Cosmetic composition for skin whitening and skin wrinkle reduction comprising protein hydrolysate isolated from black soldier fly larvae as active ingredient

Abstract

The present invention relates to a protein hydrolysate isolated from black soldier fly larvae and, more specifically, to a cosmetic composition for skin whitening and skin wrinkle reduction, comprising an anionic peptide as an active ingredient. The protein hydrolysate of black soldier fly larvae, according to the present invention, has an excellent skin absorption rate, exhibits an effect on skin whitening by inhibiting melanin synthesis and tyrosinase activity, and has an excellent effect of preventing or reducing skin wrinkles by enhancing skin elasticity through collagenase inhibitory activity and / or elastase inhibitory activity.

Description

Cosmetic composition for skin whitening and skin wrinkle improvement containing a protein hydrolysate isolated from black soldier fly larvae as an active ingredient

[0001] The present invention relates to a cosmetic composition for skin whitening and skin wrinkle improvement comprising a protein hydrolysate isolated from black soldier fly larvae, more specifically an anionic peptide, as an active ingredient.

[0002] Recently, the Food and Agriculture Organization (FAO) announced measures to promote edible insects as a future food resource as a policy to address food shortages caused by disease or environmental pollution, and interest in this is growing worldwide. In line with this, the Ministry of Agriculture, Food and Rural Affairs in Korea announced the "Five-Year Comprehensive Plan for the Promotion of the Insect Industry," and support for the insect industry has been provided accordingly.

[0003] Insects are the most diverse species on Earth, to the extent that although over 1 million species have been reported, it is estimated that over 1 million species remain unreported. Insects have been used for medicinal purposes since ancient times, and research results have reported that silkworms, grasshoppers, white-spotted flower beetle larvae (grubs), and centipedes are effective in treating chronic diseases such as diabetes, inflammatory diseases, liver disease, and arteriosclerosis.

[0004] Meanwhile, among all insects produced on an industrial scale, black soldier flies (Hermetia illucens) are a collective term for insects belonging to the family Stratiologyidae of the order Diptera, with about 1,500 species known worldwide, and in Korea, 9 genera and 14 species have been reported.

[0005] The black soldier fly larvae mentioned above are easy to mass-reare and freeze-store, so they are being applied in various fields such as feed additives, fertilizers, and biodiesel. The black soldier fly larvae mentioned above have received special attention due to their ability to grow in a wide range of organic residues and unique nutritional compositions. In particular, the nutritional suitability of black soldier fly larvae (BSF) in aquaculture and pet feed is well established.

[0006] Against this backdrop, the inventors, through diligent research efforts to further enhance the functionality of the nutritional components, particularly the protein components, contained in black soldier fly larvae, confirmed that the purified protein hydrolysate of black soldier fly larvae according to the present invention has excellent efficacy in skin whitening and skin wrinkle improvement, thereby completing the present invention.

[0007] The problem to be solved by the present invention is to provide a cosmetic composition for skin whitening and skin wrinkle improvement comprising a protein hydrolysate of black soldier fly (Hermetia illucens) larvae as an active ingredient, and a method for manufacturing the same.

[0008] To achieve the above objective, the present invention provides a cosmetic composition for skin whitening and skin wrinkle improvement comprising, as an active ingredient, a purified protein hydrolysate obtained by enzymatically reacting defatted powder of black soldier fly (Hermetia illucens) larvae with a complex of an enzyme of IUBMB enzyme nomenclature class EC 3.4.21.62, and an enzyme of IUBMB enzyme nomenclature class EC 3.4.11.1.

[0009] According to one embodiment of the present invention, the enzyme of IUBMB enzyme nomenclature class EC 3.4.21.62 may be an alkaline protease derived from Bacillus.

[0010] According to one embodiment of the present invention, the alkaline protease derived from Bacillus may be an alkaline serine endopeptidase produced by Bacillus licheniformis.

[0011] According to one embodiment of the present invention, the alkaline serine endopeptidase may be Protex 6L.

[0012] According to one embodiment of the present invention, the protein hydrolysate may have an average molecular weight of peptides within the hydrolysate of 690 Da or less, and may contain 60% or more of peptides of 499 Da or less.

[0013] According to one embodiment of the present invention, the enzyme of IUBMB enzyme nomenclature class EC 3.4.11.1 may be a complex of fungal protease and peptidase.

[0014] According to one embodiment of the present invention, the complex of the fungal-derived protease and peptidase may be a complex of the endoprotease and exopeptidase derived from Aspergillus oryzae.

[0015] According to one embodiment of the present invention, the complex of the endoprotease and exopeptidase may be a flavourzyme.

[0016] According to one embodiment of the present invention, the proteolytic enzyme may be an enzyme of IUBMB enzyme nomenclature class EC 3.4.21.62; and an enzyme of IUBMB enzyme nomenclature class EC 3.4.11.1 mixed in a weight ratio of 1:0.5 to 2.5.

[0017] According to one embodiment of the present invention, the enzyme reaction may be performed at 50 to 60 ℃ for 0.5 to 24 hours.

[0018] According to one embodiment of the present invention, the peptide contained in the purified product has an average molecular weight of 690 Da or less, and the peptide with a molecular weight of 499 Da or less may be 60% or more.

[0019] According to one embodiment of the present invention, the purified product may be an anionic peptide obtained by passing an enzymatically reacted protein hydrolysate through a cation exchange resin.

[0020] According to one embodiment of the present invention, the active ingredient may be included in the composition at a concentration of 10 to 2,000 ppm.

[0021] According to one embodiment of the present invention, the active ingredient may be prepared by a method comprising: (1) preparing an enzyme solution by mixing a complex of an enzyme of IUBMB enzyme nomenclature class EC 3.4.21.62 and an enzyme of IUBMB enzyme nomenclature class EC 3.4.11.1 as a protein hydrolyzing enzyme in purified water; (2) obtaining a reaction solution by mixing defatted powder of black soldier fly (Hermetia illucens) larvae into the enzyme solution and hydrolyzing it; (3) obtaining a supernatant by centrifuging the reaction solution; and (4) purifying the supernatant.

[0022] According to one embodiment of the present invention, the purified material may have the effect of improving wrinkles and enhancing skin elasticity through collagenase production inhibitory activity or elastase inhibitory activity.

[0023] According to one embodiment of the present invention, the purified material may exhibit tyrosinase inhibitory activity or melamine synthesis inhibitory activity.

[0024] According to one embodiment of the present invention, the cosmetic composition may be characterized by additionally including the following components based on the total weight of the cosmetic composition in addition to the purified material:

[0025] (a) Dissolved portion of purified material: 30 to 99 weight%;

[0026] (b) Thickening portion: 0.1 to 20 weight%;

[0027] (c) Solubilizing portion: 0.1 to 35 weight%;

[0028] (d) Physiologically active part: 0.01 to 35 weight%; and

[0029] (e) Moisturizing ingredient portion: 0.1 to 40 weight%.

