A composition for inhibiting lipofuscin accumulation or removing lipofuscin, containing a metalloproteinase derived from a Chryseobacterium strain.

Abstract

To provide a composition containing a metalloproteinase derived from a strain of Chryseobacterium as an active ingredient for anti-aging of the skin, skin whitening, improvement of skin texture, inhibition of lipofuscin accumulation, and / or removal of lipofuscin. [Solution] The present invention discloses a composition for anti-aging, skin whitening, or skin texture improvement, comprising a metalloproteinase derived from a strain of Chryseobacterium as an active ingredient, and / or a composition for inhibiting lipofuscin accumulation or removing lipofuscin, comprising the said metalloproteinase as an active ingredient. The composition provides anti-aging, skin whitening, or skin texture improvement effects by inhibiting lipofuscin accumulation or removing lipofuscin, and exhibits effects that prevent, improve, or treat diseases caused by lipofuscin accumulation.

Description

Technical Field

[0001] The present invention discloses a composition for skin anti-aging, skin whitening, skin texture improvement, inhibition of lipofuscin accumulation and / or removal of lipofuscin, which contains a metalloprotease derived from a Chryseobacterium strain as an active ingredient.

Background Art

[0002] Lipofuscin is a metabolite related to aging and oxidative stress and exists in the form of an insoluble pigment in cells. It is known that lipofuscin is generated by the accumulation of insufficiently decomposed substances in cells due to oxidative stress and cell deterioration. The more lipofuscin accumulates in cells, the more the cell function declines and aging is promoted.

[0003] The heart of the elderly appears brown and atrophied due to an increase in pigmentation by lipofuscin, which is called "brown atrophy of the heart". Similar pigmentation may also be seen in the liver, which is called "brown atrophy of the liver". Furthermore, cases of pigmentation by lipofuscin in the heart, liver, and kidneys have been reported in patients with wasting diseases such as cancer and chronic severe pulmonary tuberculosis. On the other hand, pigmentation such as liver spots and senile plaques is a prominent sign of skin aging, and these are phenomena caused by the accumulation of lipofuscin. Therefore, lipofuscin is also called "senile plaque in the body". Thus, inhibiting the production and accumulation of lipofuscin in skin cells is extremely important in preventing skin aging.

[0004] As mentioned earlier, lipofuscin, which is naturally produced during cellular metabolism, accumulates in cells more frequently with age, as well as due to a decline in cellular function or repeated exposure to external toxins. Furthermore, accumulated lipofuscin inhibits normal cellular function and accelerates aging. Lipofuscin is present not only in skin cells but also in the liver, kidneys, myocardium, retina, adrenal glands, nerve cells, and ganglion cells, and is known to accumulate in important organs, causing various degenerative diseases. For example, the accumulation of lipofuscin in the myocardium can degrade it and potentially cause myocardial infarction, while the accumulation of lipofuscin in the retina and nerve cells may cause macular degeneration and Alzheimer's disease. In addition, accumulation in hormone-secreting organs such as the adrenal glands, liver, and pituitary gland may inhibit the metabolic function of hormones such as growth hormone and sex hormones.

[0005] The presence of lipofuscin observed in the skin is considered a sign of aging, and research is underway to find cosmetic ingredients that can help mitigate it. However, to date, no cosmetic ingredients that directly break down lipofuscin are known, and only indirect effects through specific ingredients that reduce oxidative stress and promote skin cell regeneration are expected. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Korean Patent Publication No. 10-1887631 [Patent Document 2] Korean Patent Publication No. 10-2018-0035682 [Overview of the project] [Problems that the invention aims to solve]

[0007] In one embodiment, the present disclosure aims to provide a composition for anti-aging, skin lightening, or skin texture improvement that contains a metalloprotease derived from a strain of the genus Chryseobacterium as an active ingredient.

[0008] In another embodiment, the present disclosure aims to provide a composition for inhibiting lipofuscin accumulation or removing lipofuscin, comprising a metalloprotease derived from a strain of the genus Chryseobacterium as an active ingredient. [Means for solving the problem]

[0009] In one embodiment, the present disclosure provides a composition for anti-aging, skin lightening, or skin texture improvement, comprising a metalloprotease derived from a strain of the genus Chryseobacterium as an active ingredient.

[0010] In one exemplary implementation, the strain may be a Chryseobacterium camelliae strain.

[0011] In one exemplary implementation, the strain may be the Chryseobacterium camelliae Dolsongi-HT1 strain deposited under accession number KCCM11883P.

[0012] In one exemplary implementation, the metalloproteinase may include the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 22.

[0013] In one exemplary embodiment, the composition may provide anti-aging, skin whitening, or skin texture improvement effects by inhibiting lipofuscin accumulation or removing lipofuscin.

[0014] In another embodiment, the Disclosure provides a composition for inhibiting lipofuscin accumulation or removing lipofuscin, comprising a metalloprotease derived from a strain of the genus Chryseobacterium as an active ingredient.

[0015] In one exemplary implementation, the strain may be a Chryseobacterium camelliae strain.

