Composition for inhibiting lipofuscin deposition or scavenging lipofuscin containing metalloproteinase derived from strain of Chryseobacterium genus

Abstract

Disclosed in the present disclosure is a composition for skin aging resistance, skin whitening, or skin quality improvement containing, as an active ingredient, a metalloprotease derived from a strain of the genus Chryseobacterium, and / or a composition for inhibiting lipofuscin deposition or eliminating lipofuscin containing, as an active ingredient, the metalloprotease. The composition provides skin anti-aging, skin whitening or skin quality improving efficacy by inhibiting lipofuscin deposition or removing lipofuscin, and provides an effect of preventing, improving or treating diseases caused by lipofuscin deposition.

Description

Technical Field

[0001] This disclosure discloses a composition containing a metalloproteinase derived from a strain of the genus *Chlorella* as an active ingredient, for skin anti-aging, skin whitening, skin texture improvement, inhibition of lipofuscin deposition and / or removal of lipofuscin. Background Technology

[0002] Lipofuscin is a metabolite associated with aging and oxidative stress, existing within cells as an insoluble pigment. It is known to form due to the deposition of incompletely broken-down substances within cells caused by oxidative stress and decreased cellular function. The more lipofuscin deposited within cells, the worse the cell function and the faster the aging process.

[0003] The hearts of long-lived elderly people often appear brown and atrophied due to increased lipofuscin pigment, a phenomenon known as cardiac brown atrophy. The liver can also exhibit similar pigmentation, a condition called hepatic brown atrophy. Furthermore, cases of lipofuscin pigmentation in the heart, liver, and kidneys have been reported in cancer patients or those with chronic wasting diseases such as severe tuberculosis. On the other hand, pigmentation such as melasma and age spots are significant signs of skin aging, also caused by lipofuscin deposition. For this reason, lipofuscin is sometimes referred to as "the body's age spots." Therefore, inhibiting the production and / or deposition of lipofuscin to prevent its accumulation in skin cells is crucial for preventing skin aging.

[0004] As mentioned above, lipofuscin is a naturally occurring substance produced during cellular metabolism. It accumulates not only during aging but also when cellular function declines or when cells are repeatedly exposed to external toxins, leading to its significant deposition within cells. Furthermore, deposited lipofuscin disrupts normal cellular function and accelerates aging. Lipofuscin is not only present in skin cells but also in the liver, kidneys, myocardium, retina, adrenal glands, nerve cells, and ganglion cells. It is known that lipofuscin can deposit in vital organs and cause various degenerative diseases. For example, when lipofuscin deposits in the myocardium, it can worsen the heart muscle, leading to myocardial infarction. When it deposits in the retina or nerve cells, it can cause macular degeneration or Alzheimer's disease. In addition, when lipofuscin deposits in hormone-secreting organs such as the adrenal glands, liver, and pituitary gland, it can cause abnormalities in the metabolism of hormones such as growth hormone and sex hormones.

[0005] The appearance of lipofuscin on the skin is considered a sign of aging, and cosmetic ingredients that can help reduce it are being researched. However, to date, no known cosmetic ingredient can directly break down lipofuscin; instead, indirect effects are hoped for through certain ingredients that primarily reduce oxidative stress and promote skin cell regeneration. Summary of the Invention

[0006] Technical issues

[0007] On the one hand, the purpose of this disclosure is to provide a composition for skin anti-aging, skin lightening or skin texture improvement, which contains a metalloprotease derived from a strain of Chryseobacterium as an active ingredient.

[0008] On the other hand, the purpose of this disclosure is to provide a composition for inhibiting or removing lipofuscin deposition, which contains a metalloprotease derived from a strain of Chryseobacterium as an active ingredient.

[0009] Technical solution

[0010] On the one hand, this disclosure provides a composition for skin anti-aging, skin lightening, or skin texture improvement, which contains a metalloprotease derived from a strain of Chryseobacterium as an active ingredient.

[0011] In one exemplary implementation, the strain may be Chryseobacterium camelliae.

[0012] In one exemplary implementation, the strain may be Chryseobacterium camelliae Dolsongi-HT1, which has the accession number KCCM11883P.

[0013] In one exemplary implementation, the metalloproteinase may comprise an amino acid sequence of sequence number 20 or sequence number 22.

[0014] In one exemplary implementation, the composition may provide anti-aging, skin whitening, or skin texture improvement effects by inhibiting or removing lipofuscin deposition.

[0015] On the other hand, this disclosure provides a composition for inhibiting or removing lipofuscin deposition, which contains a metalloprotease derived from a strain of Chryseobacterium as an active ingredient.

[0016] In one exemplary implementation, the strain may be Chryseobacterium camelliae.

[0017] In one exemplary implementation, the strain may be Chryseobacterium camelliae Dolsongi-HT1, which has the accession number KCCM11883P.

[0018] In one exemplary implementation, the metalloproteinase may comprise an amino acid sequence of sequence number 20 or sequence number 22.

[0019] In one exemplary implementation, the composition may be used to prevent, improve, or treat diseases caused by lipofuscin deposition.