[0030] According to one embodiment of the present invention, the thickening agent may comprise one or more selected from sodium silicate, magnesium aluminum silicate, acrylates / C10-30 alkyl acrylate crosspolymer, ammonium acryloyldimethyltaurate / VP copolymer, sodium magnesium silicate, xanthan gum, hydroxypropyl starch phosphate, polyurethane-2, polymethyl methacrylate, and PVP polymer.

[0031] According to one embodiment of the present invention, the solubilizing agent may comprise one or more selected from ethanol, citric acid, phenoxyethanol, ethylhexyl glycerin, monooleic polyoxyethylene sorbitan, sorbitan sesquioleate, polysorbate 60, preservatives, and fragrances.

[0032] According to one embodiment of the present invention, the physiologically active part comprises Scutellaria baicalensis extract, Coptis japonica extract, Phellodendron amurense extract, Astragalus membranaceus extract, Polygonatum odoratum extract, Apricot kernel extract, Plantago asiatica extract, Bupleurum chinense extract, Coix lacryma-jobi extract, Moroheia leaf extract, red iron oxide, yellow iron oxide, adenosine, copper tripeptide-1, r-R-spider polypeptide-1, hydrolyzed collagen, niacinamide, pearl extract, gold, RH-oligopeptide-1, hyaluronic acid, elastin, glycerin, mallow extract, peppermint leaf extract, ilamascaria oil, arbutin, vitamin B3, vitamin B5, vitamin B9, vitamin C, ascorbic acid 2-glucoside, vitamin H, tocophenylacetate, acetylglutamine, acetylglucosamine, madecassoside, and It may contain one or more selected from carnosines.

[0033] According to one embodiment of the present invention, the moisturizing ingredient may comprise one or more selected from glycerin, butylene glycol, propylene glycol, sorbitol, hexylene glycol, dipropylene glycol, diglycerin, 1,2-hexanediol, panthenol, betaine, squalane, petroleum jelly, liquid paraffin, and hydrolyzed wheat gluten.

[0034] According to one embodiment of the present invention, the cosmetic composition may have any one formulation selected from a lotion, a liquid, a cream, an essence, a cosmetic ointment, a spray, a gel, a pack, a sunscreen, a makeup base, a foundation, a powder, a makeup remover, and a cleanser.

[0035] To achieve the other objectives mentioned above, the present invention provides a method for preparing a cosmetic composition for skin whitening and skin wrinkle improvement, comprising: (1) a step of preparing an enzyme solution by mixing a complex of an enzyme of IUBMB enzyme nomenclature class EC 3.4.21.62 and an enzyme of IUBMB enzyme nomenclature class EC 3.4.11.1 as a protein hydrolyzing enzyme in purified water; (2) a step of obtaining a reaction solution by mixing defatted powder of black soldier fly (Hermetia illucens) larvae into the enzyme solution and hydrolyzing it; (3) a step of obtaining a supernatant by centrifuging the reaction solution; and (4) a step of purifying the supernatant.

[0036] The protein hydrolysate of black soldier fly larva according to the present invention has excellent skin absorption rate, effectively works to whiten the skin by inhibiting melanin synthesis and tyrosinase activity, and has excellent effects in preventing or improving skin wrinkles by enhancing skin elasticity through collagenase inhibitory activity and / or elastase inhibitory activity.

[0037] The present invention will be described in detail below.

[0038] One aspect of the present invention relates to a cosmetic composition for skin whitening and skin wrinkle improvement comprising, as an active ingredient, a purified protein hydrolysate obtained by enzymatically reacting defatted powder of black soldier fly (Hermetia illucens) larvae with a complex of an enzyme of IUBMB enzyme nomenclature class EC 3.4.21.62, and an enzyme of IUBMB enzyme nomenclature class EC 3.4.11.1.

[0039] The purified product of the black soldier fly larva protein hydrolysate of the present invention exhibits skin whitening and skin wrinkle improvement activities.

[0040] In one embodiment, the enzyme of IUBMB enzyme nomenclature class EC 3.4.21.62 is an alkaline protease derived from Bacillus, such as “Alcalase” (Novozyme), “Protex 6L (also known as FoodPro Alkaline Protease)” (Genencor International BVBA), colorase N, ALK-enzyme, basylopeptidase A, basylopeptidase B, Bacillus subtilis alkaline proteinase bioprase, bioprase AL 15, bioprase APL 30, colistinase, subtilisin J, subtilisin S41, subtilisin Sendai, subtilisin GX, subtilisin E, subtilisin BL, subtilisin A (Type VIII), It may be one or more selected from genenase I, esperase, maxatase, thermoase PC 10, protease XXVII, thermoase, superase, subtilisin DY, subtilopeptidase, SP 266, savinase 8.0L, savinase 4.0T, kazusase, protease VIII, optimizease, optilean, Bacillus subtilis alkaline proteinase, protein A 3L, savinase, savinase 16.0L, savinase 32.0L EX, orientase 10B, and protease S, and preferably Bacillus licheniformis (Bacillus It may be Protex 6L, subtilisin A, or alkalase, which are alkaline serine endopeptidases produced by licheniformis, and more preferably Protex 6L.

[0041] In one embodiment, the enzyme of IUBMB enzyme nomenclature class EC 3.4.11.1 is a complex of proteases and peptidases derived from fungi, and may be one or more selected from, for example, A-LAP, acidic M17 leucine aminopeptidase, AcLAP, adipocyte-derived leucine aminopeptidase, Aminopeptidase, and "Flavourzyme" (Novozyme), and preferably may be a flavourzyme, which is a complex of endoproteases and exopeptidases derived from Aspergillus oryzae.

[0042] Meanwhile, enzymes of IUBMB enzyme nomenclature class EC 3.4.23.1, such as pepsin and aspartic proteinnase, do not exhibit significant skin whitening or skin wrinkle improvement effects even when a purified product of black soldier fly larva protein hydrolysate is prepared through enzymatic treatment. The above pepsin is an aspartic endopeptidase that selectively cleaves hydrophobic, particularly aromatic, amino acid residues.

[0043] In addition, enzymes of IUBMB enzyme nomenclature class EC 3.4.24.28 are neutral proteases derived from Bacillus, such as bacillus lysine, Bacillus metalloendopeptidase, anilozyme P10; Bacillus metalloproteinase, megateriopeptidase, and "Neutrase" (Novozyme), etc., do not show significant skin whitening and skin wrinkle improvement effects even when a purified product of black soldier fly larva protein hydrolysate is prepared through enzyme treatment.