[0016] In one exemplary implementation, the strain may be the Chryseobacterium camelliae Dolsongi-HT1 strain deposited under accession number KCCM11883P.

[0017] In one exemplary implementation, the metalloproteinase may include the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 22.

[0018] In one exemplary embodiment, the composition may prevent, improve, or treat a disease caused by lipofuscin accumulation.

[0019] In one exemplary realization, the disease resulting from lipofuscin accumulation may include at least one selected from the group consisting of sarcopenia, progeria, Alzheimer's disease, Parkinson's disease, Lou Gehrig's disease (amyotrophic lateral sclerosis; ALS), myocardial infarction, age-related macular degeneration, neuronal ceroid lipofuscinoses (NCL), acromegaly, denervation atrophy, and chronic obstructive pulmonary disease (COPD). [Effects of the Invention]

[0020] In one embodiment, the technology disclosed in the present invention has the effect of providing a component that can degrade lipofuscin. Metalloproteinases derived from Chryseobacterium strains have the effect of directly degrading lipofuscin accumulated in cells.

[0021] In another embodiment, the technology disclosed in the present invention provides a composition for anti-aging, skin lightening, or skin texture improvement that contains a metalloprotease derived from a strain of Chryseobacterium as an active ingredient.

[0022] In another embodiment, the technology disclosed in the present invention has the effect of providing a composition for inhibiting lipofuscin accumulation or removing lipofuscin, comprising a metalloprotease derived from a strain of the genus Chryseobacterium as an active ingredient. [Brief explanation of the drawing]

[0023] [Figure 1] It is a diagram showing a partial sequence of 16S rDNA of Chryseobacterium camelliae - dorsonii - HT1 strain according to an embodiment. [Figure 2] It is a diagram showing the results of evaluating keratinolytic activity according to an embodiment. [Figure 3] It is a diagram showing the results of evaluating lipofuscin - removing activity according to an embodiment.

Mode for Carrying Out the Invention

[0024] Hereinafter, the present invention will be described in detail.

[0025] In this specification, the "active ingredient" means a component that alone exhibits a desired activity or can exhibit a desired activity together with a carrier or the like that is inactive by itself.

[0026] In one aspect, the present disclosure provides a composition for skin anti - aging, skin whitening (lightening) and / or improving skin texture, which contains a metalloprotease derived from a strain of the genus Chryseobacterium as an active ingredient.

[0027] In another aspect, the present disclosure provides a method for skin anti - aging, skin whitening and / or improving skin texture, which includes applying or administering to a subject in need thereof a composition containing an effective amount of a metalloprotease derived from a strain of the genus Chryseobacterium for skin anti - aging, skin whitening and / or improving skin texture.

[0028] In another aspect, the present disclosure provides a composition containing a metalloprotease derived from a strain of the genus Chryseobacterium for use in skin anti - aging, skin whitening and / or improving skin texture.

[0029] In other embodiments, the disclosure provides uses for compositions comprising metalloproteases derived from Chryseobacterium strains for anti-aging, skin whitening, and / or improvement of skin texture.

[0030] In another embodiment, the Disclosure provides applications for compositions comprising metalloprotease derived from Chryseobacterium strains in the manufacture of compositions for anti-aging, skin whitening, and / or improving skin texture.

[0031] In another embodiment, the disclosure provides the use of a composition comprising a metalloprotease derived from a strain of Chryseobacterium in the manufacture of a composition for improving melasma.

[0032] In another embodiment, the disclosure provides the use of a composition containing a metalloprotease derived from a strain of Chryseobacterium in the manufacture of a food composition for improving melasma.

[0033] In one exemplary embodiment, the composition or metalloproteinase may provide anti-aging, skin whitening, and / or skin texture improvement effects by inhibiting lipofuscin accumulation or removing lipofuscin.

[0034] In one exemplary implementation, the anti-aging of the skin, skin whitening, and / or improvement of skin texture may be achieved by inhibiting lipofuscin accumulation or removing lipofuscin.

[0035] In one exemplary implementation, the improvement of melasma may be achieved by inhibiting lipofuscin accumulation or removing lipofuscin.

[0036] In this specification, "anti-aging" means a use that prevents, delays, and / or improves aging phenomena caused by internal factors, including genetic factors, and external factors, including ultraviolet radiation. The composition provides an effect of preventing, improving, and / or treating diseases or symptoms associated with cellular aging by reducing the level of cellular senescence. For example, the anti-aging may be the prevention, delay, and / or improvement of aging caused by lipofuscin accumulation.

[0037] In one exemplary implementation, the composition or metalloproteinase may prevent, improve and / or treat liver spots and / or melasma.

[0038] In one exemplary implementation, the composition or metalloproteinase may help to brighten, whiten, or clear the skin by inhibiting or removing lipofuscin accumulation, thereby contributing to skin whitening and / or improvement of skin tone.

[0039] In another embodiment, the Disclosure provides a composition for inhibiting lipofuscin accumulation or removing lipofuscin, comprising a metalloprotease derived from a strain of the genus Chryseobacterium as an active ingredient.

