Method of preparing ultra-low molecular weight keratin peptide

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

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.

2. 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).

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

4. 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.

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

6. 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.

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

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

9. 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.

10. 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.

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

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

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

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

15. 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.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a schematic view of a method of preparing keratin peptides,

(2) FIG. 2 illustrates the results of fractionation of keratin peptides having 1 kDa or less by ion exchange chromatography and gel filtration chromatography,

(3) FIG. 3 illustrates the results of fractionation of keratin peptides having 1 kDa or less using gel filtration chromatography,

(4) FIG. 4A and FIG. 4B illustrate the results (FIG. 4A) of the toxicity test of ultra-low molecular weight peptide of the present disclosure in human dermal fibroblasts and the results (FIG. 4B) measuring an effect of inhibiting MMP-1 expression induced by ultraviolet B (UVB), in which lane 1 was a non-treated control group, lane 2 was a group treated with 20 mJ/cm.sup.2 UVB alone, and lanes 3, 4, 5, 6, 7 and 8 were experimental groups treated with 20 mJ/cm.sup.2 ultraviolet B (UVB) and keratin peptide mixtures (No. 2 and KP 7) at 5, 10 and 20 g/mL of the present disclosure, and

(5) FIGS. 5A-5C illustrate the results (FIG. 5A) of the test on inhibiting MMP-1 activity by the synthetic keratin peptide of Table 3, the results (FIG. 5B) of the toxicity test of human dermal fibroblasts and the results (FIG. 5C) measuring an effect of inhibiting MMP-1 expression induced by ultraviolet B (UVB), in which lane 1 was a non-treated control group, lane 2 was a group treated with 20 mJ/cm.sup.2 UVB alone, and lanes 3 and 4 were experimental groups treated with 20 mJ/cm.sup.2 ultraviolet B (UVB) and synthetic keratin peptide mixtures with 10 M.

DETAILED DESCRIPTION

(6) In the following detailed description, reference is made to the accompanying drawing, which forms a part hereof. The illustrative embodiments described in the detailed description, drawing, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented here.

(7) Hereinafter, the present disclosure is described in more detail with reference to examples. However, these examples are provided to illustrate the present disclosure, and the scope of the present disclosure is not limited to these examples.

Example 1. Preparation of Keratin Peptide

(8) 1.1 Culture and Preparation of Keratin Hydrolysate

(9) For the preparation of a keratin peptide mixture, Fervidobacterium islandicum AW-1 (KCTC4680), a chicken hair degrading strain, was anaerobically cultured as a nitrogen source in a growth medium supplemented with chicken hair at 70 C. as shown in Table 1 below to obtain keratin hydrolysate.

(10) TABLE-US-00001 TABLE 1 Culture components Amount (g/L) NH.sub.4Cl 0.1 NaH.sub.2PO.sub.42H.sub.2O 0.9 MgSO.sub.47H.sub.2O 0.16 K.sub.2HPO.sub.4 1.6 Vitamin solution 10 ml (DSM 141) Trace element solution 10 ml (DSM 141) 0.1% Resazurin 1 ml Yeast extract 1.0 Chicken hair 8.0 Na.sub.2S (25%, w/v) 3 ml

(11) Then, the keratin hydrolysate was first filtered using a filter paper (5 m, No. 20, Hyundai Micro, Korea) to produce the degraded chicken hair remnant and centrifuged at 10,000g for 20 minutes at 4 C. to recover supernatant. The recovered supernatant was used as a sample for the separation and purification of functional ultra-low molecular weight keratin peptides.

(12) 1.2 Purification of Keratin Hydrolysate

(13) Ultrafiltration was used to easily fractionate ultra-low molecular weight keratin peptides having a size of 1 kDa or less from the sample. In the ultrafiltration, membranes with pore size of 10 kDa and 1 kDa were used as ultrafiltration membranes. Specifically, the sample was introduced into a filtration module equipped with a filtration membrane, and a pressure of about 10 to 30 psi was applied to allow the sample to pass through the filtration membrane so that it was filtered. First, proteins having a size of 10 kDa or more was isolated using a 10 kDa ultrafiltration membrane, and proteins having a size of 10 kDa or less flowed out from the filtration module was fractionated into a sample having a size of 1 kDa or more and a sample having a size of 1 kDa or less using an 1 kDa ultrafiltration membrane.