[0044] In addition, enzyme complexes containing a portion of the enzyme of IUBMB enzyme nomenclature class EC 3.4.24.28, such as "Protamex" (Novozyme), do not exhibit significant skin whitening and skin wrinkle improvement effects. The above Protamex is a complex of the enzyme of IUBMB enzyme nomenclature class EC 3.4.24.28 and the enzyme of IUBMB enzyme nomenclature class EC 3.4.21.62.

[0045] The above-mentioned proteolytic enzyme may be an enzyme of IUBMB enzyme nomenclature class EC 3.4.21.62; and an enzyme of IUBMB enzyme nomenclature class EC 3.4.11.1 mixed in a weight ratio of 1:0.5 to 2.5, preferably 1:1 to 3, more preferably 1:1.5 to 2.5, and a mixture in a weight ratio of 1:1.5 to 2.5 is advantageous in terms of skin whitening and skin wrinkle improvement efficacy.

[0046] In one embodiment, the purified product of the black soldier fly larva protein hydrolysate may be prepared by a method comprising: (1) preparing an enzyme solution by mixing a complex of an enzyme of IUBMB enzyme nomenclature class EC 3.4.21.62 and an enzyme of IUBMB enzyme nomenclature class EC 3.4.11.1 as a protein hydrolyzing enzyme in purified water; (2) obtaining a reaction solution by mixing defatted powder of black soldier fly (Hermetia illucens) larvae into the enzyme solution and hydrolyzing it; (3) obtaining a supernatant by centrifuging the reaction solution; and (4) purifying the supernatant.

[0047] In one embodiment, the concentration of the enzyme solution may be 0.01 to 5% (w / w), preferably 0.01 to 3% (w / w), more preferably 0.02 to 2% (w / w), more preferably 0.05 to 1.5% (w / w), and more preferably 0.05 to 1.0% (w / w).

[0048] In one embodiment, the concentration of the black soldier fly larvae defatted powder mixed into the enzyme solution may be 5 to 40% (w / w), preferably 10 to 30% (w / w), more preferably 15 to 25% (w / w), more preferably 18 to 22% (w / w), and more preferably 19 to 21% (w / w).

[0049] In one embodiment, the protein hydrolyzing enzyme may be 0.1 to 1 part by weight, preferably 0.1 to 0.5 parts by weight, and more preferably 0.2 to 0.4 parts by weight, per 100 parts by weight of the black soldier fly larva defatted powder.

[0050] In one embodiment, the hydrolysis reaction may be carried out at 50 to 60 ℃, preferably 52 to 58 ℃, more preferably 54 to 56 ℃ for 0.5 to 24 hours, preferably 0.5 to 12 hours, more preferably 0.5 to 8 hours, more preferably 0.5 to 5 hours, more preferably 1 to 5 hours, more preferably 1 to 3 hours.

[0051] In one embodiment, the purified product may be an anionic peptide obtained by passing the enzyme-reacted protein hydrolysate through a cation exchange resin.

[0052] In one embodiment, the cation exchange resin may be a weakly acidic cation exchange resin (TRILITE WCA10L, Samyang Corporation).

[0053] In one embodiment, the surface potential of the purified material may be -4 to -10 mV, preferably -5 to -9 mV, and more preferably -7 to -9 mV.

[0054] In one embodiment, the purified product has a weight-average molecular weight (Da) of 200 to 800 Da, more specifically 300 to 700 Da, and even more specifically 350 to 690 Da.

[0055] In one embodiment, the peptide contained in the purified product has an average molecular weight of 690 Da or less, and contains 60% or more of low molecular weight peptides of 499 Da or less, which is more advantageous for the skin absorption of functional ingredients.

[0056] In one embodiment, the purified product may have an amino acid content and purity of 60% or more, specifically 70% or more, and more specifically 80% or more.

[0057] The purified product of the black soldier fly larva protein hydrolysate of the present invention, prepared under the above-described conditions, can be usefully utilized in the manufacture of cosmetics for skin whitening and skin wrinkle improvement.

[0058] In one embodiment, the purified product of the black soldier fly larva protein hydrolysate of the present invention may exhibit an effect of improving skin whitening through melanin production inhibitory activity and tyrosinase inhibitory activity.

[0059] In one embodiment, the skin wrinkles may be caused by photoaging or natural aging, specifically by photoaging, and more specifically by photoaging caused by UV exposure. Additionally, the skin wrinkles may be based on the activity of MMP enzymes. Generally, skin aging is accelerated due to the increased expression and activity of MMPs, which are enzymes that degrade collagen, a major component of the skin, following UV exposure.

[0060] In one embodiment, the purified product of the black soldier fly larva protein hydrolysate of the present invention inhibits the production of collagenase (MMP) that increases due to UV stimulation and promotes the secretion of collagen whose secretion amount is reduced due to UV, thereby preventing photoaging.

[0061] In addition, the purified product of the black soldier fly larva protein hydrolysate of the present invention can exhibit the effect of improving skin wrinkles caused by photoaging and the like and enhancing skin elasticity through elastase inhibitory activity and MMP expression inhibitory activity.

[0062] Meanwhile, the inventors of the present invention tested the cumulative skin irritation of the purified black soldier fly larva protein hydrolysate through specific experiments and confirmed that it is a harmless substance to the human body. Furthermore, since the purified black soldier fly larva protein hydrolysate of the present invention has almost no toxicity or side effects, it can be used with confidence even during long-term use.

[0063] In the present invention, the meaning of "containing as an active ingredient" is that the cosmetic composition contains an effective amount to the extent that it can exhibit melanin production inhibitory activity and tyrosinase inhibitory activity related to skin whitening improvement efficacy, and / or collagenase production inhibitory activity, collagen production promoting activity, MMP expression inhibitory activity, and elastase inhibitory activity related to skin wrinkle improvement efficacy.

[0064] In one embodiment, the active ingredient may be included in the composition at a concentration of 1 to 5,000 ppm, preferably 10 to 2,000 ppm, more preferably 500 to 1,500 ppm, more preferably 800 to 1,200 ppm, and more preferably 800 to 1,000 ppm.

[0065] If the content of the above active ingredient is below the above range, it is difficult to expect an effect from the above active ingredient; if it exceeds the above range, not only is the increase in effect with increasing content minimal, but there is also a problem of reduced stability of the formulation. That is, by including the active ingredient of the present invention in the composition within the above range, excellent skin whitening and skin wrinkle improvement effects can be realized, formulation and product stability can be maintained, and excellent effects can be exhibited at an optimal content in terms of economic efficiency. In particular, the purified product of the black soldier fly larva protein hydrolysate can exhibit significant skin whitening and skin wrinkle improvement efficacy even in a trace amount of 800 to 1,000 ppm.

