adhesive protein may be used in the invention. Examples of commercially available substrate proteins include the MAPTrix sold by Kollodis BioSciences, Inc. (North Ougusta, south Carolina)^TMOr BV AMP sold by biovitco. An optional third component is a biocompatible polymer (e.g., polyethylene glycol or polyvinyl alcohol) that can be added to the composition to enhance its biocompatibility such as physical or mechanical properties (e.g., tack), enhancing the coating process.

MAPTrix developed by Kollodis BioSciences Inc. (North Ougusta, south Carolina)^TMIs a pre-designed mussel adhesive protein or a barnacle-based coating suitable for hydrophobic or hydrophilic surfaces. Pre-designed MAPTrix^TMIt is very advantageous for surface coating because its molecular backbone is alternating hydrophobic and hydrophilic domains. Our BV AMPs are based on this amphipathic adhesion protein recombinantly functionalized with antimicrobial peptides.

Recently, the inventors have developed and tested antimicrobial adhesive protein coated textiles and cosmetic puffs for their ability to inhibit microbial growth on the surface of the textiles and cosmetic puffs (see korean patent application KR2017 0084822 and US patent application US 15/953,055, which are incorporated herein by reference for all purposes as if fully set forth herein).

MAPTrix^TMIs a fusion protein comprising: a first peptide of mussel foot protein FP-5 selected from the group consisting of seq ID no: 8-11, or a group consisting of sequence ID numbers: 15, a barnacle-derived adhesion protein; and at least one second peptide selected from the group consisting of: selected from the group consisting of sequence ID numbers: 1-3, mussel FP-1, mussel FP-2 (sequence ID No.: 4), selected from the group consisting of sequence ID No.: 5-6 of the group consisting of mussel FP-3, mussel FP-4 (SEQ ID NO: 7), mussel FP-6 (SEQ ID NO: 12) and fragments thereof, and a second peptide attached to the C-terminus, N-terminus or both C-and N-terminus of FP-5. Preferably, the first and second electrodes are formed of a metal,the second peptide is a peptide comprising the sequence ID number: 3, FP-1 of the amino acid sequence of 3. More preferably, the fused mussel adhesive protein is a fusion protein comprising the sequence ID no: 13 or 14. The recombinant mussel adhesive protein should be treated with tyrosinase to convert tyrosine residues to DOPA residues for strong adhesion. To convert tyrosine residues to DOPA, recombinant mussel adhesive protein was dissolved at 0.1 mg/ml in 0.1M sodium acetate buffer with 0.1M ascorbic acid, pH 5.5. Mushroom tyrosinase was added to a final concentration of 150U/ml and the mixture was incubated under oxygen conditions at 300rpm for 1 hour. As previously described, the enzyme activity was stopped with 0.2ml of 6N HCl per ml of reaction (DS Hwang et al, Appl Environ Microbiol.2004; 70(6): 3352-.

Any suitable biologically derived or synthetically designed antimicrobial peptide may be used in the present invention. Most synthetic or natural antimicrobial peptides are generally cationic, with some examples having a net charge of up to + 9. The cationic and hydrophobic components of the Antimicrobial Peptide mix to make it well suited for interacting with and interfering with the cytoplasmic membranes of microorganisms that normally exhibit an anionic surface, lipid-rich, such as phosphatidylglycerol or cardiolipin (see William C. Wimley, descriptive of Antimicrobial Peptide Action with the Interfacial activity model, ACS Chem biol. 2010; 5(10): 905-.

The binding of the antimicrobial peptide to the microbial membrane is significant due to electrostatic interactions, whereas the binding of the antimicrobial peptide to the neutral phosphatidylcholine/cholesterol/sphingomyelin-rich surface of animal plasma membranes is weaker. After 1 hour or more of exposure to AMP, the microbial membrane structure is completely disrupted.