[0020] In one exemplary implementation, the disease caused by the lipofuscin deposition may include one or more selected from the group consisting of sarcopenia, progeria, Alzheimer's disease, Parkinson'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).

[0021] Invention Effects

[0022] On the one hand, the technology disclosed in this disclosure has the effect of providing a material capable of decomposing lipofuscin. Metalloproteinases derived from *Chlorella* strains provide the effect of directly decomposing intracellularly deposited lipofuscin.

[0023] On the other hand, the technology disclosed in this disclosure has the effect of providing a composition for skin anti-aging, skin lightening or skin texture improvement containing a metalloprotease derived from a strain of Chryseobacterium as an active ingredient.

[0024] On another aspect, the technology disclosed in this disclosure has the effect of providing a composition for inhibiting or removing lipofuscin deposition by containing a metalloprotease derived from a strain of Chryseobacterium as an active ingredient. Attached Figure Description

[0025] Figure 1 The partial 16S rDNA sequence of *Cryptotympany* strain Shiduochatian-HT1 according to one embodiment is shown.

[0026] Figure 2 The results of keratin degradation activity evaluation according to one embodiment are shown.

[0027] Figure 3 The results of the lipofuscin scavenging activity evaluation according to one embodiment are shown. Detailed Implementation

[0028] This disclosure will now be described in detail.

[0029] In this article, "active ingredient" refers to an ingredient that exerts the desired activity on its own, or in combination with a carrier or other inactive component.

[0030] On the one hand, this disclosure provides a composition for skin anti-aging, skin lightening and / or skin texture improvement, which contains a metalloprotease derived from a strain of Chryseobacterium as an active ingredient.

[0031] On the other hand, this disclosure provides a method for skin anti-aging, skin whitening and / or skin texture improvement, the method comprising applying or administering to a subject in need a composition containing a metalloprotease derived from a strain of Chryseobacterium in an amount effective for skin anti-aging, skin whitening and / or skin texture improvement.

[0032] On the other hand, this disclosure provides a composition containing a metalloprotease derived from a strain of Chryseobacterium for use in skin anti-aging, skin whitening and / or skin texture improvement.

[0033] On the other hand, this disclosure provides the use of a composition containing a metalloprotease derived from a strain of Chryseobacterium for skin anti-aging, skin whitening and / or skin texture improvement.

[0034] On the other hand, this disclosure provides the use of a composition containing a metalloprotease derived from a strain of Chryseobacterium in the preparation of a composition for skin anti-aging, skin whitening and / or skin texture improvement.

[0035] On the other hand, this disclosure provides the use of a composition containing a metalloprotease derived from a strain of Chryseobacterium in the preparation of a composition for improving melasma.

[0036] On the other hand, this disclosure provides the use of a composition containing a metalloprotease derived from a strain of Chryseobacterium in the preparation of a food composition for improving melasma.

[0037] In one exemplary implementation, the composition or metalloproteinase can provide anti-aging, skin whitening, and / or skin texture improvement effects by inhibiting or clearing lipofuscin deposition.

[0038] In one exemplary implementation, the skin anti-aging, skin whitening, and / or skin texture improvement can be achieved by inhibiting lipofuscin deposition or removing lipofuscin.

[0039] In one exemplary implementation, the improvement of melasma may be achieved by inhibiting lipofuscin deposition or removing lipofuscin.

[0040] In this document, "anti-aging" refers to the use of methods to prevent, delay, and / or improve aging phenomena caused by internal factors, including genetic factors, and external factors, including ultraviolet radiation. The composition can reduce cellular senescence levels, thereby providing effects of prevention, improvement, and / or treatment of diseases or symptoms related to cellular senescence. For example, the anti-aging effect can be the prevention, delay, and / or improvement of aging caused by lipofuscin deposition.

[0041] In one exemplary implementation, the composition or metalloproteinase can prevent, improve, and / or treat liver spots and / or melasma.

[0042] In one exemplary implementation, the composition or metalloproteinase can inhibit or remove lipofuscin deposition, thereby making the skin clean, bright, or transparent, and contributing to whitening and / or skin tone improvement.

[0043] On the other hand, this disclosure provides a composition for inhibiting or removing lipofuscin deposition, which contains a metalloprotease derived from a strain of Chryseobacterium as an active ingredient.

[0044] On the other hand, this disclosure provides a method for inhibiting or removing lipofuscin deposition, the method comprising applying or administering to a subject in need a composition containing a metalloprotease derived from a strain of Chryseobacterium in an amount effective for inhibiting or removing lipofuscin deposition.

[0045] In one exemplary implementation, the method can improve or treat diseases caused by lipofuscin deposition.

[0046] In one exemplary implementation, the method can have an anti-aging effect on the object.

[0047] In one exemplary implementation, the method can brighten the skin tone of an object.

[0048] In one exemplary implementation, the method can improve the skin texture of an object.

[0049] On the other hand, this disclosure provides a composition containing a metalloprotease derived from a strain of Chryseobacterium for use in inhibiting or removing lipofuscin deposition.

[0050] On the other hand, this disclosure provides the use of a composition containing a metalloprotease derived from a strain of Chryseobacterium for inhibiting or removing lipofuscin deposition.