(14) For the fractionated keratin peptide sample having a size of 1 kDa or less, keratin peptides were purified by two methods: i) purification by ion exchange chromatography. The fractionated sample was separated and purified depending on an ionic character by ion exchange chromatography using a Biologic duo-flow FPLC system (Bio-rad) and Macro-prep DEAE support (Bio-Rad) (50 mM Tris-HCl buffer, pH 7.5, 0 M-1 M NaCl). The results are illustrated in FIG. 2. As illustrated in FIG. 2, two peaks were observed at 210 nm, and the relative cationic Fr. 4-47 were pooled and lyophilized. For desalt and separation based on the molecular weight, the lyophilized sample was subjected to gel filtration chromatography (20 mM Tris-HCl buffer, pH 7.0, 100 mM NaCl) using Bio-rad biologic duo-flow FPLC system and superdex 30 pg (GE). Four peaks were observed at 210 nm. Fr. 16-29, expected to include a keratin peptide compared to the reference material, were pooled and lyophilized. Hereinafter, the keratin peptide obtained by the above method is referred to as No. 2;

(15) ii) purification by gel filtration chromatography. The fractionated sample was subjected to gel filtration chromatography in the same manner as described in i). The results are illustrated in FIG. 3. As illustrated in FIG. 3. Fr. 17-26 were pooled and lyophilized. Hereinafter, the keratin peptide obtained by the above method is referred to as KP7.

(16) The absorbance of the samples was measured at 280 nm and 210 nm in order to measure the amounts of protein and peptide upon performing chromatography.

(17) To confirm the desalting and molecular weight of the finally separated and purified No. 2 and KP7 samples, gel filtration chromatography was performed using Superdex peptide 10/300 GL (GE). It was confirmed to be an ultra-low molecular weight keratin peptide having a molecular weight of 1 kDa or less (See FIGS. 2 and 3). Further, they were used in the following examples.

Example 2. Cytotoxicity Test of Keratin Peptide in Human Dermal Fibroblasts and Effect of Inhibiting MMP-1 Expression Induced by UVB

(18) MTT assay was performed using human dermal fibroblasts to examine the cytotoxicity of the keratin peptide prepared by the method of Example 1 as described above.

(19) First, human fibroblasts were divided into 96-well plates in a density of 510.sup.3 cells/well, and each of them was cultured using a medium supplemented with Dulbecco's Modified Eagle's Medium (DMEM) including 10% fetal bovine serum (FBS) and penicillin-streptomycin (GIBCO Invitrogen, Auckland, NZ) under a condition of 37 C. and 5% CO.sub.2 for 6 hours. 200 mg of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) powder was dissolved in 40 mL of PBS and filtered to prepare 5 mg/mL MTT solution. After 6 hours therefrom, the medium including the prepared keratin peptide was added, and after 72 hours, 20 L of 5 mg/ml MTT solution was added. After culture for 3 hours, medium and MTT solution were removed, and 200 L of dimethyl sulfoxide (DMSO) was added and mixed at room temperature for 30 minutes. The absorbance of the reaction mixture was measured at 570 nm using a microplate reader (Sunrise-Basic Tecan, Austria), and the cell survival rate thereof was calculated according to the following Equation 1. The result of calculation of cell survival rate is illustrated in FIG. 4A.
Cell survival rate(%)=100((absorbance of non-treated control groupabsorbance of sample-added group)/absorbance of non-treated control group100)[Equation 1]

(20) As illustrated in FIG. 4A, the keratin peptide of the present disclosure exhibited no cytotoxicity up to a concentration of 100 g/mL.