[0040] In another embodiment, the Disclosure provides a method for inhibiting lipofuscin accumulation or removing lipofuscin, comprising applying or administering a composition containing a metalloprotease derived from a strain of Chryseobacterium effective in inhibiting lipofuscin accumulation or removing lipofuscin to a subject requiring such treatment.

[0041] In one exemplary implementation, the method may improve or treat a disease caused by lipofuscin accumulation.

[0042] In one exemplary implementation, the method may have an anti-aging effect on the subject.

[0043] In one exemplary implementation, the method may lighten the skin color of the subject.

[0044] In one exemplary implementation, the method may improve the texture of the target skin.

[0045] In another embodiment, the Disclosure provides a composition comprising a metalloprotease derived from a strain of the genus Chryseobacterium for use in inhibiting lipofuscin accumulation or removing lipofuscin.

[0046] In another embodiment, the disclosure provides uses for compositions comprising metalloproteases derived from Chryseobacterium strains for inhibiting lipofuscin accumulation or removing lipofuscin.

[0047] In another embodiment, the disclosure provides applications for compositions comprising metalloprotease derived from Chryseobacterium strains in the manufacture of lipofuscin accumulation inhibitory or lipofuscin removal compositions.

[0048] In one exemplary implementation, the composition or metalloproteinase may prevent, improve, or treat a disease caused by lipofuscin accumulation.

[0049] In one exemplary implementation, the composition or metalloproteinase may have an anti-aging effect on the subject.

[0050] In one exemplary embodiment, the composition or metalloproteinase may lighten the skin color of the subject.

[0051] In one exemplary implementation, the composition or metalloproteinase may improve the texture of the target skin.

[0052] In one exemplary realization, the disease resulting from lipofuscin accumulation may include at least one selected from the group consisting of sarcopenia, progeria, Alzheimer's disease, Parkinson's disease, Lou Gehrig's disease (amyotrophic lateral sclerosis; ALS), myocardial infarction, age-related macular degeneration, neuronal ceroid lipofuscinoses (NCL), acromegaly, denervation atrophy, and chronic obstructive pulmonary disease (COPD).

[0053] In one exemplary implementation, the strain may be a Chryseobacterium camelliae strain.

[0054] In one exemplary implementation, the strain may be the Chryseobacterium camelliae Dolsongi-HT1 strain deposited under accession number KCCM11883P.

[0055] In one exemplary implementation, the strain may be isolated from green tea leaves.

[0056] In one exemplary implementation, the Chryseobacterium camelliae dolsoni-HT1 strain may have the 16S rDNA subsequence sequence of Sequence ID No. 3.

[0057] In one exemplary implementation, the metalloproteinase may include the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 22.

[0058] In one exemplary implementation, the metalloproteinase may consist of the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 22.

[0059] Peptides necessary for releasing peptides and proteins produced within a cell to the outside of the cell are called signal peptides or signal sequences. These play a role in guiding proteins produced within the cell to the cell wall and ultimately promoting secretion across the cell membrane. When proteins are secreted outside the cell, the signal peptide is cleaved, and the remaining portion forms a structure that performs its function.

[0060] The metalloproteinase relating to this disclosure may be represented by the amino acid sequence of SEQ ID NO: 22, which includes the signal sequence of SEQ ID NO: 21. Sequence ID 21 MKKNFNFCCLAGAIAVFSLTA Sequence ID 22 MKKNFNFCCLAGAIAVFSLTACNDNSTEDNNLLQQPQAESAKVEQPNEREKACYYVDQNWNSSAVLKTTLKTSTDTNFMNGQMTKIASLWGRNNPTLRFVDDPSSPNSTYNAISYSTGKIYYGYAIYYDAKNKGGDIVNAMILAHEYG HQLQYIFGLPSVNESTARPNELEADGFAGYYLRRPEGYNKTQFSEIAAAYEFAQSIGDYATTSPGHHGTPQRRSAVRLGFLLGQYNLSASDFDYNFFYYYQGVLNGTYKMGKNSRNPELDAYMMQYIDELRKIQSGEISAEEFKQLK

[0061] In one exemplary implementation, the composition containing the metalloproteinase derived from the Chryseobacterium strain may be a cosmetic composition, an oral composition, a non-therapeutic oral composition, or a pharmaceutical composition.

[0062] In one exemplary implementation, the composition comprising the metalloproteinase derived from the Chryseobacterium strain may be applied, ointed, or administered to a target in the form of a cosmetic composition, an oral composition, a non-therapeutic oral composition, or a pharmaceutical composition.

[0063] According to one exemplary implementation, the composition containing the metalloproteinase derived from the Chryseobacterium strain may be a cosmetic composition.

[0064] The cosmetic composition may further contain functional additives and components commonly found in cosmetic compositions. The functional additives may include components selected from the group consisting of water-soluble vitamins, oil-soluble vitamins, high molecular weight peptides, high molecular weight polysaccharides, sphingolipids, and seaweed extracts. Other components that may be included include oils and fats, moisturizers, emollients, surfactants, organic and inorganic pigments, organic powders, UV absorbers, preservatives, bactericides, antioxidants, plant extracts, pH adjusters, alcohols, dyes, fragrances, blood circulation promoters, cooling agents, antiperspirants, and purified water.