[0066] In one embodiment, the cosmetic composition for skin whitening and skin wrinkle improvement of the present invention may be any one formulation selected from lotion, emulsion, cream, essence, cosmetic ointment, spray, gel, pack, sunscreen, makeup base, foundation, powder, makeup remover, and cleanser.

[0067] In one embodiment, the cosmetic composition for skin whitening and skin wrinkle improvement of the present invention may additionally include the following components based on the total weight of the cosmetic composition in addition to the above-mentioned purified material:

[0068] (a) Dissolved portion of purified material: 30 to 99 weight%;

[0069] (b) Thickening portion: 0.1 to 20 weight%;

[0070] (c) Solubilizing portion: 0.1 to 35 weight%;

[0071] (d) Physiologically active part: 0.01 to 35 weight%; and

[0072] (e) Moisturizing ingredient portion: 0.1 to 40 weight%.

[0073] In one embodiment, the thickening agent may be one or more selected from sodium silicate, magnesium aluminum silicate, acrylates / C10-30 alkyl acrylate crosspolymer, ammonium acryloyldimethyltaurate / VP copolymer, sodium magnesium silicate, xanthan gum, hydroxypropyl starch phosphate, polyurethane-2, polymethyl methacrylate, and PVP polymer, but is not limited thereto.

[0074] In one embodiment, the solubilizing agent may be one or more selected from ethanol, citric acid, phenoxyethanol, ethylhexyl glycerin, monooleic polyoxyethylene sorbitan, sorbitan sesquioleate, polysorbate 60, preservatives, and fragrances, but is not limited thereto.

[0075] In one embodiment, the physiologically active part is one or more selected from Scutellaria baicalensis extract, Coptis japonica extract, Phellodendron amurense extract, Astragalus membranaceus extract, Polygonatum odoratum extract, Apricot kernel extract, Plantago asiatica extract, Bupleurum chinense extract, Coix lacryma-jobi extract, Moroheia leaf extract, red iron oxide, yellow iron oxide, adenosine, copper tripeptide-1, r-R-spider polypeptide-1, hydrolyzed collagen, niacinamide, pearl extract, gold, RH-oligopeptide-1, hyaluronic acid, elastin, glycerin, mallow extract, peppermint leaf extract, ilamascaria oil, arbutin, vitamin B3, vitamin B5, vitamin B9, vitamin C, vitamin H, tocophenylacetate, acetylglutamine, acetylglucosamine, madecassoside, and carnosine. It is possible, but is not limited to this.

[0076] In one embodiment, the moisturizing ingredient may be one or more selected from glycerin, butylene glycol, propylene glycol, sorbitol, hexylene glycol, dipropylene glycol, diglycerin, 1,2-hexanediol, panthenol, betaine, squalane, petroleum jelly, liquid paraffin, and hydrolyzed wheat gluten, but is not limited thereto.

[0077] The cosmetic composition of the present invention may be used alone or by overlapping application, or by overlapping application with other cosmetic compositions other than the present invention. Furthermore, the cosmetic composition according to the present invention, which has excellent skin whitening and wrinkle improvement effects, may be used according to conventional methods of use, and the frequency of use may be varied depending on the user's skin condition or preference.

[0078]

[0079] Another aspect of the present invention relates to a method for preparing a cosmetic composition for skin whitening and skin wrinkle improvement, comprising: (1) preparing an enzyme solution by mixing a complex of an enzyme of IUBMB enzyme nomenclature class EC 3.4.21.62 and an enzyme of IUBMB enzyme nomenclature class EC 3.4.11.1 as a protein hydrolyzing enzyme in purified water; (2) obtaining a reaction solution by mixing defatted powder of black soldier fly (Hermetia illucens) larvae into the enzyme solution and hydrolyzing it; (3) obtaining a supernatant by centrifuging the reaction solution; and (4) purifying the supernatant.

[0080] As the “protein hydrolyzing enzyme,” “purified product of black soldier fly larva protein hydrolysate and method for preparing the same,” “skin whitening,” “skin wrinkles,” and “cosmetic composition” of the present invention have already been described above, their description is omitted to avoid excessive duplication.

[0081]

[0082] The present invention will be described in detail below by way of examples, but the present invention is not limited by the following examples.

[0083]

[0084] <Example>

[0085] Preparation Example: Preparation of defatted powder of black soldier fly larvae

[0086] (a) Black soldier fly larvae were purchased from a professional breeding farm and fasted for 3 days to completely expel excrement, and then washed three times with running water while still raw. Afterward, the black soldier fly larvae were boiled in water at 70–100°C, and the boiled larvae were crushed to an average size of 400–500 µm, centrifuged (4,000 rpm, 15 min) to separate the oil, and dried to produce dried black soldier fly larvae powder.

[0087] (b) 10 times the weight of the dried powder of the black soldier fly larvae was mixed with distilled water and left for 2 hours, then centrifuged at 1,000 rpm for 30 minutes to recover the supernatant.

[0088] (c) Five times the weight of isopropanol was mixed into the supernatant and stirred vigorously for 3 minutes, then left to stand for 15 minutes to separate the aqueous layer (protein layer) and the isopropanol layer (lipid layer). Five times the weight of n-hexane was mixed into the aqueous layer and stirred vigorously for 3 minutes, then left to stand for 15 minutes to separate the aqueous layer (protein layer) and the n-hexane layer (lipid-soluble / insoluble material layer). The protein in the aqueous layer was recovered and then freeze-dried to produce black soldier fly larva defatted powder.

[0089]

[0090] Example 1: Preparation of purified black soldier fly larva protein hydrolysate

[0091] (a) FoodPro in 500 g of purified water ⓡ 0.1 g of alkaline protease (also known as 'Protex 6L') (Genencor international BVBA, Denmark), and flavourzyme ⓡ An enzyme solution was prepared by mixing 0.2 g of Novozymes.

[0092] (b) 100 g of black soldier fly larvae defatted powder was mixed with 500 g of the enzyme solution above, and the mixture was hydrolyzed for 2 hours while stirring at 55 ℃ and 100 rpm.

[0093] (c) The above reaction solution was heated at 90°C for 20 minutes to inactivate the enzyme, then centrifuged at 6,000 rpm for 20 minutes, and the supernatant was recovered by filtering through a 0.2 µm filter paper.

[0094] (d) A weakly acidic cation exchange resin (TRILITE WCA 10L, Samyang Corporation) was swollen in 10 times its weight of an aqueous solution of 1N acetic acid and then packed into an open column tube. Afterward, the resin was activated by flowing 10 times its weight of an aqueous solution of 1M HCl through it, and then washed by flowing 10 times its weight of distilled water through it.

[0095] Subsequently, the supernatant obtained in step (c) above was loaded into an open column (maximum resin volume of 20 g / L), and distilled water at a weight of 10 times the resin was continuously flowed at an appropriate rate to elute it. The eluted eluent was freeze-dried to obtain a final purified product (anionic peptide).