The present invention provides an antibacterial adhesive protein coating composition for cosmetic puffs, in which microbial contamination is sufficiently suppressed without using any other antibacterial agent such as silver nanoparticles or antibiotics. Microbial contamination is a major problem in cosmetic adapters (adaptors), such as cosmetic puffs. Contamination may come from the user and their surroundings. The antimicrobial adhesive protein-coated surface can prevent such microbial contamination by providing an antimicrobial peptide that acts only on the surface of the microbial membrane and not on the plasma membrane of human cells. The antimicrobial coating composition comprises a mussel adhesive protein as an adhesive protein, which mussel adhesive protein is functionalised with one or two antimicrobial peptides which can be recombinantly introduced into the C-terminus, N-terminus or both the C-terminus and N-terminus of the adhesive protein. The antimicrobial peptide may be selected from the group consisting of KLWKKWAKKWLKLWKA (SEQ ID NO: 16), FALALKALKKL (SEQ ID NO: 17), ILRWPWPWRRK (SEQ ID NO: 18), AKRHHGYKRKFH (SEQ ID NO: 19), KWKLFKKIGAVLKVL (SEQ ID NO: 20), LVKLVAGIKKFLKWK (SEQ ID NO: 21), IWSILAPLGTTLVKLVAGIGQQKRK (SEQ ID NO: 22), GIGAVLKVLTTGLPALISWI (SEQ ID NO: 23), SWLSKTAKKGAVLKVL (SEQ ID NO: 24), KKLFKKILKYL (SEQ ID NO: 25), GLKKLISWIKRAAQQG (SEQ ID NO: 26), GWLKKIGKKIERVGQHTRDATIQGLGIAQQAANVAATAR (SEQ ID NO: 27).

In one embodiment, a coating composition for an antimicrobial cosmetic puff is provided comprising a mussel adhesive protein functionalized with antimicrobial peptide KLWKKWAKKWLKLWKA (seq ID No.: 16) to inhibit bacterial growth on the surface of the cosmetic puff. In another embodiment, a coating composition for an antimicrobial cosmetic puff is provided comprising mussel adhesive proteins functionalized with antimicrobial peptide KLWKKWAKKWLWLKLWKA (seq ID No. 16) and AKRHHGYKRKFH (seq ID No. 19), respectively, to inhibit the broad-range growth of bacteria on the surface of the cosmetic puff.

Methods for preparing the antimicrobial adhesive proteins are disclosed in PCT/KR2016/008677, KR 10-2015-0179898 and PCT/KR2017/002980 patent applications, which are incorporated herein by reference in their entirety.

The invention also relates to an antibacterial coating composition for a cosmetic puff. According to the present invention, there is provided an antibacterial cosmetic puff coated with an antibacterial adhesive protein. The antimicrobial coating composition may be applied to any cosmetic puff made from: polyurethane (PUR), acrylonitrile butadiene rubber (NBR), Styrene Butadiene Rubber (SBR), Natural Rubber (NR), styrene-isoprene-styrene (SIS), styrene-ethylene-butylene-styrene (SEBS), polyvinyl alcohol, nitrile rubber, butyl rubber, chloroprene rubber, or combinations thereof. In one embodiment, a method for automatically solidifying a composition of antimicrobial adhesive proteins is provided. The antimicrobial mussel adhesive protein is enzymatically treated to convert tyrosine residues to DOPA residues, then freeze-dried and the DOPA-containing antimicrobial adhesive protein is dissolved in distilled water at a concentration of 0.1 to 3% (wt/wt), more preferably 0.1 to 0.3% (wt/wt).

In another embodiment, a method of rapidly solidifying a composition of antimicrobial adhesive proteins without regard to pH is provided.

In the prior art, it is well known that DOPA autooxidizes to dopaquinone when the pH of a polymer or protein containing DOPA residues rises above about 5. It is well known that this autooxidation is faster at higher pH. In protein binder based compositions containing DOPA, low pH will cause the binder to set too slowly. Previous strategies to increase the speed of adhesion were to increase the pH (as disclosed in WO 2003/051418), to decrease the pH and use concentrated solutions (as disclosed in WO 2004/005421), or to add periodate ions (as disclosed in WO 2003/080137).

A polydopamine coating produced by mussel adhesive protein was prepared in a buffered polydopamine solution at pH 8.5. The incubation time for coating various substrates in polydopamine is about 18 hours, which is a very inefficient method (See See Sileika et al, Antibacterial performance of polypamine-modified polymer surface treating and active component, ACS application. Mater. interfaces,2011,3(12), 4602-materials 4610). For fast curing, the rate of auto-polymerization of dopamine increases with increasing temperature from 20 ℃ to 60 ℃. (see Zhou P et al, (2014) Rapid-disposed polypamine coating a right and a visorous polishing: formation, chromatography and biochemical evaluation. plos ONE 9(11): e 113087).

For rapid solidification, rapid evaporation of the buffer is required to promote autoxidation of DOPA to dopaquinone for the crosslinking reaction. As the buffer solution evaporated, we obtained a concentrated DOPA-containing solution. In addition, heating the solution containing concentrated DOPA can cause rapid solidification thereof.