[0051] On the other hand, this disclosure provides the use of a composition containing a metalloprotease derived from a strain of Chryseobacterium in the preparation of a composition for inhibiting or removing lipofuscin deposition.

[0052] In one exemplary implementation, the composition or metalloproteinase can prevent, improve, and / or treat diseases caused by lipofuscin deposition.

[0053] In one exemplary implementation, the composition or metalloproteinase may have an anti-aging effect on the object.

[0054] In one exemplary implementation, the composition or metalloproteinase can brighten the skin tone of an object.

[0055] In one exemplary implementation, the composition or metalloproteinase can improve the skin texture of the subject.

[0056] In one exemplary implementation, the disease caused by the lipofuscin deposition may include one or more selected from the group consisting of sarcopenia, progeria, Alzheimer's disease, Parkinson'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).

[0057] In one exemplary implementation, the strain may be Chryseobacterium camelliae.

[0058] In one exemplary implementation, the strain can be Chryseobacterium camelliae Dolsongi-HT1, which has the accession number KCCM11883P.

[0059] In one exemplary implementation, the strain can be isolated from green tea leaves.

[0060] In one exemplary implementation, the *Aureobacterium tea-tea* strain *Tea field-HT1* may have a 16S rDNA partial sequence of sequence number 3.

[0061] In one exemplary implementation, the metalloproteinase may comprise an amino acid sequence of sequence number 20 or sequence number 22.

[0062] In one exemplary implementation, the metalloproteinase may be composed of an amino acid sequence of sequence number 20 or sequence number 22.

[0063] Peptides required to transport intracellular peptides or proteins to the extracellular space are called signal peptides or signal sequences. These guide intracellular proteins toward the cell wall, ultimately helping them to be secreted across the cell membrane. When a protein is secreted extracellularly, the signal peptide is cleaved, and the remaining portion forms a structure to perform its function.

[0064] According to this disclosure, a metalloproteinase can be represented by an amino acid sequence of sequence number 22, which includes a signal sequence of sequence number 21.

[0065] Serial number 21

[0066] MKKNFNFCCLAGAIAVFSLTA

[0067] Serial number 22

[0068] MKKNFNFCCLAGAIAVFSLTACNDNSTEDNNLLQQPQAESAKVEQPNEREKACYYVDQNWNSSAVLKTTLKTSTDTNFMNGQMTKIASLWGRNNPTLRFVDDPSSPNSTYNAISYSTGKIYYGYAIYYDAKNKGGDIVNAMILAHEYG HQLQYIFGLPSVNESTARPNELEADGFAGYYLRRPEGYNKTQFSEIAAAYEFAQSIGDYATTSPGHHGTPPQRRSAVRLGFLLGQYNLSASDFDYNFFYYYQGVLNGTYKMGKNSRNPELDAYMMQYIDELRKIQSGEISAEEFKQLK

[0069] In one exemplary implementation, the composition containing a metalloproteinase derived from the *Chlorella* strain can be a cosmetic composition, an oral composition, a non-therapeutic oral composition, or a pharmaceutical composition.

[0070] In one exemplary implementation, a composition containing a metalloproteinase derived from the *Chlorella* strain can be applied, applied, or administered to an object as a cosmetic composition, an oral composition, a non-therapeutic oral composition, or a pharmaceutical composition.

[0071] According to an exemplary implementation, a composition containing a metalloproteinase derived from the *Chlorella* strain can be a cosmetic composition.

[0072] The cosmetic composition may also contain functional additives and ingredients included in general cosmetic compositions. As functional additives, they may include ingredients selected from the group consisting of water-soluble vitamins, oil-soluble vitamins, high-molecular-weight peptides, high-molecular-weight polysaccharides, sphingolipids, and seaweed extract. Other included formulation ingredients may include oils, moisturizers, lubricants, surfactants, organic and inorganic pigments, organic powders, ultraviolet absorbers, preservatives, bactericides, antioxidants, plant extracts, pH adjusters, ethanol, pigments, fragrances, blood circulation promoters, cooling agents, antiperspirants, and purified water.

[0073] The dosage form of the cosmetic composition is not particularly limited and can be appropriately selected according to the purpose. For example, it can be formulated into one or more dosage forms selected from the group consisting of lotion, toner, astringent, emulsion, milk lotion, moisturizing lotion, nourishing lotion, massage cream, nourishing cream, moisturizing cream, hand cream, foundation, serum, nourishing essence, mask, soap, facial foam, cleansing milk, cleansing cream, body lotion, and body cleanser, but is not limited thereto.

[0074] When the dosage form of the composition is a paste, cream, or gel, the carrier component may include animal fiber, plant fiber, wax, paraffin, starch, astragalus gum, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc, or zinc oxide, etc.

[0075] When the composition is in the form of a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder may be used as the carrier component. In particular, when the dosage form is a spray, it may further contain propellants such as chlorofluorocarbons, propane / butane, or dimethyl ether.

[0076] When the dosage form of the composition is a solution or emulsion, a solvent, solvator or emulsifier is used as the carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylethylene glycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid esters of sorbitan anhydride.