[0096]

[0097] Example 2: Use of strongly acidic cation exchange resin

[0098] The procedure was carried out in the same manner as in Example 1, but the final purified product was obtained by using TRILITE MC-08H, a strong acidic cation exchange resin, instead of the weak acidic cation exchange resin in step (d).

[0099]

[0100] Example 3: Weight ratio of alkali protease and flavozyme 1:1

[0101] The procedure was carried out in the same manner as in Example 1, but the alkali protease and flavozyme from step (a) were each mixed in an amount of 0.15 g (weight ratio of 1:1) to obtain the final purified product.

[0102]

[0103] Example 4: Use of Alcalase and Flavozyme Complex Enzyme

[0104] The same procedure as in Example 1 was followed, but a purified product of black soldier fly larva protein hydrolysate was prepared using an alkalase and flavozyme complex enzyme instead of an alkali protease (Protex 6L) and flavozyme complex enzyme.

[0105]

[0106] Comparative Example 1: Omission of purification process

[0107] The black soldier fly larva protein hydrolysate was prepared by carrying out the same procedure as in Example 1, but omitting the purification process in step (d).

[0108]

[0109] Comparative Example 2: Alkaline protease alone

[0110] The same procedure as in Example 1 was followed, but an alkaline protease (Protex 6L) was used alone to prepare a purified product of black soldier fly larva protein hydrolysate.

[0111]

[0112] Comparative Example 3: Flavozyme alone

[0113] The same procedure as in Example 1 was carried out, but using Flavozyme alone, a purified product of black soldier fly larva protein hydrolysate was prepared.

[0114]

[0115] Comparative Example 4: Alcalase alone

[0116] The same procedure as in Example 1 was followed, but a purified product of black soldier fly larva protein hydrolysate was prepared using alkalase alone instead of the complex enzyme of alkali protease (Protex 6L) and flavozyme.

[0117]

[0118] <Test Example>

[0119] Statistical analysis of all test examples was performed using MINITAB version 21.1.1.0 (USA). Differences in mean values ​​between experimental groups (p < 0.05) were determined using one-way ANOVA with Duncan's post-hoc test. Each of the following experiments was repeated at least three times, and the experimental data were expressed as mean ± standard deviation (SD). In each data set, *, **, and *** indicate a significant difference (Tukey's test) at P < 0.05, P < 0.01, and P < 0.001, respectively, compared to the control group, and #, ##, and ### indicate a significant difference (Tukey's test) at P < 0.05, P < 0.01, and P < 0.001, respectively, compared to the negative or positive control group. The defatted black soldier fly larva powder of the preparation example was used as the control group.

[0120]

[0121] Test Example 1: Surface potential measurement

[0122] We intended to measure the surface potential of the black soldier fly larva protein hydrolysate or its purified product (hereinafter referred to as "hydrolysate") according to the above examples and comparative examples. Specifically, the surface potential of each sample was measured using a zeta potential analyzer, which is a measuring instrument for dynamic light scattering and electrophoretic light scattering, and is shown in Table 1 below.

[0123] Surface Potential (mV) Control Group - 0.11 Example 1 - 8.84 Example 2 - 5.65 Example 3 - 5.79 Example 4 - 5.27 Comparative Example 1 - 0.83 Comparative Example 2 - 3.27 Comparative Example 3 - 3.42 Comparative Example 4 - 4.16

[0124] Looking at Table 1 above, it can be seen that the hydrolysate according to the embodiment of the present invention showed better separation of low molecular weight anionic peptides compared to the hydrolysate according to the comparative example. In particular, it can be seen that the hydrolysate using a weak acidic cation exchange resin according to Example 1 showed better separation of low molecular weight anionic peptides compared to the hydrolysate using a strong acidic cation exchange resin according to Example 2.

[0125]

[0126] Test Example 2: Average molecular weight, distribution by molecular weight, and amino acid composition

[0127] 2-1: Average molecular weight

[0128] Gel permeation chromatography (GPC) is widely used to predict the average molecular weight distribution (Mw) of macromolecular compounds such as proteins. It is known that this GPC method can measure relatively effectively, especially in the case of low molecular weight compounds (molecular weight of about 40,000 or less), among macromolecular materials.

[0129] The molecular weight of the hydrolysates according to the above examples and comparative examples was investigated. The investigation method involved centrifuging each sample, collecting the supernatant, and analyzing it using gel permeation chromatography (GPC; Agilent 1100 HPLC, Ultrahydrogel™ 120, 250, 500, 1000 columns). The average molecular weight was calculated by estimating the molecular weight (Mi) from the retentin volume (VR) of the GPC data and calculating the relative number of molecules of a given molecular weight (Ni = Hi / Mi) from the peak height (Hi). The weight-averaged molecular weight (Mw) was calculated using the following formula, and the results are shown in Table 2. Here, Ni is the number of molecules or moles with a weight of Mi in the polymer sample, and Mi is the weight of a specific molecule in the sample.

[0130] Mw = ∑NiMi 2 / ∑NiMi

[0131] Classification Weight-Average Molecular Weight (Da) Control Group 54,259 Example 1523 Example 2611 Example 3642 Example 4674 Comparative Example 1704 Comparative Example 2726 Comparative Example 3750 Comparative Example 4815

[0132] Looking at Table 2 above, it can be seen that the average molecular weight of the hydrolysates of the examples or comparative examples is significantly reduced compared to the control group.

[0133] 2-2: Peptide Distribution by Molecular Weight of Protein Hydrolysates

[0134] After centrifuging each hydrolysate sample according to the above examples and comparative examples, the supernatant was taken, and the weight-average molecular weight distribution was investigated based on the distribution data obtained by analyzing the weight-average molecular weight (Mw) by gel permeation chromatography. The results are shown in Table 3.

[0135] Molecular Weight Size (%) 10 kDa or more 5 ~ 9.9 kDa 1 ~ 4.9 kDa 500 ~ 990 Da 499 Da or less total Control Group 23 16 22 24 15 100 Example 10 00 18 8 2100 Example 20 00 37 6 3100 Example 30 00 29 7 1100 Example 40 00 35 6 5 100 Comparative Example 10 0 23 26 6 100 Comparative Example 20 15 5 8 36 100 Comparative Example 30 9 11 6 11 9 100 Comparative Example 40 7 12 5 3 28 100

[0136] Looking at Table 3 above, unlike the control group and comparative example, no peptides of 1 kDa or higher were found in the hydrolysate of the example. In particular, the hydrolysate of Example 1 showed that peptides of 499 Da or lower accounted for more than 80%, which appears to be advantageous for the skin absorption of functional ingredients when applied to the manufacture of cosmetics.