[0065] The cosmetic composition is not particularly limited in its formulation and may be appropriately selected depending on the purpose. For example, it may be manufactured as at least one formulation selected from the group consisting of lotion, skin softener, toner, astringent, emulsion, lotion, moisturizing lotion, nourishing lotion, massage cream, nourishing cream, moisturizing cream, hand cream, foundation, serum, nourishing essence, pack, soap, facial cleansing foam, facial cleansing lotion, facial cleansing cream, body lotion, and body soap, but is not limited to these.

[0066] If the formulation of the composition is a paste, cream, or gel, animal fibers, plant fibers, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc, or zinc oxide may be used as the carrier component.

[0067] When the formulation of the composition is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder may be used as the carrier component, and especially in the case of a spray, a propellant such as hydrochlorofluorocarbon, propane / butane, or dimethyl ether may be further included.

[0068] When the formulation of the above composition is a solution or emulsion, a solvent, solvating agent, or emulsifying agent is used as the carrier component, for example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerin aliphatic ester, polyethylene glycol, or fatty acid ester of sorbitan.

[0069] When the formulation of the above composition is a suspension, the carrier component may be a liquid diluent such as water, ethanol, or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, or polyoxyethylene sorbitan ester, or microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, or tragacanth.

[0070] When the formulation of the above composition is a surfactant-containing cleanser, the carrier component may be an aliphatic alcohol sulfate, an aliphatic alcohol ether sulfate, a sulfosuccinate monoester, isethionate, imidazolium derivative, methyl taurate, sarcosinate, fatty acid amide ether sulfate, alkylamide betaine, aliphatic alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, ethoxylated glycerin fatty acid ester, etc.

[0071] According to one exemplary implementation, the composition comprising the metalloproteinase derived from the Chryseobacterium strain may be an oral composition or a non-therapeutic oral composition.

[0072] The oral composition or non-therapeutic oral composition may be a liquid or solid formulation, and components commonly used in the art may be added without difficulty to the formulation or intended use, as selected by those skilled in the art, and a synergistic effect may occur when applied simultaneously with other raw materials.

[0073] The liquid formulation may contain various flavorings and natural carbohydrates as additional components, similar to ordinary beverages. Examples of natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. Suitable flavorings include natural flavorings (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavorings (e.g., saccharin, aspartame, etc.). The proportion of natural carbohydrates may generally be about 1 to 20 g per 100 mL of the composition, or 5 to 12 g in a uniform form.

[0074] The oral composition or non-therapeutic oral composition may, in one embodiment, contain various supplements, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, coloring agents and flavor enhancers (such as cheese and chocolate), pectin acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, and carbonating agents used in carbonated beverages. In another embodiment, it may contain fruit pulp for the production of natural fruit juices and vegetable beverages. The components may be used independently or in combination. The proportion of the additives may vary, but is generally selected in the range of 0.001 to about 20 parts by weight per 100 parts by weight of the composition.

[0075] According to one exemplary implementation, the composition containing the metalloproteinase derived from the Chryseobacterium strain may be a pharmaceutical composition.

[0076] The pharmaceutical composition may further contain pharmaceutical adjuvants such as preservatives, stabilizers, hydrates or emulsifiers, salts and / or buffers for osmotic pressure adjustment, and other therapeutically useful substances, and may be formulated in various oral or parenteral forms according to conventional methods.

[0077] The oral preparations mentioned above include, for example, tablets, pills, hard and soft capsules, liquids, suspensions, emulsifiers, syrups, powders, granules, pellets, etc. These preparations may contain surfactants, diluents (e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, and glycine), lubricants (e.g., silica, talc, stearic acid and its magnesium or calcium salts, and polyethylene glycol). Tablets may further contain binders such as aluminum magnesium silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, and polyvinylpyrrolidine, and may optionally contain pharmaceutical additives such as disintegrants, absorbents, colorants, flavorings, and sweeteners, such as starch, agar, alginic acid or its sodium salt. The tablets may be manufactured by conventional mixing, granulation, or coating methods.

[0078] Furthermore, the form of parenteral administration may be a transdermal formulation, such as an injection, infusion, ointment, lotion, gel, cream, spray, suspension, emulsion, or patch, but is not limited to these.

[0079] The determination of the dosage of the active ingredient is within the scope of those skilled in the art, and the daily dose of the drug varies depending on various factors such as the progression of the disease, the time of onset, age, health condition, and complications of the patient to be treated. However, when using an adult as a reference, in one embodiment, the composition may be administered at a dose of 1 μg / kg to 200 mg / kg, and in other embodiments, at a dose of 50 μg / kg to 50 mg / kg, divided into 1 to 3 doses per day.

[0080] The aforementioned pharmaceutical composition may be a topical skin preparation, and the term "topical skin preparation" is a general term that may include any preparation applied to the skin, and may encompass a variety of pharmaceutical formulations.