[0077] When the composition is in the form of a suspension, liquid diluents such as water, ethanol or propylene glycol can be used as the carrier component; suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitan ester and polyoxyethylene sorbitan anhydride ester; microcrystalline cellulose; aluminum hydroxide; bentonite, agar or tragacanth gum, etc.

[0078] When the formulation of the composition is a surfactant-containing detergent, the carrier component may include fatty alcohol sulfates, fatty alcohol ether sulfates, sulfosuccinate monoesters, hydroxyethyl sulfonates, imidazoline derivatives, methyl taurine, sarcosinates, fatty acid amide ether sulfates, alkylamide betaine, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives, or ethoxylated glycerol fatty acid esters, etc.

[0079] According to an exemplary implementation, the composition containing a metalloproteinase derived from the *Chlorella* strain can be an oral composition or a non-therapeutic oral composition.

[0080] The oral or non-therapeutic oral composition may be in liquid or solid dosage form. Those skilled in the art can easily select and add commonly used ingredients according to the dosage form or purpose of use. When used with other raw materials, a synergistic effect can be obtained.

[0081] The liquid formulation can be like a typical beverage, and may include various flavoring agents or natural carbohydrates as additional ingredients. 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. As flavoring agents, natural flavoring agents (such as tau martin and stevia extracts, such as levofloxacin A and glycyrrhizin) and synthetic flavoring agents (such as saccharin and aspartame) can be advantageously used. The proportion of natural carbohydrates is typically about 1 to 20 g per 100 ml of the composition, and in some cases, about 5 to 12 g.

[0082] On the one hand, the oral or non-therapeutic oral composition may contain various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, coloring agents, and thickeners (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, and carbonating agents used in carbonated beverages. On the other hand, it may contain fruit pulp used to make natural fruit juices and vegetable beverages. The ingredients may be used alone or in combination. The proportions of the additives may vary, but are generally selected from 0.001 to about 20 parts by weight per 100 parts by weight of the composition.

[0083] According to one exemplary implementation, a composition containing a metalloproteinase derived from the *Chlorella* strain can be a pharmaceutical composition.

[0084] The pharmaceutical composition may further contain pharmaceutical adjuvants such as preservatives, stabilizers, wettable powders or emulsification promoters, salts and / or buffers for adjusting osmotic pressure, and other therapeutically useful substances, and may be formulated into various oral or non-oral dosage forms according to conventional methods.

[0085] Oral medications include, for example, tablets, pills, hard capsules and soft capsules, liquids, suspensions, emulsions, syrups, powders, granules, pellets, and balls. These dosage forms may contain surfactants, diluents (e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, and glycine), and lubricants (e.g., silica, talc, stearic acid and its magnesium or calcium salts, and polyethylene glycol). Tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth gum, methylcellulose, sodium carboxymethyl cellulose, and polyvinylpyrrolidone, and may, depending on the application, contain pharmaceutical additives such as disintegrants, absorbents, colorants, flavorings, and sweeteners, such as starch, agar, alginate, or their sodium salts. The tablets can be prepared by conventional mixing, granulation, or coating methods.

[0086] In addition, the non-oral administration form can be a transdermal dosage form, such as, but not limited to, injections, drops, ointments, lotions, gels, creams, sprays, suspensions, oils, patches, etc.

[0087] The dosage of the active ingredient is determined within the skill of a person skilled in the art. The daily dose of the drug depends on various factors such as the degree of disease progression, onset time, age, health status, and complications of the subject. However, when based on adults, the composition can be administered in doses of 1 μg / kg to 200 mg / kg or 50 μg / kg to 50 mg / kg, divided into one to three doses per day.

[0088] The pharmaceutical composition may be a topical skin preparation, which is a general term for any preparation that may be applied externally to the skin and may include pharmaceuticals in various dosage forms.

[0089] In one exemplary implementation, the composition containing a metalloproteinase derived from the *Chlorobacterium* strain may contain 0.0001 to 99.99% by weight of a metalloproteinase derived from the *Chlorobacterium* strain, based on the total weight of the composition.

[0090] In another exemplary implementation, the composition containing a metalloproteinase derived from the *Chlorobacterium* strain may contain, based on the total weight of the composition, 0.0001% or more, 0.0005% or more, 0.001% or more, 0.005% or more, 0.01% or more, 0.05% or more, 0.1% or more, or 0.5% or more, and less than 20% or less, 15% or less, 10% or less, 5% or less, 1% or less, 0.5% or less, 0.1% or less, 0.05% or less, 0.01% or less, 0.005% or less, or 0.001% or less of a metalloproteinase derived from the *Chlorobacterium* strain.

[0091] For example, a composition containing a metalloproteinase derived from the *Chlorobacterium* strain may contain, based on the total weight of the composition, 0.0001 to 1 wt%, 0.0001 to 0.5 wt%, 0.0001 to 0.1 wt%, 0.0001 to 0.05 wt%, 0.0001 to 0.01 wt%, 0.0001 to 0.005 wt%, 0.0001 to 0.001 wt%, 0.0005 to 0.001 wt%, or 0.001 to 0.005 wt% of a metalloproteinase derived from the *Chlorobacterium* strain.