[0137] 2-3: Amino acid composition of protein hydrolysate

[0138] 1 mL of a 7% 5-sulfosalicylic acid aqueous solution was added to 1 mL of each hydrolysate sample according to the above examples and comparative examples and maintained for 2 hours to precipitate the protein, followed by centrifugation (4,500 rpm, 10 min). The obtained supernatant was diluted with sample dilution buffer (pH 2.2), filtered through a membrane filter (0.45 µm), and then analyzed using an automatic amino acid analyzer from Sykam GmbH (Gewerbering, Eresing, Germany; cation separation column LCA K06 / NA). The results are shown in Table 4.

[0139] Amino Acids (Weight%) Control Group Example 1 Example 2 Example 3 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 ASP 4.5 3 7.65 *** 6.54 * 6.04 * 5.86 * 5.075.545.21THR0.531.58 ** 1.26 * 1.02 * 0.91.04 * 1.23 * 1.27 * SER0.120.560.420.250.240.40.420.47GLU11.658.56 ** 9.54 * 7.53 *** 9.79 * 7.14 *** 8.45 ** 11.29PRO3.9211.71 *** 10.11 ** 8.47 * 4.783.483.683.72GLY2.915.91 ** 5.08 ** 5.29 ** 3.72.892.642.93ALA3.096.07 *** 5.63 ** 5.52 ** 4.95 * 3.634.164.87 *VAL2.258.83 *** 7.85 ** 7.46 ** 4.223.463.823.63ISOL1.426.04 *** 5.13 ** 4.63 ** 2.542.32.752.07LEU1.598.11 *** 6.62 ** 5.86 * 4.24 * 3.683.883.45TYR0.613.57 *** 2.88 ** 2.65 ** 1.41 * 0.571.23 * 0.57PHE1.254.78 ** 4.02 * 3.84 * 2.192.182.011.2HIS3.563.883.743.283.312.563.241.49 * LYS1.854.74 *** 4.12 ** 3.85 ** 3.71 ** 3.25 * 2.89 * 1.44ARG2.023.72 ** 3.432.942.722.152.630.53CYS0.451.29 *** 1.26 * 1.04 * 0.790.610.850.57MET0.211.09 *** 0.95 * 0.82 * 0.630.480.540.46total (순도)41.9688.09 *** 78.58 ** 70.49 ** 55.98 * 44.8949.9645.17

[0140] Looking at Table 4 above, it can be seen that the total amino acid content and purity of the protein hydrolysate according to the example are significantly higher than those of the control group and comparative example. In particular, the total amino acid content and purity of the protein hydrolysate according to Example 1 were found to be significantly higher than those of Example 2 and Example 3.

[0141]

[0142] Test Example 3: Cytotoxicity

[0143] To investigate the effect of the sample obtained in the above example on cell viability by the MTS assay, an experiment was performed as follows using the method described in the existing literature (Choi JHet al., Flowers of Inula japonica attenuate inflammatory responses, Immune Network 2010, 10(5), 145-152).

[0144] First, 5×10 CCD-986sk cells in a 96-well plate 4 Cells were dispensed at a density of cell / mL and cultured under 5% CO2 and 37°C conditions. After 24 hours, samples according to the examples were treated at different concentrations (0.1, 0.2, 0.5, 1.0 mg / mL) and cultured for another 24 hours. Subsequently, the supernatant was removed, and 1 μg / mL of MTT solution was added and left for 2 hours. Then, 100 μL of DMSO was added per well to dissolve the formazan crystals for 20 minutes, and the absorbance was measured at 590 nm using an ELISA plate reader. Cell viability was calculated using Equation 1 below, and the results are shown in Table 5 below as a relative ratio to the control group.

[0145] [Mathematical Formula 1]

[0146] Cell viability (%) = (Absorbance of sample-treated group / Absorbance of control group) × 100

[0147] Sample (mg / mL) Cell Viability (%) Control Group (Untreated Group) 100 Example 10.1 114.9 0.2 119.6 0.5 139.3 1.0 128.3 Example 20.1 105.7 0.2 102.1 0.5 109.4 1.0 102.6 Example 30.1 115.2 0.2 109.8 0.5 103.7 1.0 96.1 Example 40.1 112.4 0.2 117.6 0.5 121.3 1.0 113.5 Comparative Example 10.1 84.2 0.2 80.3 0.5 75.4 1.0 73.6

[0148] Looking at Table 5 above, it can be seen that the sample according to the embodiment of the present invention does not exhibit significant toxicity up to a concentration of 1.0 mg / mL (1,000 ppm). On the other hand, it was confirmed that the sample according to Comparative Example 1, which did not undergo the purification process of the present invention, exhibited significant toxicity at a concentration of 0.5 mg / mL or higher.

[0149]

[0150] Test Example 4: Skin whitening activity

[0151] 4-1: Melanin production inhibitory activity

[0152] 5×10⁶ B16-F10 melanoma cells in a 6-well plate 41 mL of medium was seeded at a concentration of cells / mL and cultured for 24 hours. Subsequently, all medium was removed from the well plates and washed with PBS to completely remove the medium. Then, α-MSH was added to each well plate at a concentration of 200 nM, and samples according to the examples and comparative examples were added at a concentration of 1 mg / mL, respectively. The plates were then cultured for 72 hours in a 5% CO2 incubator at 37°C. Next, the medium was removed and washed with PBS, after which cells were secured using trysine-EDTA and centrifuged at 1000 rpm for 3 minutes. After removing the supernatant, 1N NaOH dissolved in 10% DMSO was added to lyse the cells. Melanin was then lysed in a heat block at 80°C for 1 hour, and the amount of melanin was determined by measuring the absorbance at 475 nm. Intracellular protein levels were measured using a BCA protein assay kit (Pierce, USA), and the protein was quantified according to the BSA standard curve. The amount of melanin was calculated according to a standard curve using a melanin standard substance. Subsequently, the inhibition rate of intracellular melanin production was calculated according to the following formula. In this case, the negative control group was a test group treated with α-MSH at a concentration of 200 nM, and the positive control group was a test group treated with arbutin at a concentration of 100 μg / mL.

[0153] [Mathematical Formula 2]

[0154] Intracellular melanin production inhibition rate (%) = 100 - [(Amount of melanin relative to protein amount in sample-treated group) / (Amount of melanin relative to protein amount in negative control group) × 100]

[0155] Classification Melanin Production Inhibition Rate (%) Negative Control (α-MSH) 0.0 Positive Control (Arbutin) 44.8 ## Example 165.0 ### Example 243.2 ## Example 342.7 ## Example 445.4 ## Comparative Example 110.5 Comparative Example 218.3 #Comparative Example 315.9 Comparative Example 419.1 #

[0156] Looking at Table 6 above, it can be seen that the sample according to the embodiment of the present invention has a significantly higher melanin production inhibitory effect compared to the comparative example, and inhibits melanin production to a level similar to or higher than that of the positive control group. In particular, the sample according to Example 1 of the present invention was found to have a significantly higher melanin production inhibitory effect compared to Examples 2 to 4 and the positive control group.