[0081] In one exemplary embodiment, the composition containing the metalloproteinase derived from the Chryseobacterium strain may contain 0.0001 to 99.99% by weight of the metalloproteinase derived from the Chryseobacterium strain, based on the total weight of the composition.

[0082] In another exemplary embodiment, the composition containing the metalloproteinase derived from the Chryseobacterium strain may contain, with respect to the total weight of the composition, 0.0001% by weight or more, 0.0005% by weight or more, 0.001% by weight or more, 0.005% by weight or more, 0.01% by weight or more, 0.05% by weight or more, 0.1% by weight or more, or 0.5% by weight or more of the metalloproteinase derived from the Chryseobacterium strain, and may contain 20% by weight or less, 15% by weight or less, 10% by weight or less, 5% by weight or less, 1% by weight or less, 0.5% by weight or less, 0.1% by weight or less, 0.05% by weight or less, 0.01% by weight or less, 0.005% by weight or less, or 0.001% by weight or less.

[0083] For example, the composition containing the metalloproteinase derived from the Chryseobacterium strain may contain, with respect to the total weight of the composition, 0.0001 to 1% by weight, 0.0001 to 0.5% by weight, 0.0001 to 0.1% by weight, 0.0001 to 0.05% by weight, 0.0001 to 0.01% by weight, 0.0001 to 0.005% by weight, 0.0001 to 0.001% by weight, 0.0005 to 0.001% by weight, or 0.001 to 0.005% by weight of the metalloproteinase derived from the Chryseobacterium strain.

[0084] The present disclosure will be described in more detail below with reference to examples. These examples are merely illustrative and it will be obvious to those with ordinary skill in the art that the scope of the present disclosure is not limited in any way by these examples.

[0085] [Example 1] Preparation of culture solution for Chryseobacterium strains In this embodiment, a culture medium of the Chryseobacterium strain was prepared as follows, using the Chryseobacterium camelliae strain as the Chryseobacterium strain. The Chryseobacterium camelliae strain deposited under accession number KCCM11883P was the Chryseobacterium camelliae dorsoni-HT1 strain. The strain can be obtained according to the isolation and identification method described below, as described in Patent Document 2. This document is incorporated herein by reference in its entirety.

[0086] (1) Isolation of microorganisms from green tea To select microorganisms with plant-derived keratin-degrading activity, microorganisms were cultured at 30°C or 37°C using a minimal liquid medium (M9 medium containing 0.5% soluble keratin: CaCl2 0.015 g / L, Na2HPO4 6.78 g / L, KH2PO4 3 g / L, NaCl 0.5 g / L, MgSO4 0.5 g / L) with keratin as the carbon and nitrogen sources.

[0087] The aforementioned method for selecting microorganisms using minimal culture medium is a method for selecting target microorganisms based on their growth rate. Since microorganisms with low keratinase activity are naturally removed during the culture stage, it is possible to select active microorganisms that possess high-activity enzymes. This suggests that microorganisms that can grow in minimal culture medium possess keratinase activity.

[0088] Therefore, microorganisms with keratin-degrading activity were isolated from green tea grown in the Dolseongi tea garden on Jeju Island, South Korea, using a limited culture medium.

[0089] (2) Identification of isolated microorganisms To identify the isolated microorganisms possessing keratinase activity, their species and genus names were determined by analyzing the DNA sequences encoding the 16S ribosomal RNA sub-sequences of the microorganisms.

[0090] Microorganisms were cultured in liquid medium, and then their gene DNA was extracted. This DNA was then amplified by PCR (polymerase chain reaction) using the 27F(5'-AGAGTTTGATCMTGGCTCAG-3'; SEQ ID NO: 1) / 1492R(5'-TACGGYTACCTTGTTACGACTT-3'; SEQ ID NO: 2) primer, which is commonly used for bacterial identification, and its sequence was analyzed.

[0091] Sequence analysis revealed 99% homology to the 16S rRNA partial sequence of Chryseobacterium camelliae strain THG C4-1 (KACC16985; Sequence accession no. (16S rRNA) JX843771) and 98% homology to the 16S rRNA partial sequence of Chryseobacterium taiwanense.

[0092] Therefore, the novel microbial strain isolated and cultured was confirmed to be Chryseobacterium camelliae by molecular phylogenetic analysis based on its 16S rDNA sequence, and was named Chryseobacterium camelliae Dolsongi-HT1. The novel microbial strain was deposited with the Korea Center for Microbial Conservation (KCCM), the depositary institution, on September 1, 2016, under accession number KCCM11883P. Figure 1 shows the 16S rDNA partial sequence of the Chryseobacterium camelliae Dolsongi-HT1 strain (SEQ ID NO: 3).

[0093] (3) Production of microbial culture solution Chryseobacterium camelliae dolsoni-HT1 strain, derived from green tea, was seed-cultured for 15 hours. Subsequently, 10 mL of the cultured strain was inoculated into 1 L of optimal medium (1% Tryptone / 1% NaCl / 0.5% Skim milk) and cultured at 30°C and 200 rpm for 15 hours. After culturing, a culture solution from which microorganisms had been removed was obtained.