[0092] On the one hand, this disclosure includes at least the following implementation examples.

[0093] Example 1. Use of a composition containing a metalloprotease derived from a strain of Chryseobacterium for preparing a composition for skin anti-aging, skin lightening or skin texture improvement.

[0094] Implementation Example 2. An application, according to Implementation Example 1, wherein the strain is Chryseobacterium camelliae.

[0095] Example 3. An application, according to Example 1 or Example 2, wherein the strain is Chryseobacterium camelliae Dolsongi-HT1 strain with accession number KCCM11883P.

[0096] Implementation Example 4. An application, according to any one of Implementation Examples 1 to 3, wherein the metalloproteinase comprises an amino acid sequence of sequence number 20 or sequence number 22.

[0097] Implementation Example 5. An application, according to any one of Implementation Examples 1 to 4, wherein the skin anti-aging, skin whitening, or skin texture improvement is achieved by inhibiting or removing lipofuscin deposition.

[0098] Example 6. An application according to any one of Examples 1 to 5, wherein the composition containing the metalloproteinase derived from the *Chlorobacterium* strain contains 0.0001 to 20% by weight of the metalloproteinase derived from the *Chlorobacterium* strain, based on the total weight of the composition.

[0099] Example 7. Use of a composition containing a metalloprotease derived from a strain of Chryseobacterium for preparing a composition for inhibiting or removing lipofuscin deposition.

[0100] Example 8. An application according to the application of Example 7, wherein the strain is Chryseobacterium camelliae.

[0101] Example 9. An application, according to Example 7 or Example 8, wherein the strain is Chryseobacterium camelliae Dolsongi-HT1 strain with accession number KCCM11883P.

[0102] Implementation Example 10. An application, according to any one of Implementation Examples 7 to 9, wherein the metalloproteinase comprises an amino acid sequence of sequence number 20 or sequence number 22.

[0103] Example 11. An application, according to any one of Examples 7 to 10, wherein the composition is used to prevent, improve or treat diseases caused by lipofuscin deposition.

[0104] Implementation Example 12. An application, according to any one of Implementation Examples 7 to Implementation Examples 11, wherein the disease caused by lipofuscin deposition includes one or more selected from the group consisting of sarcopenia, progeria, Alzheimer's disease, Parkinson'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).

[0105] Implementation Example 13. An application, according to any one of Implementation Examples 7 to Implementation Examples 12, wherein the composition containing the metalloproteinase derived from the said Chlorobacterium strain contains 0.0001 to 20% by weight of the metalloproteinase derived from the Chlorobacterium strain based on the total weight of the composition.

[0106] The present disclosure will now be described in more detail through embodiments. These embodiments are merely examples of the present disclosure, and those skilled in the art should understand that the scope of the present disclosure should not be construed as limited to these embodiments.

[0107] Example 1. Preparation of culture medium for Chlorella strains

[0108] In this embodiment, *C. tea* strain was used as the *C. tea* strain, and a culture medium for the *C. tea* strain was prepared as follows. The *C. tea* strain with accession number KCCM11883P, *C. tea* strain Shiduo Chatian-HT1, was used. As described in Korean Patent Publication No. 10-2018-0035682, the strain can be obtained according to the isolation and identification methods described below. The full text of that document is incorporated herein by reference.

[0109] (1) Isolation of microorganisms in green tea

[0110] To screen for microorganisms with plant-derived keratin-degrading activity, the microorganisms were cultured at 30°C or 37°C in a liquid minimal medium (M9 medium containing 0.5% soluble keratin: 0.015 g / L CaCl2, 6.78 g / L Na2HPO4, 3 g / L KH2PO4, 0.5 g / L NaCl, and 0.5 g / L MgSO4) with keratin as the carbon and nitrogen source.

[0111] The microbial screening method based on the aforementioned minimal culture medium is a method for screening desired microorganisms according to their growth rate. Microorganisms with low keratinase activity are naturally removed during the culture stage, thus enabling the screening of active microorganisms containing highly active enzymes. Microorganisms that can grow in the minimal culture medium indicate that they possess keratinase activity.

[0112] Therefore, microorganisms with keratinogenic activity were isolated from green tea grown in the Seokdo tea fields of Jeju Island, South Korea, using limited culture media.

[0113] (2) Identification of isolated microorganisms

[0114] To identify the isolated microorganisms with keratinase activity, the DNA sequence encoding the partial 16S ribosomal RNA sequence of the microorganisms was analyzed to determine their species and genus.

[0115] After culturing the microorganisms in liquid culture medium, genomic DNA was extracted from the microorganisms and amplified by PCR (polymerase chain reaction) using primers commonly used for bacterial identification: 27F (5'-AGAGTTTGATCMTGGCTCAG-3', sequence number 1) / 1492R (5'-TACGGYTACCTTGTTACGACTT-3', sequence number 2). The sequence was then analyzed.

[0116] Base sequence analysis showed that it had 99% homology with the partial 16S rRNA sequence of *Chryseobacterium teaensis* strain THG C4-1 (KACC 16985; Sequence accession no. (16S rRNA) JX843771) and 98% homology with the partial 16S rRNA sequence of *Chryseobacterium taiwanense*.