[0157] 4-2: Tyrosinase Inhibitory Activity

[0158] 1.5 × 10⁶ MNT-1 melanoma cells in a 6-well plate 5 Cells were seeded at a concentration of cells / mL and cultured for 24 hours. Subsequently, α-MSH at 200 nM and samples according to the examples and comparative examples at a concentration of 1 mg / mL, respectively, were added to each well plate and cultured for 72 hours in a 5% CO2 incubator at 37°C. Next, the cells were centrifuged at 13,000 rpm for 1 minute, and the supernatant was removed. Then, 300 μl of 0.5% Triton X-100 solution was added to the cell pellet to lyse the cells. The mixture was centrifuged again at 13,000 rpm for 3 minutes, and the supernatant was recovered. 100 μl of the recovered cell lysis supernatant was mixed with 1 mM L-DOPA solution and reacted at 37°C for 1 hour. The absorbance of the reaction mixture was measured at 450 nm using an ELISA reader. Tyrosinase activity was calculated as a percentage of the absorbance of the negative control. At this time, the negative control group is a test group treated with α-MSH at a concentration of 200 nM, and the positive control group is a test group treated with arbutin at a concentration of 100 μg / mL.

[0159] Classification Tyrosinase Activity (% of Negative Control) Tyrosinase Inhibitory Activity (%) Negative Control (α-MSH) 100 - Positive Control (Arbutin) 51.52 ###48.48 ### Example 149.56 ### 50.44 ### Example 272.45 ## 27.55 ## Example 368.24 ## 31.76 ## Example 465.37 ## 34.63 ## Comparative Example 191.168.84 Comparative Example 288.7211.28 Comparative Example 385.3914.61 Comparative Example 489.2410.76 Comparative Example 587.3312.67

[0160] Looking at Table 7 above, it can be seen that the sample according to the embodiment of the present invention has a significantly higher tyrosinase activity inhibitory effect compared to the comparative example, and inhibits tyrosinase activity to a level similar to or higher than that of the positive control group. In particular, the sample according to Example 1 of the present invention was found to have a significantly higher tyrosinase activity inhibitory effect compared to Examples 2 to 4 and the positive control group.

[0161]

[0162] Test Example 5: Skin wrinkle improvement activity

[0163] 5-1: Collagenase production inhibitory activity

[0164] 1.5 × 10⁶ CCD-986sk cells in a 96-well plate 5 Dispensed at a concentration of cells / mL and incubated for 24 hours in a 37°C, 5% CO₂ incubator, then irradiated with UVA at 1 J / cm² 2The samples were treated with [the appropriate medium]. Subsequently, all medium was removed and washed with PBS to completely remove the medium. Then, samples according to the examples and comparative examples, diluted with serum-free medium, were treated at a concentration of 1 mg / mL each and cultured for 24 hours. Afterward, the medium was removed, cell lysis buffer was added to each well, reacted at 150 rpm for 10 minutes, and then centrifuged to collect the supernatant. The amount of protein in the collected supernatant was measured using a BCA protein assay kit, and the protein was quantified according to the BSA standard curve. In addition, the amount of collagenase was measured using a Human-MMP-1 ELISA kit, and the collagenase content was quantified according to the collagenase standard curve. Subsequently, the inhibition rate (%) of intracellular collagenase production was calculated according to the following formula. In this case, the negative control group was the untreated sample group, and the positive control group was the test group treated with TGF-1β at a concentration of 100 ng / mL.

[0165] [Mathematical Formula 3]

[0166] Intracellular collagenase production inhibition rate (%) = 100 - [(Amount of collagenase (MMP-1) relative to protein amount of sample-treated group (pg / µg)] / (Amount of collagenase (MMP-1) relative to protein amount of negative control group (pg / µg)] × 100]

[0167] Classification Collagenase production inhibition rate (%) Control group 0.0 Positive control group (TGF-1β) 45.5 ## Example 161.9 ### Example 247.13 ## Example 341.26 ## Example 444.8 ## Comparative Example 125.82 # Comparative Example 219.28 Comparative Example 324.36 # Comparative Example 420.24

[0168] Looking at Table 8 above, it can be seen that the sample according to the embodiment of the present invention has a significantly higher collagenase production inhibitory effect compared to the comparative example, and inhibits collagenase production to a level similar to or higher than that of the positive control group. In particular, the sample according to Example 1 of the present invention was found to have a significantly higher collagenase production inhibitory effect compared to Examples 2 to 4 and the positive control group.

[0169] 5-2: Collagen production-promoting activity

[0170] 1.5 × 10⁶ CCD-986sk cells in a 96-well plate 5 The cells were dispensed at a concentration of cells / mL and incubated for 24 hours in a 37°C, 5% CO₂ incubator, after which the medium was completely removed by washing with PBS. Subsequently, samples according to the examples and comparative examples, diluted with serum-free medium, were treated at a concentration of 1 mg / mL each and incubated for 24 hours. Afterward, the collagen content of the recovered culture medium was determined using a Procollagen type I Peptide EIA kit and quantified according to the collagen standard curve. Additionally, with the culture medium removed, cell lysis buffer was added to each well and reacted at 150 rpm for 10 minutes, followed by centrifugation to collect the supernatant. The amount of protein in the recovered supernatant was measured using a BCA protein assay kit and quantified according to the BCA standard curve. Subsequently, the intracellular collagen production rate (%) was calculated according to the following formula. In this case, the positive control group was the test group treated with L-ascorbic acid at a concentration of 100 μM.

[0171] [Mathematical Formula 4]

[0172] Intracellular collagen production rate (%) = [(Amount of collagen relative to protein in sample-treated group (ng / µg)] / (Amount of collagen relative to protein in control group (ng / µg)] × 100]

[0173] Classification Collagen Production Rate (%) Control Group 100 Positive Control Group (TGF-1β) 112.1 * Example 1139.6 *** Example 2117.4 ** Example 3121.6 ** Example 4123.7 ** Comparative Example 1 104.8 Comparative Example 2 106.4 Comparative Example 3 106.5 Comparative Example 4 107.3

[0174] Looking at Table 9 above, it can be confirmed that the sample according to the embodiment of the present invention has a significantly higher collagen production-promoting effect compared to the comparative example and the positive control group. In particular, the sample according to Example 1 of the present invention was found to have a significantly higher collagen production-promoting effect compared to Examples 2 to 4.