Any alcohol-containing buffer can be used to prepare the antimicrobial adhesive protein coating solution for rapid evaporation. For example, ethanol, propanol, isopropanol may be used in the present invention. Preferably, a buffer containing ethanol is used to prepare an antibacterial adhesive protein coating composition for allowing the cosmetic puff to be rapidly coated. The percentage of alcohol ranges from 10% to 90%. Preferably, the ethanol content in the buffer is in the range of 50% to 70%. In one embodiment, an antimicrobial composition is provided comprising 0.05 mg of antimicrobial binding protein dissolved in 70% ethanol buffer to make a coated cosmetic puff within 30 minutes. When heated to 60 ℃, the incubation time for applying the cosmetic puff is reduced to 5 minutes without loss of antimicrobial activity.

The present invention provides a buffered antimicrobial adhesive protein as a coating composition, comprising a buffer solution based on a sodium salt such as sodium acetate or sodium bicarbonate, wherein the antimicrobial adhesive protein is dissolved at a concentration of 0.02 to 0.1 mg/ml.

The present invention also provides a buffered solution comprising an alcohol comprising at least one salt that can facilitate an antimicrobial coating. Some salts (e.g., sodium acetate) cause the mussel adhesive protein to aggregate into nano-to micro-sized particles that are coated onto the surface of the cosmetic puff. The rapid evaporation of the coating solution allows the antimicrobial adhesive protein particles to be rapidly applied to the surface of the cosmetic puff. The above salt is selected from sodium acetate (CH)₃COONa), sodium bicarbonate (NaHCO)₃) Sodium carbonate (Na)₂CO₃) Sodium phosphate (Na)₃PO₄) Sodium hydroxide (NaOH), potassium acetate, potassium carbonate, potassium bicarbonate, potassium phosphate, and potassium hydroxide. In one embodiment, the concentration of the anti-microbial adhesion protein in the coating composition is 0.02 to 0.1 mg/ml, wherein the anti-microbial adhesion protein is dissolved in 10mM sodium acetate buffer (wherein ethanol is 50 wt%).

The present invention also provides a composition comprising an antimicrobial adhesive protein and L-dopamine dissolved in a base buffer or an alcohol-containing buffer. In one embodiment, the L-dopamine is dissolved in the antimicrobial adhesin solution, wherein the concentration of the L-dopamine ranges from 0.001 mg/ml to 0.1 mg/ml.

The present invention provides a method for applying an antimicrobial adhesive protein coating to the surface of a cosmetic puff, said method comprising the steps of:

a) providing an antimicrobial coating composition comprising an antimicrobial binding protein dissolved in an alcohol buffer, wherein the concentration of said polymer in said composition ranges from 0.01 to 0.2 mg/ml, more preferably from 0.02 to 0.1 mg/ml,

b) applying a coating composition to the surface to be coated to obtain an antimicrobial coating on at least a portion of the surface of the cosmetic puff, and

c) drying the antimicrobial adhesive protein-coated cosmetic puff.

The present invention provides a method for rapidly applying an antibacterial coating on the surface of a cosmetic puff which is rapidly cured within 10 minutes. The present invention provides a method for applying an antimicrobial adhesive protein coating to the surface of a cosmetic puff, said method comprising the steps of:

a) providing a composition comprising an alcoholic solution of an antimicrobial adhesion protein and wherein the concentration of said polymer in said composition ranges from 0.01 to 0.2 mg/ml, more preferably from 0.02 to 0.1 mg/ml,

b) applying the composition to the surface of a cosmetic puff, and

c) the cosmetic puff coated with the adhesive was dried at 60 ℃.

The following examples are provided to illustrate preferred embodiments of the present invention and are not intended to limit the scope of the invention by the particular embodiments described herein, which are presented for illustrative purposes only. Functionally equivalent products, compositions and methods, as described herein, are clearly within the scope of the present invention.

Examples of the invention

Example 1 construction of expression vectors encoding mussel adhesive protein fused to antibacterial peptide

The expression vector is designed to introduce an antimicrobial peptide at the C-terminus of the recombinant mussel adhesive protein. Nucleic acids encoding antimicrobial peptides were purchased from Cosmogentec co. The constructed expression vector was transfected into E.coli BL21(DE3), and Table 1 lists the antimicrobial peptides introduced at the C-terminus of mussel adhesive protein.