[0117] Therefore, through molecular systematic taxonomy analysis based on the 16S rDNA base sequence, the novel microbial strain isolated and cultured was confirmed to be Chryseobacterium camelliae, and named Chryseobacterium camelliae Dolsongi-HT1. On September 1, 2016, the novel microbial strain was deposited at the Korean Center for Microbial Culture Collection (KCCM) with the accession number KCCM11883P. Figure 1 The image shows a partial 16S rDNA sequence of the *Aureobacterium teaii* strain Shiduochatian-HT1, sequence number 3.

[0118] (3) Preparation of microbial culture medium

[0119] The *Aureobacterium teaense* strain Shiduo Chatian-HT1, derived from green tea, was seeded for 15 hours. Then, 10 mL of the seeded strain was inoculated into 1 L of optimal culture medium (1% tryptone / 1% sodium chloride / 0.5% skim milk) and incubated at 30°C and 200 rpm for 15 hours. After incubation, the culture medium containing the microorganisms was obtained.

[0120] Example 2. Enzyme purification and sequence analysis from the culture medium of Chlorella strains.

[0121] Proteins with a size greater than 10 kDa were obtained from the culture medium of the *Corydalis* strain prepared in Example 1 by ultrafiltration using a 10 kDa cut-off filter membrane. To screen for proteins with proteolytic activity, proteins were separated using an ion exchange resin (Q-sepharose® HP column, Cytica, Marlborough, MA, USA), and the proteins contained in the active fraction were analyzed using a mass spectrometer (Q Exactive™ Plus mass spectrometer, Thermo Fisher Scientific, Waltham, MA, USA). More than 120 proteins were analyzed.

[0122] Since the whole genome analysis of *Cryptotympany* strain Shiduo Chatian-HT1 was completed, eight proteins, named proteases, were screened from the proteins analyzed by comparing the whole genome with the protein analysis results. These eight screened proteins were then prepared into recombinant proteins, and their keratinogenic activities were compared.

[0123] First, the gene sequences of the eight proteins listed in Table 1 were amplified by PCR using forward and reverse primers. Each enzyme was then expressed using the Bacillus subtilis Secretion protein expression system (TaKaRa®). The pBE-S vector was used for recombinant protein preparation, and the gene was introduced into the vector using an In-Fusion cloning system. The vector containing the gene was then introduced into the host Bacillus subtilis RIK1285. For protein expression, the expression strain was cultured at 37°C for 24 hours. After culture, the microbial cells were removed, the culture medium was recovered, concentrated by ultrafiltration, and then freeze-dried to obtain the sample.

[0124] Table 1

[0125]

[0126] Keratin activity was determined using 0.5% (wt / v) keratin and a Tris-HCl buffer at pH 8.0. When keratin is broken down by keratin, it releases azure dye, turning the reaction solution blue. Therefore, absorbance at 595 nm was measured to compare activity (absorbance 0.01 = 1 unit).

[0127] This result confirmed keratinase activity in the two protein samples, MetalloP and ContP1 (samples 5 and 6), thus confirming that MetalloP and ContP1 can provide skin texture improvement through exfoliation (see reference). Figure 2 In particular, MetalloP exhibited significantly high keratinase activity. On the other hand, ContP2, as protein sample 4, had the highest content among the purified proteins but showed no keratinase activity whatsoever.

[0128] As described above, enzymes with keratinase activity were purified from the *Cryptotympany* strain *Tea* 'Shiduo Chatian-HT1, isolated from green tea leaves. The sequences were compared with the NCBI database, confirming that MetalloP and ContP1 belong to the metalloprotease family. The purified enzyme MetalloP was confirmed to have sequence number 20. The sequence of MetalloP can be represented by sequence number 22, which includes a signal sequence suitable for the expression strain, including sequence number 21. The sequence of ContP1 can be represented by sequence number 24, which includes a signal sequence including sequence number 23. The sequence of ContP2 can be represented by sequence number 26, which includes a signal sequence including sequence number 25.

[0129] Serial number 20

[0130] CNDNSTEDNNLLQQPQAESAKVEQPNEREKACYYVDQNWNSSAVLKTTLKTSTDTNFMNGQMTKIASLWGRNNPTLRFVDDPSSPNSTYNAISYSTGKIYYGYAIYYDAKNKGGDIVNAMILAHEYGHQLQYIFGLP SVNESTARPNELEADGFAGYYLRRPEGYNKTQFSEIAAAYEFAQSIGDYATTSPGHHGTPPQRRSAVRLGFLLGQYNLSASDFDYNFFYYYQGVLNGTYKMGKNSRNPELDAYMMQYIDELRKIQSGEISAEEFKQLK