(21) In order to measure the expression level of MMP-1 in the cells, the keratin peptides of the present disclosure were dissolved in sterilized water at a concentration of 5, 10, and 20 g/mL in human-derived human dermal fibroblast. Then, ultraviolet ray B was irradiated with 20 mJ/cm.sup.2 using Vilber Lourmat (BioLink Crosslinker, France), cultured for 24 hours, and then cell proteins were recovered. At this time, the medium was removed before ultraviolet irradiation, and then washed with phosphate buffered saline (PBS) solution to remove serum components in the medium and then irradiated with ultraviolet ray B. The cells were washed twice with PBS solution to recover the proteins, and then the cells adhered to the bottom were recovered using a lysis buffer and centrifuged at 14,000 rpm for 10 minutes. After each supernatant was recovered, the protein concentration of each cell supernatant was quantified according to the method of use with a protein assay kit (Bio-Rad, USA) using bovine serum albumin (BSA) as the reference material. About 10 mg protein from each of the protein extracts was denatured and separated by 8% gel sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane at 120 mA for 2 hours. Then, the cells were immersed in a TBS-T solution including 5% skim milk for 6 hours to block non-specific proteins, washed once with distilled water and twice with TBS-T. At this time, Matrix Metalloprotease-1 (MMP-1) monoclonal antibody (Neo Markers, Fremont, Calif.) was diluted with a TBS-T solution at a ratio of 1:1,000 to be used and reacted at 4 C. overnight. After the primary antibody reaction, the cells were washed twice with TBS-T for 15 minutes. Horseradishperoxidase (HRP)-bound anti-rabbit IgG (Santacruz, USA) was diluted at a ratio of 1:5,000 to be used as the secondary antibody and reacted at a room temperate for 1 hour and 30 minutes. After the reaction, the cells were washed with TBS-T 4 times for 5 minutes and reacted with ECL substrate (Amersham TM, UK) for 5 minutes. Then, the cells were sensitized to X-ray film to analyze changes in expression of the cell aging-related protein. The results of the above experiment are illustrated in FIG. 4B.

(22) As illustrated in FIG. 4B, when No. 2 was treated at a concentration of 10 g/mL or more and KP7 was treated at a concentration of 5 g/mL or more, it was confirmed that the expression of MMP-1 was significantly inhibited compared to the only UVB-treated group.

(23) Therefore, the keratin peptide of the present disclosure has no toxicity to skin cells and has an excellent effect of inhibiting MMP-1 expression even under a condition of ultraviolet irradiation so that it can be used as a cosmetic or food composition for preventing, improving and treating skin aging and skin wrinkles. In particular, it was confirmed that the keratin peptide could be used for improving skin aging and skin wrinkles caused by UVB.

(24) In addition, in the prior patent (Patent Application No. 10-2014-0092000), it was confirmed that the groups treated keratin peptide of 1 kDa or less at a concentration of 200 g/ml and 400 g/ml, respectively, exhibited about 12% and 41% of an effect of inhibiting MMP-1 expression compared to only UVB-treated group, but No. 2 and KP7 inhibited the expression of MMP-1 at a lower concentration as compared with the former. This indicates that the keratin peptide prepared by the method of purifying ultra-low molecular weight keratin peptide using Example 1 of the present disclosure has a high effect of preventing, improving and treating skin aging and skin wrinkles.

Example 3. Identification and Synthesis of Amino Acid Sequence of Keratin Peptide

(25) The peptides were identified using LC-MS/MS in order to confirm amino acid sequences of the keratin peptides having such activity in No. 2 and KP 7 in which the anti-aging ability and the wrinkle improving ability were confirmed as described above.

(26) Specifically, a feather keratin sequence of chicken (Gallus gallus ver. 5.0) was obtained from Genbank to construct a database for keratin peptide mapping. By transcript analysis of AW-1 strain, selection of proteases involved in degrading the chicken hair and peptide cleavage sites were confirmed, and a database was constructed for analysis of LC-MS/MS results based thereon. As the mass spectrometer, ESI-Q-TOF (Thermo (Dionex) UHPLC Ultimate 3000, ABsciex Triple TOF 5600+) was used. The keratin peptides whose separation, purification and anti-aging ability were evaluated by the method were identified to reveal 16 kinds derived from No. 2 and 8 kinds derived from KP7, which were ultra-low molecular weight peptide having a molecular weight of 1 kDa or less (at least 445.3 Da to at most 710.3 Da). Those are shown in Table 2 below.