[ Table 1]

Antibacterial peptide sequence for introducing mussel adhesive protein

Example 2 preparation of antibacterial mussel adhesive protein

2.1 protein expression

Coli BL21(DE) was cultured in LB medium (5g/L yeast extract, 10g/L trypsin and 10g/L NaCl) and IPTG was added to a final concentration of 1mM when the optical density of the culture broth was 0.6 at 600nm, thereby inducing the expression of recombinant antibacterial peptide-fused mussel adhesive protein. The E.coli BL21(DE) culture was centrifuged at 13,000rpm for 4 to 10 minutes to obtain a cell aggregate, which was stored at-80 ℃.

The cell aggregates were resuspended in 100. mu.l of SDS-PAGE buffer (0.5M Tris-HCl, 10% glycerol, 5% SDS, 5% β -mercaptoethanol, 0.25% bromophenol blue at pH 6.8), denatured by boiling for 5 minutes at 100 ℃ for SDS-PAGE analysis, samples were electrophoresed on 15% SDS-polyacrylamide gels, and protein bands were detected using Coomassie blue staining.

2.2 purification

The cell aggregates of example 2.1 were stirred with lysis buffer containing 2.4g/L sodium dihydrogen phosphate, 5.6g/L disodium hydrogen phosphate, 10mM EDTA and 1% Triton X-100 and disrupted using a high pressure homogenizer. The lysate was centrifuged through a centrifugal filter unit at 9,000rpm for 20 minutes to obtain an insoluble protein complex containing mussel adhesive protein. The surfactant adhesive protein eluted from the insoluble complex was centrifuged at 25% (v/v) acetic acid concentration under the same conditions (9,000rpm, 20 minutes) to obtain a supernatant. The obtained supernatant was centrifuged at pH 12.8 under the same conditions (9,000rpm, 20 minutes) by adding 10N NaOH. The supernatant was neutralized with acetic acid at pH 6-7 and then centrifuged under the same conditions to obtain precipitated mussel adhesive protein. And dissolving the precipitate in distilled water, and freeze-drying to obtain the freeze-dried mussel adhesive protein with the purity of 90%.

2.3 enzymatic conversion of tyrosine residues to DOPA residues

The lyophilized surfactant mussel adhesive protein is dissolved in 0.1M acetate buffer containing 20mM ascorbic acid and 20mM sodium borate to a concentration of 1 mg/ml, and then the surfactant adhesive protein solution is saturated with oxygen for 30 minutes by adding oxygen to the solution. After adding 40-1. mu.g tyrosinase per antimicrobial adhesion protein (preferably 40-1. mu.g per adhesion protein), it was shaken under oxygen for one hour. After one hour, the chemical modification reaction was terminated by adding 5% acetic acid to the solution. The terminated surfactant adhesive protein solution is freeze-dried to obtain a lyophilized powder. By this process, the tyrosine residue of the adhesion protein is modified to DOPA.

EXAMPLE 3 preparation of antibacterial mussel adhesive protein coated cosmetic powder puff

The antibacterial adhesive protein prepared in example 2.3 was dissolved in distilled water to form antibacterial coating solutions of 1. mu.g/ml, 5. mu.g/ml and 10. mu.g/ml, respectively. The polyurethane-based cosmetic puff was applied by 1) spray coating and 2) dip coating, and then dried on a clean bench at room temperature. For dip coating, cosmetic puff samples were immersed in the antimicrobial coating solution for 30 minutes, 6 hours, and 12 hours, respectively, and then dried on a clean bench at room temperature.

EXAMPLE 4 antimicrobial Activity assay of antimicrobial cosmetic powder puff

To evaluate the antibacterial effect of the antibacterial cosmetic puff prepared in example 3 on SA (staphylococcus aureus), a puff containing 1 × 10 was prepared⁴CFU/ml Staphylococcus aureus PBS, and these organisms were inoculated into each sample for 18 h. Antibacterial evaluation was performed according to KS K0693 as a standard protocol. As a control sample, a commercial puff manufactured and sold by innisfreeco.

[ Table 2]

Summary of antimicrobial activity of antimicrobial coated puffs under various conditions

Condition	Sample (BioVit)	Control (Innisfree)
			As received	99.9％	99.9％
Water washing,. times.3	99.9％	99.9％
			Cleaning with detergent,. times.3	93.9％	78.9％

In the comparative experiment, it was observed that the antibacterial adhesive protein-coated puff showed excellent antibacterial activity and could solve the durability problem, compared to the commercial product of Innisfree. Existing products (e.g., the Innisfree product) are typically silver nanoparticle based products, and the continuous release of silver nanoparticles from cosmetic puffs can cause durability problems.