[0094] [Example 2] Enzyme purification and sequence analysis from culture medium of Chryseobacterium strains. From the culture medium of the Chryseobacterium strain prepared in Example 1, proteins with a size of 10 kDa or larger were obtained by ultrafiltration using a 10 kDa cut-off size filter. To select proteins with proteolytic activity, the proteins were isolated using ion exchange resin (Q-sepharose(R) HP column, Cytica, Marlborough, MA, USA) and analyzed by mass spectrometer (Q Exactive). TM Proteins in the active fraction were analyzed using a Plus mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). A total of 120 proteins were analyzed.

[0095] Since the whole genome of the Chryseobacterium cameliae dolsoni-HT1 strain had already undergone whole genome analysis, eight proteins named proteases were selected from the analyzed proteins by comparing the whole genome analysis results with the protein analysis results. The eight selected proteins were converted into recombinant proteins as follows, and their keratin-degrading activity was compared.

[0096] First, the gene sequences of the eight proteins listed in Table 1 below were amplified by PCR using forward and reverse primers, and then each enzyme was expressed using the Bacillus subtilis Secrection protein expression system (TaKaRa(R)). The vector used for the recombinant protein production was the pBE-S vector, and the genes were introduced into the vector using the In-Fusion Cloning system. The gene transfer vector was introduced into the host B. subtilis RIK1285. To express the protein, the expression strain was cultured at 37°C for 24 hours. After culturing, the microbial cells were removed, the culture medium was collected, concentrated by ultrafiltration, and then freeze-dried to obtain the sample.

[0097] [Table 1]

[0098] Keratinase activity was measured using 0.5% (wt / v) keratin azure and pH 8.0 Tris-HCl buffer. When keratin azure is broken down by keratinase, the azure dye is released, and the reaction solution turns blue. Therefore, to compare the activity levels, the absorbance at 595 nm was measured (absorbance 0.01 = 1 unit).

[0099] As a result, keratinase activity was observed in MetalloP and ContP1 from two protein samples, numbers 5 and 6, confirming that MetalloP and ContP1 have an effect of improving skin texture through exfoliation (see Figure 2). In particular, MetalloP showed remarkably high keratinase activity. On the other hand, ContP2 from protein sample number 4 had the highest content among the purified proteins, but showed no keratinase activity at all.

[0100] As described above, enzymes possessing keratinase activity were purified from the Chryseobacterium camelliae dolsoni-HT1 strain isolated from green tea leaves, and their sequences were compared with the NCBI database. This confirmed that MetalloP and ContP1 are enzymes belonging to the metalloprotease series. The purified enzyme MetalloP was confirmed to have the sequence of Sequence ID No. 20. The sequence of MetalloP can be represented by Sequence ID No. 22, which includes the signal sequence of Sequence ID No. 21, which is suitable for the expressing strain. The sequence of ContP1 can be represented by Sequence ID No. 24, which includes the signal sequence of Sequence ID No. 23. The sequence of ContP2 can be represented by Sequence ID No. 26, which includes the signal sequence of Sequence ID No. 25. Sequence ID 20 CNDNSTEDNNLLQQPQAESAKVEQPNEREKACYYVDQNWNSSAVLKTTLKTSTDTNFMNGQMTKIASLWGRNNPTLRFVDDPSSPNSTYNAISYSTGKIYYGYAIYYDAKNKGGDIVNAMILAHEYGHQLQYIFGLP SVNESTARPNELEADGFAGYYLRRPEGYNKTQFSEIAAAYEFAQSIGDYATTSPGHHGTPPQRRSAVRLGFLLGQYNLSASDFDYNFFYYYQGVLNGTYKMGKNSRNPELDAYMMQYIDELRKIQSGEISAEEFKQLK Sequence ID 21 MKKNFNFCCLAGAIAVFSLTA Sequence ID 22 MKKNFNFCCLAGAIAVFSLTACNDNSTEDNNLLQQPQAESAKVEQPNEREKACYYVDQNWNSSAVLKTTLKTSTDTNFMNGQMTKIASLWGRNNPTLRFVDDPSSPNSTYNAISYSTGKIYYGYAIYYDAKNKGGDIVNAMILAHEYG HQLQYIFGLPSVNESTARPNELEADGFAGYYLRRPEGYNKTQFSEIAAAYEFAQSIGDYATTSPGHHGTPQRRSAVRLGFLLGQYNLSASDFDYNFFYYYQGVLNGTYKMGKNSRNPELDAYMMQYIDELRKIQSGEISAEEFKQLK Sequence ID 23 MKKLLLGALMLGFMSACNS Sequence ID 24 MKKLLLGALMLGFMSACNSDNIANQHEQSDNLVSPTGGSALQRGCASEEVRKIALQNSPELRQRFSALEAQTEKFENDLKLGKVLSDGSVEIPVVVNVLYRTSSENLSDSRIAEQIAVLNADYAGTNSDVSKIPSEFQSVKSGDVKVKFRLANTVRKSTTKTSWS TNDAMKRSSSGGIDATSPSNYLNIWVVGNMGQILGYATFPESAGMWNDGVVIAAPYFGKTGASSPFNLGRTATHEVGHYLNLRHIWGDANCGNDLVADTPTQTGANSGKPNYPLYNTCSGVQRSVMFMNYMDYVDDAAMFMFSAGQRTRMQSVVASSGPRSGLRLY Sequence ID 25 MKTMLKSILTVALLWSAVS Sequence ID 26 MKTMLKSILTVALLWSAVSCNTNEMSDINSSAQTQTSNSGNFAAKASDNPENPLNNWEECGKKHNVILTYVITYQKTLDNSGLTNSEIADKCIMASNNYFNQKYAQDYNNTQSPFTVAKIRSILNDGDNKFSNVIQGMNA SDKVKAKTSELVNMMYNLASTDKNTEYAEVKARIVDFEAAGIMNDASLSQAEKDQLLQTTSIARYSSYLWSNTIDHSDSVSSVTGKKKWWKWVVVAVCDFAGGAAGTAAGGGVLSVAGAIAGAAGASSGGAALVDWISPDAP