(27) TABLE-US-00002 TABLE 2 Theoretical No. Sample Sequence molecular weight 1 No. 2, KP7 GGFGL 449.2 2 No. 2 GGFGI 449.2 3 No. 2 FGGFG 483.2 4 No. 2 GFGGF 483.2 5 No. 2 GPTPL 483.3 6 No. 2 GLGSR 488.3 7 No. 2 PISGGF 576.3 8 No. 2 SFPQN 591.3 9 No. 2 GGFGGFG 597.3 10 No. 2 FPQNT 605.3 11 No. 2 SSGGFGI 623.3 12 No. 2 SSGGFGL 623.3 13 No. 2 SGGFGGF 627.3 14 No. 2 GFGGFGL 653.3 15 No. 2 PISSGGF 663.3 16 No. 2 GGFGGFGL 710.3 17 KP7 GLSGL 445.3 18 KP7 SGGFGI 536.3 19 KP7 GGFGGF 540.2 20 KP7 GVPISS 558.3 21 KP7 SGGFGF 570.2 22 KP7 IQPSPV 639.4 23 KP7 GVPISSGG 672.3 24 KP7 SFPQNT 692.3

(28) Further, for confirming sequences of peptides having anti-aging ability, 9 kinds of peptides were synthesized based on the results of identifying peptides as shown in Table 3 below.

(29) TABLE-US-00003 TABLE 3 No. Name Sequence 1 2n7-1 SGGFG 2 2n7-2 PISS 3 2n7-3 GGFGGFGI 4 2n7-4 GFGGF 5 2n7-5 FPQN 6 KP7-1 GLSGL 7 KP7-2 IQPSPV 8 2-1 GPTPL 9 2-2 GLGSR

Example 4. Evaluation of MMP-1 Enzyme Activity Inhibition in In Vitro Assay of Synthetic Peptide

(30) MMP-1 Fluorometric Drug Discovery Kit manufactured by Enzo company was used to evaluate an ability to inhibit Matrix metalloproteinase-1 (MMP-1) enzyme activity in the peptide synthesized in Example 3. N-Isobutyl-N-(4-methoxyphenylsulfonyl) glycyl hydroxamic acid (NNGH) which is known to inhibit MMP-1 activity was used as a positive control group, and NNGH was treated at a concentration of 0.65 M. As a sample-added group, a mixture solution in which MMP-1 enzyme and keratin peptides were mixed at 1, 5 and 10 g/mL was reacted at 37 C. for 5 minutes. The substrate was added to the reaction mixture, fluorescence thereof was measured at Ex/Em=540/590 nm, and the ability to inhibit MMP-1 activity was evaluated according to the following Equation 2. The results of the ability of inhibiting MMP-1 activity are illustrated in FIG. 5A.
Ability to inhibit MMP-1activity(%)=Fluorescence intensity of sample-added group/Fluorescence intensity of non-treated group100[Equation 2]

(31) As illustrated in FIG. 5A, it was confirmed that the synthetic peptides inhibited the activity of MMP-1 enzyme compared to the group treated with the only MMP-1 enzyme at concentrations of 10 M in 2n7-2, 2n7-4, 2n7-5, KP7-1, KP7-2, 2-1 and 2-2. In particular, it was confirmed that peptide 2n7-4, KP7-1, KP7-2, 2-1 and 2-2 had a significant effect of inhibiting MMP-1 activity.

Example 5. Cytotoxicity Test in Human Fibroblasts and Inhibitory Effect of MMP-1 Expression Induced by Ultraviolet B

(32) In order to examine the cytotoxicity of the peptides synthesized in Example 3, MTT assay using human dermal fibroblast was performed in the same manner as in Example 2. The results of the experiment are illustrated in FIG. 5B.

(33) As illustrated in FIG. 5B, the synthetic peptide of the present disclosure did not exhibit cytotoxicity up to a concentration of 100 g/mL.

(34) In order to measure the expression of MMP-1 in cells, synthetic peptides 2-1 and 2-2 were dissolved in DMSO and treated at a concentration of 10 M in the cultured human dermal fibroblasts derived from human. The method was carried out in the same manner as in Example 2 as described above. The results of measurement of MMP-1 expression are illustrated in FIG. 5C.

(35) As illustrated in FIG. 5C, it was confirmed that when synthetic peptides 2-1 and 2-2 were treated at a concentration of 10 M, the expression of MMP-1 was inhibited by about 24% and 35%, respectively, compared to the group treated with the only UVB.

(36) From the foregoing, it will be appreciated that various embodiments of the present disclosure have been described herein for purposes of illustration and that various modifications may be made without departing from the scope and spirit of the present disclosure. Accordingly, the various embodiments disclosed herein are not intended to be limiting, with the true scope and spirit being indicated by the following claims.