EXAMPLE 5 high-speed antimicrobial coating composition

Various coating conditions were prepared for two different concentrations of antimicrobial adhesion protein and are summarized in table 3 below.

[ Table 3]

70ml of pure ethanol (Sigma Aldrich, St.Louis, USA) and 30ml of distilled water were mixed to prepare 70 vol% ethanol solution. 5 mg of the anti-microbial adhesin having the sequence KLWKKWAKKWLKLWKA (SEQ ID NO: 16) at the C-terminus was dissolved in 50ml of 70 vol% ethanol buffer to prepare a 0.1 mg/ml anti-microbial adhesin coating solution, which was then diluted to 0.05 mg/ml and 0.025 mg/ml by adding another 70 vol% ethanol buffer. Dopamine hydrochloride 98% was purchased from sigmaldrich (st louis usa) and used as such. Dissolving L-dopamine in 70 vol% ethanol buffer solution to prepare 0.01 mg/ml, 0.1 mg/ml and 1 mg/ml dopamine solution respectively. First, a dopamine solution (1ml) was applied to the hydrophobic surface of a polystyrene petri dish to increase wettability, and the antibacterial mussel adhesive protein was rapidly introduced onto the dopamine coated surface of the hydrophobic polystyrene petri dish. The antimicrobial adhesive protein coating solution (3ml) was added to each petri dish for coating. After 10 minutes of incubation, the coating solution was removed from the petri dish and the coated petri dish was dried at room temperature for 30 minutes. The resulting dried petri dishes were used for further antimicrobial activity analysis. The antimicrobial effect of the antimicrobial coated surface is shown in fig. 2, where the growth of e.coli and s.aureus bacteria is completely inhibited.

EXAMPLE 6 antimicrobial Activity assay of coated surfaces

The coated surfaces prepared in example 5 were evaluated for antimicrobial activity using the gram negative bacteria (e.coli) and the gram positive bacteria Staphylococcus Aureus (SA). According to JIS Z2801: 2000 the antimicrobial activity of the coated surfaces was determined as described by Haldar et al in Nature Protocols 2007, 2(19), 2412. Basically, JIS Z2801: 2000 is generally expressed as the antimicrobial value calculated from the difference between the Log10 number of Colony Forming Units (CFU) on the treated surface and the Log10 number of Colony Forming Units (CFU) measured on the untreated surface.

As shown by e.coli in fig. 4A and SA in fig. 4B, growth of both bacteria was completely inhibited, indicating that the addition of dopamine did not affect the antimicrobial efficacy, whereas dopamine coated surfaces might contribute/accelerate the coating of the surface of interest with the antimicrobial adhesion protein.

EXAMPLE 7 antimicrobial coating of powder puff surface at Room temperature

An antimicrobial coating solution and a dopamine solution were prepared as described in example 5.

5ml of the prepared coating solution was dispensed into separate 60mm by 15mm polystyrene petri dishes, respectively. A cosmetic puff based on polyurethane foam manufactured and sold by Dessin is purchased from a convenience store of seoul, korea. Each cosmetic puff was immersed in the dopamine solution and then immediately immersed in the antimicrobial coating solution for 10 seconds, 30 seconds, 60 seconds, and 180 seconds, respectively. After dip coating, all puffs were dried at 60 ℃ for 10 minutes.

As shown in fig. 4, no color change was observed in the applied cosmetic puff.

EXAMPLE 8 antibacterial Activity assay of high-speed antibacterial cosmetic puff

A series of cosmetic puffs coated with antibacterial adhesive protein prepared according to example 7 were subjected to antibacterial testing in accordance with JIS Z2801.

After inoculating the puff with E.coli, the bacteria were counted for Colony Forming Units (CFU). As shown in fig. 5, the naked cosmetic puff (control) did not inhibit the growth of bacteria, while the antibacterial cosmetic puff showed significant inhibition of the growth of bacteria in a short application time.

Even if the growth of bacteria could not be completely inhibited in this test, the antibacterial efficacy could be enhanced by increasing the coating time or increasing the concentration of the antibacterial adhesion protein, as shown in example 6.