[0101] [Example 3] Evaluation of lipofuscin-degrading activity of metalloproteinases derived from Chryseobacterium strains. The enzymes MetalloP, ContP1, and ContP2, which were overexpressed in recombinant bacterial strains prepared in Example 2, were used to treat lipofuscin-inducing cells as follows, and their lipofuscin removal effects were evaluated. ContP1 is an enzyme with approximately 50% of the keratin-degrading effect compared to MetalloP, and ContP2 is an enzyme with no keratin-degrading effect.

[0102] Fibroblasts were grown to 90% confluent, and then cultured in growth medium with 40 μM leupeptin, 45 μM FeCl3, and 10 μM H2O2 for 10 days to accumulate lipofuscin within the cells. The culture medium was changed every two days. After 10 days of culture, each sample was treated with a 10 ppm solution for 7 days. The degree of lipofuscin degradation was measured using a confocal laser scanning microscope (LSM 980, Carl Zeiss, Oberkochen, Germany) equipped with a FITC (fluorescein isothiocyanate) 540 / 40 filter.

[0103] As a result, as shown in Figure 3, lipofuscin removal was observed in the experimental groups (MetalloP, ContP1) in which cells with accumulated lipofuscin were treated with metalloproteinases. In particular, in the experimental group treated with MetalloP, the accumulated lipofuscin was completely removed, confirming that MetalloP exhibited the most superior lipofuscin degradation activity.

[0104] The following describes examples of formulations of compositions relating to one aspect of this disclosure, but these are applicable to a variety of other formulations. This is not intended to limit the disclosure, but rather to provide specific examples.

[0105] [Formulation Example 1] Softening Lotion A softening lotion was prepared by mixing 0.01% by weight of the metalloproteinase MetalloP obtained in Example 2, 3% by weight of glycerin, 2% by weight of butylene glycol, 2% by weight of propylene glycol, 0.1% by weight of carboxyvinyl polymer, 10% by weight of ethanol, 0.1% by weight of triethanolamine, a small amount of preservative, a small amount of pigment, a small amount of fragrance, and the remainder of purified water, according to a conventional manufacturing method.

[0106] [Example of formulation 2] Nourishing lotion 0.01% by weight of the metalloproteinase MetalloP obtained in Example 2, 4% by weight of beeswax, 1.5% by weight of polysorbate 60, 0.5% by weight of sorbitan sesquiole, 5% by weight of liquid paraffin, 5% by weight of squalane, 5% by weight of caprylic / capric triglyceride, 3% by weight of glycerin, 3% by weight of butylene glycol, 3% by weight of propylene glycol, 0.1% by weight of carboxyvinyl polymer, 0.2% by weight of triethanolamine, a small amount of preservative, a small amount of pigment, a small amount of fragrance, and the remainder of purified water were mixed together, and a nourishing lotion was produced according to a normal manufacturing method.

[0107] [Formulation Example 3] Nutritional Cream A nourishing cream was prepared by mixing 0.01% by weight of the metalloproteinase MetalloP obtained in Example 2, 10% by weight of beeswax, 1.5% by weight of polysorbate 60, 0.5% by weight of sorbitan sesquiole, 10% by weight of liquid paraffin, 5% by weight of squalane, 5% by weight of caprylic / capric triglyceride, 5% by weight of glycerin, 3% by weight of butylene glycol, 3% by weight of propylene glycol, 0.2% by weight of triethanolamine, a small amount of preservative, a small amount of pigment, a small amount of fragrance, and the remainder of purified water, according to a conventional manufacturing method.

[0108] [Formulation Example 4] Pack A pack was prepared by mixing 0.01% by weight of the metalloproteinase MetalloP obtained in Example 2, 13% by weight of polyvinyl alcohol, 0.2% by weight of sodium carboxymethylcellulose, 0.1% by weight of allantoin, 5% by weight of ethanol, 0.3% by weight of nonylphenyl ether, a small amount of preservative, a small amount of pigment, a small amount of fragrance, and the remainder of purified water, and following a conventional manufacturing method.