[0131] Serial number 21

[0132] MKKNFNFCCLAGAIAVFSLTA

[0133] Serial number 22

[0134] MKKNFNFCCLAGAIAVFSLTACNDNSTEDNNLLQQPQAESAKVEQPNEREKACYYVDQNWNSSAVLKTTLKTSTDTNFMNGQMTKIASLWGRNNPTLRFVDDPSSPNSTYNAISYSTGKIYYGYAIYYDAKNKGGDIVNAMILAHEYGHQLQYIFGLPSVNESTARPNELEADGFAGYYLRRPEGYNKTQFSEIAAAYEFAQSIGDYATTSPGHHGTPPQRRSAVRLGFLLGQYNLSASDFDYNFFYYYQGVLNGTYKMGKNSRNPELDAYMMQYIDELRKIQSGEISAEEFKQLK

[0135] Sequence No. 23

[0136] MKKLLLGALMLGFMSACNS

[0137] Sequence No. 24

[0138] MKKLLLGALMLGFMSACNSDNIANQHEQSDNLVSPTGGSALQRGCASEEVRKIALQNSPELRQRFSALEAQTEKFENDLKLGKVLSDGSVEIPVVVNVLYRTSSENLSDSRIAEQIAVLNADYAGTNSDVSKIPSEFQSVKSGDVKVKFRLANTVRKSTTKTSWSTNDAMKRSSSGGIDATSPSNYLNIWVVGNMGQILGYATFPESAGMWNDGVVIAAPYFGKTGASSPFNLGRTATHEVGHYLNLRHIWGDANCGNDLVADTPTQTGANSGKPNYPLYNTCSGVQRSVMFMNYMDYVDDAAMFMFSAGQRTRMQSVVASSGPRSGLRLY

[0139] Sequence No. 25

[0140] MKTMLKSILTVALLWSAVS

[0141] Sequence No. 26

[0142] MKTMLKSILTVALLWSAVSCNTNEMSDINSSAQTQTSNSGNFAAKASDNPENPLNNWEECGKKHNVILTYVITYQKTLDNSGLTNSEIADKCIMASNNYFNQKYAQDYNNTQSPFTVAKIRSILNDGDNKFSNVIQGMNA SDKVKAKTSELVNMMYNLASTDKNTEYAEVKARIVDFEAGIMNDASLSQAEKDQLLQTTSIARYSSYLWSNTIDHSDSVSSVTGKKKWWKWVVVAVCDFAGGAAGTAAGGGVLSVAGAIAGAAGASSGGAALVDWISPDAP

[0143] Example 3. Evaluation of the lipofuscin-degrading activity of metalloproteinases derived from *Chlorobacterium* strains.

[0144] MetalloP, ContP1, and ContP2 are enzymes prepared and overexpressed by the recombinant strain in Example 2. MetalloP, ContP1, and ContP2 were then introduced into lipofuscin-induced cells to evaluate the lipofuscin scavenging efficacy of these enzymes. ContP1 is an enzyme with approximately 50% more keratinogenic efficacy than MetalloP, while ContP2 is an enzyme without keratinogenic efficacy.

[0145] Fibroblasts were grown to 90% confluent status, and then cultured for 10 days with 40 μM leupeptin, 45 μM FeCl3, and 10 μM H2O2 added to the growth medium to induce intracellular deposition of lipofuscin. 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 determined using a confocal laser scanning microscope (LSM 980, Carl Zeiss, Oberkochen, Germany) equipped with a fluorescein isothiocyanate (FITC) 540 / 40 filter.

[0146] The result, such as Figure 3 As shown, in the experimental groups (MetalloP, ContP1) treated with metalloproteinases in cells with lipofuscin deposition, lipofuscin was cleared. In particular, the experimental group treated with MetalloP completely cleared the deposited lipofuscin, thus confirming that MetalloP has the best lipofuscin-degrading activity.

[0147] Examples of dosage forms of compositions according to one aspect of this disclosure are described below, but various other dosage forms are also applicable. This is not intended to limit the disclosure, but is merely for illustration.

[0148] Dosage form example 1. Toner

[0149] Mix 0.01% by weight of the metalloproteinase MetalloP obtained in Example 2, 3% by weight of glycerol, 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, trace amounts of preservatives, trace amounts of pigments, trace amounts of fragrances, and the remainder of purified water to prepare a skin softening lotion according to conventional preparation methods.

[0150] Dosage form example 2. Nourishing toner

[0151] The following ingredients were mixed: 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 sesquioleate, 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, trace amounts of preservatives, trace amounts of pigments, trace amounts of fragrances, and the remainder of purified water, and a nourishing toner was prepared according to conventional preparation methods.

[0152] Dosage form example 3. Nourishing cream

[0153] The following ingredients were mixed: 0.01 wt% of the metalloproteinase MetalloP obtained in Example 2, 10 wt% of beeswax, 1.5 wt% of polysorbate 60, 0.5 wt% of sorbitan sesquioleate, 10 wt% of liquid paraffin, 5 wt% of squalane, 5 wt% of caprylic / capric triglyceride, 5 wt% of glycerin, 3 wt% of butylene glycol, 3 wt% of propylene glycol, 0.2 wt% of triethanolamine, trace amounts of preservatives, trace amounts of pigments, trace amounts of fragrances, and the remainder of purified water, and the mixture was prepared into a nourishing cream according to conventional preparation methods.