EXAMPLE 9 durability test

The antibacterial coated cosmetic puff prepared according to example 7 was cut into 4cm x 4cm and exposed to different number of washing cycles (3 and 10) with or without detergent. A commercially available antibacterial cosmetic puff was used as a control. One water wash cycle corresponds to one home wash with a washing machine.

The antibacterial activity was measured according to the procedure described in example 8 and the results are shown in the figure; 5A is a puff without an antimicrobial coating, 5B is an antimicrobial adhesive protein coated puff, and 5C is a commercial antimicrobial puff. The puff without the antimicrobial coating showed bacterial growth, while the two antimicrobial cosmetic puffs inhibited bacterial growth. After washing with water, the antibacterial adhesive protein-coated puff showed significant antibacterial activity even after 10 washes, while the commercial antibacterial puff showed less antibacterial activity, depending on the number of washing cycles.

<110> Bye Ouwei Tech Ltd

<120> biocompatible composition for rapid synthesis of antibacterial cosmetic puff

And method for preparing the same

<130>2018-OPA-1460/PCT

<150>KR 10-2017-0084822

<151>2017-07-04

<160>27

<170>KoPatentIn 3.0

<210>1

<211>10

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> model peptides derived from tandem repeat decapeptide of foot protein 1 (FP-1, Mytilus edulis)

<400>1

Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys

1 5 10

<210>2

<211>20

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> 2-fold repetitive sequence derived from podoprotein 1 (FP-1, edible mussel)

<400>2

Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro

1 5 10 15

Pro Thr Tyr Lys

20

<210>3

<211>60

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> 6-fold repetitive sequence derived from podoprotein 1 (FP-1, edible mussel)

<400>3

Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro

1 5 10 15

Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys

20 25 30

Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr

35 40 45

Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys

50 55 60

<210>4

<211>39

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> partial sequence of Pedopodium protein type 2 (FP-2, Mytilus galloprovincialis, Calif.)

<400>4

Glu Val His Ala Cys Lys Pro Asn Pro Cys Lys Asn Asn Gly Arg Cys

1 5 10 15

Tyr Pro Asp Gly Lys Thr Gly Tyr Lys Cys Lys Cys Val Gly Gly Tyr

20 25 30

Ser Gly Pro Thr Cys Ala Cys

35

<210>5

<211>52

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> foot protein type 3 (FP-3, edible mussel)

<400>5

Ala Asp Tyr Tyr Gly Pro Lys Tyr Gly Pro Pro Arg Arg Tyr Gly Gly

1 5 10 15

Gly Asn Tyr Asn Arg Tyr Gly Gly SerArg Arg Tyr Gly Gly Tyr Lys

20 25 30

Gly Trp Asn Asn Gly Trp Lys Arg Gly Arg Trp Gly Arg Lys Tyr Tyr

35 40 45

Glu Phe Glu Phe

50

<210>6

<211>46

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> foot protein type 3 (FP-3, Mediterranean mussel: mgfp-3A)

<400>6

Ala Asp Tyr Tyr Gly Pro Lys Tyr Gly Pro Pro Arg Arg Tyr Gly Gly

1 5 10 15

Gly Asn Tyr Asn Arg Tyr Gly Arg Arg Tyr Gly Gly Tyr Lys Gly Trp

20 25 30

Asn Asn Gly Trp Lys Arg Gly Arg Trp Gly Arg Lys Tyr Tyr

35 40 45

<210>7

<211>60

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> partial sequence from foot protein type 4 (California mussel)

<400>7

Gly His Val His Arg His Arg Val Leu His Lys His Val His Asn His

1 5 10 15

Arg Val Leu His Lys His Leu His Lys His Gln Val Leu His Gly His

20 25 30

Val His Arg His Gln Val Leu His Lys His Val His Asn His Arg Val

35 40 45

Leu His Lys His Leu His Lys His Gln Val Leu His

50 55 60

<210>8

<211>75

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> foot protein type 5 (FP-5, edible mussel)

<400>8

Ser Ser Glu Glu Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Ala Tyr His

1 5 10 15

Tyr His Ser Gly Gly Ser Tyr His Gly Ser Gly Tyr His Gly Gly Tyr

20 25 30

Lys Gly Lys Tyr Tyr Gly Lys Ala Lys Lys Tyr Tyr Tyr Lys Tyr Lys

35 40 45

Asn Ser Gly Lys Tyr Lys Tyr Leu Lys Lys Ala Arg Lys Tyr His Arg

50 5560

Lys Gly Tyr Lys Lys Tyr Tyr Gly Gly Ser Ser

65 70 75

<210>9

<211>76

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> foot protein 5 (FP-5, edible mussel)

