# US 10,945,940 B2 · Method of preparing ultra-low molecular weight keratin peptide

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- Number: US 10,945,940 B2
- Document ID: US10945940B2
- Status: Granted
- Kind: B2
- Application number: 15/771,747
- Filed: 2016-10-31
- Published: 2018-12-06
- Granted: 2021-03-16
- Earliest claimed: 2015-10-29
- Assignees: Kyungpook National University Industry-Academic Cooperation Foundation
- Applicants: Kyungpook National University Industry-Academic Cooperation Foundation
- Inventors: Dong Woo Lee; Nam Joo Kang; Yong Jik Lee; Hyeon Su Jin; In Hyuk Yeo; Kyeong Seop Song; Jae-Eun Lee
- Web page: https://patentgrep.com/patent/US10945940B2

## CPC

- A61K 8/65: Collagen; Gelatin; Keratin; Derivatives or degradation products thereof
- A23L 33/18: Peptides; Protein hydrolysates
- A61Q 19/08: Anti-ageing preparations
- B01D 15/34: Size-selective separation, e.g. size-exclusion chromatography; Gel filtration; Permeation
- B01D 15/361: {Ion-exchange}
- C07K 1/16: by chromatography
- C07K 1/18: Ion-exchange chromatography
- C07K 1/34: by filtration, ultrafiltration or reverse osmosis
- C07K 1/36: by a combination of two or more processes of different types
- C07K 14/4741: {Keratin; Cytokeratin}
- C12P 21/06: produced by the hydrolysis of a peptide bond, e.g. hydrolysate products (preparing foodstuffs by protein hydrolysis A23J3/00)
- A23V 2002/00: Food compositions, function of food ingredients or processes for food or foodstuffs
- B01D 61/145: {Ultrafiltration}
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A23L: FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF (preservation thereof A23B)
- A61Q: SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- B01D: SEPARATION (separating solids from solids by wet methods B03B, B03D; by pneumatic jigs or tables B03B; by other dry methods B07; magnetic or electrostatic separation of solid materials from solid materials or fluids, separation by high-voltage electric fields B03C; centrifuges, vortex apparatus B04; presses per se for squeezing-out liquid from liquid-containing material B30B9/02; treatment of water C02F, e.g. softening by ion-exchange C02F1/42; {arrangements of air intake cleaners in gas turbine plants F02C7/05} ; arrangements or mounting of filters in air-conditioning, air-humidification or ventilation F24F13/28)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- A23V: INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION

## Abstract

The present disclosure relates to methods of preparing an ultra-low molecular weight keratin peptide and use thereof. In particular, the present disclosure relates to a method of preparing an ultra-low molecular weight keratin peptide using culturing a microorganism having keratinolytic activity in a medium including keratin, ultrafiltration, ion exchange chromatography and gel filtration chromatography, a peptide prepared by the method, and a cosmetic and food composition for preventing or improving skin aging or skin wrinkles including the same. According to the method of preparing a keratin peptide of the present invention, it is possible to eco-friendly biologically treat waste resources and efficiently purify and recover anti-aging functional ultra-low molecular weight keratin peptides. In addition, the keratin peptide of the present disclosure breaks down collagen to have abilities to inhibit MMP-1 expression and activity, which is an enzyme that causes skin aging, which has an excellent effect on anti-skin aging and skin wrinkle improvement and has no toxicity to skin cells. It is suitable for use as a cosmetic, pharmaceutical or food composition for preventing, improving or treating skin aging or skin wrinkles, thereby being effectively used for the efficient and rapid production and development of high value-added functional cosmetic substances.

## Claims

### Claim 1

A method for preventing or improving skin aging or skin wrinkles, the method comprising the step of treating a skin of subject with a cosmetic composition comprising a peptide comprising at least one sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 9.

### Claim 2 (depends on 1)

The method of claim 1, wherein the peptide is prepared by a method comprising the steps of:
(1) obtaining keratin hydrolysates by culturing a microorganism having keratinolytic activity in a culture medium including keratin;
(2) fractionating a protein having a molecular weight of 100 Da to 1000 Da in the keratin hydrolysates obtained from step (1);
(3) purifying the protein by performing ion exchange chromatography, gel filtration chromatography or a combination thereof on the fractionated product obtained from step (2); and
(4) purifying and desalting the protein by performing gel filtration chromatography on the purified protein obtained from step (3).

### Claim 3 (depends on 2)

The method of claim 2, wherein the microorganism in step (1) is Fervidobacterium islandicum AW-1 (KCTC4680).

### Claim 4 (depends on 2)

The method of claim 2, wherein the keratin in step (1) is derived from a feather, hair, leather, a nail, a claw, a horn or a hoof of a bird or a mammal.

### Claim 5 (depends on 2)

The method of claim 2, wherein the keratin in step (1) is derived from a feather of a bird.

### Claim 6 (depends on 5)

The method of claim 5, wherein the feather is present in the culture medium in an amount of 5 g/L to 15 g/L.

### Claim 7 (depends on 2)

The method of claim 2, wherein the culture in step (1) is an anaerobic culture.

### Claim 8 (depends on 2)

The method of claim 2, wherein the culture in step (1) is a static anaerobic culture.

### Claim 9 (depends on 2)

The method of claim 2, wherein the keratin peptide has an ability to inhibit matrix metalloproteinase-1 (MMP-1) expression or ability to inhibit MMP-1 activity.

### Claim 10 (depends on 2)

The method of claim 2, wherein the culture in step (1) is an anaerobic culture performed under a temperature condition of 60° C. to 90° C.

### Claim 11 (depends on 2)

The method of claim 2, wherein the fraction in step (2) is performed by ultrafiltration.

### Claim 12 (depends on 1)

The method of claim 1, wherein the skin aging is caused by ultraviolet rays.

### Claim 13 (depends on 1)

The method of claim 1, wherein the skin wrinkles is caused by ultraviolet rays.

### Claim 14 (depends on 1)

The method of claim 1, wherein the cosmetic composition comprising a peptide comprises SEQ ID NO: 9.

### Claim 15 (depends on 1)

The method of claim 1, wherein the cosmetic composition comprises a first peptide comprising SEQ ID NO: 9 and a second peptide selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 8.