[0109] [Example of formulation 5] Topical drug (patch) A topical drug (patch) was manufactured according to the composition shown in Table 2 below, using a standard manufacturing method.

[0110] [Table 2]

[0111] [Formulation Example 6] Powder In Example 2, 2 g of the metalloproteinase MetalloP was mixed with 1 g of lactose, and then the mixture was filled into an airtight bag to produce a powder.

[0112] [Formulation Example 7] Tablets 100 mg of the metalloproteinase MetalloP obtained in Example 2, 100 mg of corn starch, 100 mg of lactose, and 2 mg of magnesium stearate were mixed, and then tablets were manufactured by compressing them according to a standard manufacturing method.

[0113] [Formulation Example 8] Capsules 100 mg of the metalloproteinase MetalloP obtained in Example 2, 100 mg of corn starch, 100 mg of lactose, and 2 mg of magnesium stearate were mixed, and then the mixture was filled into gelatin capsules according to a standard manufacturing method to produce capsules.

[0114] [Formulation Example 9] Pill In Example 2, 1 g of the metalloproteinase MetalloP, 1.5 g of lactose, 1 g of glycerin, and 0.5 g of xylitol were mixed, and then pills were prepared according to a standard method to contain 4 g per pill.

[0115] [Formulation Example 10] Granules 150 g of the metalloproteinase MetalloP obtained in Example 2, 50 mg of soybean extract, 200 mg of glucose, and 600 mg of starch were mixed, then 100 mg of 30% ethanol was added, and the mixture was dried at 60°C to form granules, which were then filled into bags to produce a granular preparation.

[0116] [Formulation Example 11] Drinkable Medication 50 mg of the metalloproteinase MetalloP obtained in Example 2, 10 g of glucose, 0.6 g of citric acid, and 25 g of liquid oligosaccharide were mixed, and then 300 mL of purified water was added. 200 mL of this mixture was then filled into each bottle. After filling the bottles, the drink was sterilized at 130°C for 4-5 seconds to produce the beverage.

[0117] [Formulation Example 12] Caramel 50 mg of the metalloproteinase MetalloP obtained in Example 2, 1.8 g of corn syrup, 0.5 g of skim milk powder, 0.5 g of soy lecithin, 0.6 g of butter, 0.4 g of hydrogenated vegetable oil, 1.4 g of sugar, 0.58 g of margarine, and 20 mg of salt were mixed and molded to produce caramel.

[0118] Although specific parts of this disclosure have been described in detail above, it will be clear to those with ordinary skill in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of this disclosure. Therefore, the substantial scope of this disclosure should be defined by the appended claims and their equivalents.

[0119] [Accession Number] Depository name: Korea Microbial Conservation Center Accession number: KCCM11883P Entrustment date: September 1, 2016

Claims

1. A composition for anti-aging, skin whitening, or skin texture improvement, containing a metalloproteinase derived from a strain of the genus Chryseobacterium as an active ingredient.

2. The composition for skin anti-aging, skin whitening, or skin texture improvement according to claim 1, wherein the bacterial strain is the Chryseobacterium camelliae strain.

3. The composition for skin anti-aging, skin whitening, or skin texture improvement according to claim 1, wherein the strain is the Chryseobacterium camelliae Dolsongi-HT1 strain deposited under accession number KCCM11883P.

4. The composition for skin anti-aging, skin whitening, or skin texture improvement according to claim 1, wherein the metalloproteinase comprises the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO:

22.

5. The composition for skin anti-aging, skin whitening, or skin texture improvement according to claim 1, wherein the composition provides skin anti-aging, skin whitening, or skin texture improvement effects by inhibiting lipofuscin accumulation or removing lipofuscin.

6. A composition for inhibiting lipofuscin accumulation or removing lipofuscin, containing a metalloproteinase derived from a strain of the genus Chryseobacterium as an active ingredient.

7. The lipofuscin accumulation inhibitory or lipofuscin removal composition according to claim 6, wherein the strain is a strain of Chryseobacterium camelliae.

8. The lipofuscin accumulation inhibitory or lipofuscin removal composition according to claim 6, wherein the strain is the Chryseobacterium camelliae Dolsongi-HT1 strain deposited under accession number KCCM11883P.

9. The composition for inhibiting lipofuscin accumulation or removing lipofuscin according to claim 6, wherein the metalloproteinase comprises the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO:

22.

10. The composition for inhibiting lipofuscin accumulation or removing lipofuscin according to claim 6, wherein the composition prevents, improves, or treats diseases caused by lipofuscin accumulation.

11. Diseases caused by the accumulation of lipofuscin include sarcopenia, progeria, Alzheimer's disease, Parkinson's disease, Lou Gehrig's disease (amyotropic lateral sclerosis; ALS), myocardial infarction, age-related macular degeneration, neuronal ceroid lipofuscinosis (NCL), acromegaly, and denervation. A lipofuscin accumulation inhibitory or lipofuscin removal composition according to claim 10, comprising at least one selected from the group consisting of astrophy and chronic obstructive pulmonary disease (COPD).

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