<400>9

Ser Ser Glu Glu Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Thr Tyr His

1 5 10 15

Tyr His Ser Gly Gly Ser Tyr His Gly Ser Gly Tyr His Gly Gly Tyr

20 25 30

Lys Gly Lys Tyr Tyr Gly Lys Ala Lys Lys Tyr Tyr Tyr Lys Tyr Lys

35 40 45

Asn Ser Gly Lys Tyr Lys Tyr Leu Lys Lys Ala Arg Lys Tyr His Arg

50 55 60

Lys Gly Tyr Lys Lys Tyr Tyr Gly Gly Gly Ser Ser

65 70 75

<210>10

<211>71

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> foot protein 5 (FP-5, Mytilus coruscus)

<400>10

Tyr Asp Asp Tyr Ser Asp Gly Tyr Tyr Pro Gly Ser Ala Tyr Asn Tyr

1 5 10 15

Pro Ser Gly Ser His Trp His Gly His Gly Tyr Lys Gly Lys Tyr Tyr

20 25 30

Gly Lys Gly Lys Lys Tyr Tyr Tyr Lys Phe Lys Arg Thr Gly Lys Tyr

35 40 45

Lys Tyr Leu Lys Lys Ala Arg Lys Tyr His Arg Lys Gly Tyr Lys Lys

50 55 60

His Tyr Gly Gly Ser Ser Ser

65 70

<210>11

<211>76

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Mytilus edulis adhesive protein foot protein type 5 from (Mediterranean mussel)

<400>11

Ser Ser Glu Glu Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Thr Tyr His

1 5 10 15

Tyr His Ser Gly Gly Ser Tyr His Gly Ser Gly Tyr His Gly Gly Tyr

20 25 30

Lys Gly Lys Tyr Tyr Gly Lys Ala Lys Lys Tyr Tyr Tyr Lys Tyr Lys

35 40 45

Asn Ser Gly Lys Tyr Lys Tyr Leu Lys Lys Ala Arg Lys Tyr His Arg

50 55 60

Lys Gly Tyr Lys Lys Tyr Tyr Gly Gly Gly Ser Ser

65 70 75

<210>12

<211>99

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> mussel adhesive protein foot protein type 6

<400>12

Gly Gly Gly Asn Tyr Arg Gly Tyr Cys Ser Asn Lys Gly Cys Arg Ser

1 5 10 15

Gly Tyr Ile Phe Tyr Asp Asn Arg Gly Phe Cys Lys Tyr Gly Ser Ser

20 25 30

Ser Tyr Lys Tyr Asp Cys Gly Asn Tyr Ala Gly Cys Cys Leu Pro Arg

35 40 45

Asn Pro Tyr Gly Arg Val Lys Tyr Tyr Cys Thr Lys Lys Tyr Ser Cys

50 55 60

Pro Asp Asp Phe Tyr Tyr Tyr Asn Asn Lys Gly Tyr Tyr Tyr Tyr Asn

65 70 75 80

Asp Lys Asp Tyr Phe Asn Cys Gly Ser Tyr Asn Gly Cys Cys Leu Arg

85 90 95

Ser Gly Tyr

<210>13

<211>194

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Mixed mussel adhesive protein (based on FP-151, MEFP-5: Kollodis)

<400>13

Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro

1 5 10 15

Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys

20 25 30

Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr

35 40 45

Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ser Ser Glu Glu

50 55 60

Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Ala Tyr His Tyr His Ser Gly

65 70 75 80

Gly Ser Tyr His Gly Ser Gly Tyr His Gly Gly Tyr Lys Gly Lys Tyr

85 90 95

Tyr Gly Lys Ala Lys Lys Tyr Tyr Tyr Lys Tyr Lys Asn Ser Gly Lys

100105 110

Tyr Lys Tyr Leu Lys Lys Ala Arg Lys Tyr His Arg Lys Gly Tyr Lys

115 120 125

Tyr Tyr Gly Gly Ser Ser Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys

130 135 140

Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro

145 150 155 160

Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys

165 170 175

Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr

180 185 190

Tyr Lys

<210>14

<211>196

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Mixed mussel adhesive protein (based on FP-151, MGFP-5)