[0154] Dosage form example 4. Facial mask

[0155] The following ingredients were mixed: 0.01% by weight of the metalloproteinase MetalloP obtained in Example 2, 13% by weight of polyvinyl alcohol, 0.2% by weight of sodium carboxymethyl cellulose, 0.1% by weight of allantoin, 5% by weight of ethanol, 0.3% by weight of nonylphenyl ether, trace amounts of preservative, trace amounts of pigment, trace amounts of fragrance, and the remainder of purified water, and the mixture was prepared into a face mask according to conventional preparation methods.

[0156] Dosage form example 5. Topical medications (patch tablets)

[0157] Prepare topical medications (patch tablets) according to conventional methods based on the components listed in Table 2 below.

[0158] Table 2.

[0159]

[0160] Dosage form example 6. Powder

[0161] 2g of the metalloproteinase MetalloP obtained in Example 2 and 1g of lactose were mixed and then filled into an airtight packaging bag to make a powder.

[0162] Dosage form example 7. Tablets

[0163] 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 compressed into tablets using conventional methods.

[0164] Dosage form example 8. Capsules

[0165] 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 filled into gelatin capsules using conventional methods to prepare capsules.

[0166] Dosage form example 9. Pills

[0167] After mixing 1g of the metalloproteinase MetalloP obtained in Example 2, 1.5g of lactose, 1g of glycerol, and 0.5g of xylitol, the mixture was prepared into pills of 4g each using conventional methods.

[0168] Dosage form example 10. Granules

[0169] Mix 150g of the metalloproteinase MetalloP obtained in Example 2, 50mg of soybean extract, 200mg of glucose, and 600mg of starch, add 100mg of 30% ethanol, dry at 60°C to form granules, and then fill into small bags to make granules.

[0170] Dosage form example 11. Beverage preparation

[0171] Mix 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, then add 300 ml of purified water, filling each bottle with 200 ml of the mixture. After filling, sterilize at 130°C for 4-5 seconds to produce a beverage.

[0172] Dosage form example 12. Caramel

[0173] 50 mg of the metalloproteinase MetalloP obtained in Example 2, 1.8 g of corn syrup, 0.5 g of skim milk, 0.5 g of soy lecithin, 0.6 g of butter, 0.4 g of hydrogenated vegetable oil, 1.4 g of white sugar, 0.58 g of margarine, and 20 mg of salt were mixed and shaped to make caramel.

[0174] Specific portions of this disclosure have been described in detail above; however, those skilled in the art should understand that these specific descriptions are merely preferred embodiments and are not intended to limit the scope of this disclosure. Therefore, the essential scope of this disclosure is defined by the claimed technical solutions and their equivalents.

[0175] [Collection Information]

[0176] Strain name: Chryseobacterium camelliae Dolsongi-HT1

[0177] Name of depositary institution: Korean Center for Microbial Preservation (45 Hongje-nae 2ga-gil, Seodaemun-gu, Seoul, South Korea, 120-861, Korea)

[0178] Collection Number: KCCM11883P

[0179] Date of deposit: September 1, 2016

Claims

1. Use of a composition containing a metalloproteinase derived from a strain of *Chlorella* for preparing a composition for skin anti-aging, skin whitening or skin texture improvement.

2. The use according to claim 1, wherein, The strain in question is *Aureobacterium teaense*.

3. The use according to claim 1, wherein, The strain is the *Aureobacterium teaense* strain Shiduo Chatian-HT1, with accession number KCCM11883P.

4. The use according to claim 1, wherein, The metalloproteinase contains an amino acid sequence of sequence number 20 or sequence number 22.

5. The use according to claim 1, wherein, The skin anti-aging, skin whitening, or skin texture improvement are achieved by inhibiting or removing lipofuscin deposition.

6. The use according to claim 1, wherein, The composition containing a metalloproteinase derived from the aforementioned *Chlorobacterium* strain contains 0.0001 to 20% by weight of a metalloproteinase derived from a *Chlorobacterium* strain, based on the total weight of the composition.

7. Use of a composition containing a metalloproteinase derived from a strain of *Chlorobacterium* for preparing a composition for inhibiting or removing lipofuscin deposition.

8. The use according to claim 7, wherein, The strain in question is *Aureobacterium teaense*.

9. The use according to claim 7, wherein, The strain is the *Aureobacterium teaense* strain Shiduo Chatian-HT1, with accession number KCCM11883P.

10. The use according to claim 7, wherein, The metalloproteinase contains an amino acid sequence of sequence number 20 or sequence number 22.

11. The use according to claim 7, wherein, The composition is used to prevent, improve or treat diseases caused by lipofuscin deposition.

12. The use according to claim 11, wherein, Diseases caused by the aforementioned lipofuscin deposition include one or more selected from the group consisting of sarcopenia, progeria, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, myocardial infarction, age-related macular degeneration, neuronal ceroid lipofuscin deposition, acromegaly, denervation atrophy, and chronic obstructive pulmonary disease.

13. The use according to claim 7, wherein, The composition containing a metalloproteinase derived from the aforementioned *Chlorobacterium* strain contains 0.0001 to 20% by weight of a metalloproteinase derived from a *Chlorobacterium* strain, based on the total weight of the composition.

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