<400>14

Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro

1 5 10 15

Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys

20 25 30

Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr

35 40 45

Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ser Ser Glu Glu

50 55 60

Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Thr Tyr His Tyr His Ser Gly

65 70 75 80

Gly Ser Tyr His Gly Ser Gly Tyr His Gly Gly Tyr Lys Gly Lys Tyr

85 90 95

Tyr Gly Lys Ala Lys Lys Tyr Tyr Tyr Lys Tyr Lys Asn Ser Gly Lys

100 105 110

Tyr Lys Tyr Leu Lys Lys Ala Arg Lys Tyr His Arg Lys Gly Tyr Lys

115 120 125

Lys Tyr Tyr Gly Gly Gly Ser Ser Ala Lys Pro Ser Tyr Pro Pro Thr

130 135 140

Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser

145 150 155 160

Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys

165 170 175

Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro

180 185 190

Pro Thr Tyr Lys

195

<210>15

<211>56

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> adhesion peptides derived from Semibalanus balanoides

<400>15

Met Arg Val Ile Leu Phe Ala Met Leu Ile Gly Gly Ser Leu Ala Cys

1 5 10 15

Gln Asn Arg Leu Glu Thr Leu Val Gln Glu Ala Thr Gly Asn Ala Gly

20 25 30

Asp Leu Ser Thr Asn Val His Glu Glu Cys Asn Ser Gln Val Gly Thr

35 40 45

Phe Asn Ala Val His Ala Pro Gln

50 55

<210>16

<211>16

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> antimicrobial peptide (KLWKKWAKKWLKLWKA)

<400>16

Lys Leu Trp Lys Lys Trp Ala Lys Lys Trp Leu Lys Leu Trp Lys Ala

1 5 10 15

<210>17

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> antimicrobial peptide (FALALKALKKL)

<400>17

Phe Ala Leu Ala Leu Lys Ala Leu Lys Lys Leu

1 5 10

<210>18

<211>12

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> antimicrobial peptide (ILRWPPWWRRK)

<400>18

Ile Leu Arg Trp Pro Trp Trp Pro Trp Arg Arg Lys

1 5 10

<210>19

<211>12

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> antimicrobial peptide (AKRHHGYKRKFH)

<400>19

Ala Lys Arg His His Gly Tyr Lys Arg Lys Phe His

1 5 10

<210>20

<211>15

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> antimicrobial peptide (KWKLFKKIGAVLKVL)

<400>20

Lys Trp Lys Leu Phe Lys Lys Ile Gly Ala Val Leu Lys Val Leu

1 5 10 15

<210>21

<211>15

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> antimicrobial peptide (LVKLVAGIKKFLKWK)

<400>21

Leu Val Lys Leu Val Ala Gly Ile Lys Lys Phe Leu Lys Trp Lys

1 5 10 15

<210>22

<211>25

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> antimicrobial peptide (IWSILAPLGTTLVKLVAGIGQQKRK)

<400>22

Ile Trp Ser Ile Leu Ala Pro Leu Gly Thr Thr Leu Val Lys Leu Val

1 5 10 15

Ala Gly Ile Gly Gln Gln Lys Arg Lys

20 25

<210>23

<211>20

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> antimicrobial peptide (GIGAVLKVLTTGLPALISWI)

<400>23

Gly Ile Gly Ala Val Leu Lys Val Leu Thr Thr Gly Leu Pro Ala Leu

1 5 10 15

Ile Ser Trp Ile

20

<210>24

<211>16

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> antimicrobial peptide (SWLSKTAKKGAVLKVL)

<400>24

Ser Trp Leu Ser Lys Thr Ala Lys Lys Gly Ala Val Leu Lys Val Leu

1 5 10 15

<210>25

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> antimicrobial peptide (KKLFKKILKYL)

<400>25

Lys Lys Leu Phe Lys Lys Ile Leu Lys Tyr Leu

1 5 10

<210>26

<211>16

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> antimicrobial peptide (GLKKLISWIKRAAQQG)

<400>26

Gly Leu Lys Lys Leu Ile Ser Trp Ile Lys Arg Ala Ala Gln Gln Gly

1 5 10 15

<210>27

<211>39

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> antibacterial peptide

(GWLKKIGKKIERVGQHTRDATIQGLGIAQQAANVAATAR)

<400>27

Gly Trp Leu Lys Lys Ile Gly Lys Lys Ile Glu Arg Val Gly Gln His

1 5 10 15

Thr Arg Asp Ala Thr Ile Gln Gly Leu Gly Ile Ala Gln Gln Ala Ala

20 25 30

Asn Val Ala Ala Thr Ala Arg

35