[0175] 5-3: Inhibitory activity of MMP1 gene expression increased by UVB

[0176] 5 × 10⁶ CCD-986sk cells in a 24-well plate 5 Subculture was performed using a cell / dish. After 1 day, the medium was removed, 1 mL of PBS was added, and UVB at 10 mJ / cm² was irradiated. Subsequently, samples according to the examples and comparative examples, diluted in serum-free DMEM medium, were treated at a concentration of 1 mg / mL each and cultured for 24 hours. Afterward, the medium was removed, total RNA was isolated from the cells, and cDNA was synthesized using reverse transcriptase. Using this as a template, RT-PCR (CFX Connect Real-Time PCR Detection System, Bio-Rad Co.) was performed to measure MMP-1 gene expression levels. Relative gene expression levels were analyzed using image analysis software, and the degree of MMP-1 gene expression was quantified by beta-actin for quantitative comparison.

[0177] Classification UVB Treatment MMP-1 Expression Level (%) Control -100 Negative Control +346.5 *** Example 1 + 114.9 ###Example 2 + 164.5 ## Example 3+172.0 ## Example 4+183.5 ## Comparative Example 1 + 277.7 # Comparative Example 2+264.3 # Comparative Example 3 + 217.0 # Comparative Example 4 + 255.6 #

[0178] Looking at Table 10 above, it can be confirmed that the sample according to the embodiment of the present invention has a significantly higher MMP1 expression inhibitory activity compared to the comparative example, which is increased by UVB. In particular, the sample according to Example 1 was found to have a significantly higher MMP1 expression inhibitory activity compared to Examples 2 to 4.

[0179] 5-4: Elastase Inhibitory Activity

[0180] Elastase inhibitory activity was performed according to the manufacturing protocol of the EnzCheck Elastase Assay Kit (Molecular Probes Inc., Eugene, OR, USA).

[0181] First, a stock solution of 1 mg / mL Elastin was prepared and diluted so that the final concentration was 25 μg / mL when 50 μL of Elastin was added to the well. Next, a stock solution of 100 U / mL Elastase enzyme was prepared and diluted so that the final concentration was 0.5 U / mL when 100 μL was added to the well. Additionally, the samples according to the Examples and Comparative Examples were prepared by diluting to a final concentration of 2% (w / v). The fluorescence intensity of the samples in each well was measured using a fluorescence microplate reader (Fluoroskan, Thermo Fisher) at a wavelength of 485 nm and an emission wavelength of 520 nm. The percentage of elastase inhibitory activity was calculated as follows: Inhibition Rate (%) = (1 - S / C) x 100, where "S" and "C" represent the fluorescence intensity of the sample-treated group and the positive control group, respectively. At this time, an Elastase Inhibitor (ascorbic acid, 2% w / v) was used as a positive control.

[0182] Classification Elastase Inhibition Rate (%) Control Group (Untreated) 32.8 Positive Control Group (Ascorbic Acid) 100.0 *** Example 187.9 *** Example 263.5 ** Example 365.7 ** Example 470.8 ** Comparative Example 148.3 Comparative Example 239.6 Comparative Example 343.3 Comparative Example 445.7

[0183] Looking at Table 11 above, it can be confirmed that the sample according to the embodiment of the present invention has a significantly higher elastase activity inhibitory effect compared to the comparative example. In particular, the sample according to Example 1 of the present invention was found to have a significantly higher elastase activity inhibitory effect compared to Examples 2 to 4.

[0184]

[0185] Preparation Examples and Comparative Preparation Examples: Preparation of Cosmetic Compositions

[0186] Cosmetic compositions in the form of creams were prepared using protein hydrolysates according to the examples and comparative examples, with the compositions shown in Table 12 below. In Table 12 below, Preparation Examples 1 to 4 are cosmetic compositions prepared by applying samples according to Examples 1 to 4, respectively, and Comparative Preparation Examples 1 to 4 are cosmetic compositions prepared by applying samples according to Comparative Examples 1 to 4, respectively.

[0187] Ingredient Content (Weight%) Control Group - Samples according to Examples and Comparative Examples 1,000 ppm Thickening Part: Sodium Silicate 2.0, Xanthan Gum 0.5 Solubilizing Part: Polysorbate 60 2.5, Sorbitan Sesquioleate 1.5 Physiologically Active Part: Vitamin C 0.0 8, Limonene Oil 0.1~30, Tocophenyl Acetate 0.1 Moisturizing Part: Glycerin 4.0, Vaseline 7.0, Squalane 3.0, Propylene Glycol 36.0 Purified Material Dissolving Part: Betaine 5.0, Purified Water To 100

[0188]

[0189] Test Example 6: Human Patch Test

[0190] The human patch test was conducted in accordance with the ethical regulations based on the Declaration of Helsinki and the Guidelines for Human Application Testing of Cosmetics of the Ministry of Food and Drug Safety. It was performed on 32 healthy female subjects aged 20 to 60 years (average age 31.34 ± 3.06 years) without specific skin diseases or allergies, using a method devised by Frosch & Kligman (J. Am. Acad. Dermatol, 1(1) 35 (1979)). First, the patch site was disinfected with 70% ethanol, and then a Van der Bend (Van der Bend, Netherlands) containing 2 mL of the cosmetic composition of the example was applied. After 48 h, the patch was removed, and the test site was marked with a skin marker (Chemotechnique Diagnostics AB, Sweden). The reactivity of each test site was evaluated after 20 min and 24 h. The evaluation criteria for the primary skin irritation test were based on the judgment criteria of the PCPC Guidelines presented in Table 13 below, and the degree of skin reaction (average reactivity) evaluated twice was scored according to Equation 5 below and shown in Table 14 below.

[0191] Response Clinical Symptoms (Response) 0 No response 1 Faint or mild erythema 2 Moderate erythema; and barely detectable edema and / or papule formation at the patch boundary 3 Moderate erythema; and edema spread throughout the patch site 4 Severe erythema; and severe edema; or blistering 5 Severe reaction spreading beyond the patch site

[0192] [Mathematical Formula 5]

[0193] Average Responsiveness = [(Responsiveness × Number of subjects who responded) / (Total number of subjects × Maximum score (5 points)) × 100] × 1 / 2

[0194] Number of subjects showing a differentiation response (persons) Response rate 24 hours 48 hours Average Control group 00.00.00.0 Preparation example 100.00.00.0 Preparation example 200.00.00.0 Preparation example 300.00.00.0 Preparation example 400.00.00.0

[0195] Looking at Table 14 above, as a result of evaluating the degree of skin irritation by applying the composition according to the manufacturing example of the present invention, all 32 subjects showed a negative reaction with a reaction grade of 0.0, so the composition of the present invention is judged to be a non-irritating substance in terms of primary irritation to human skin.

[0196]

[0197] Test Example 7: Confirmation of wrinkle improvement effect

[0198] We intended to confirm the skin wrinkle improvement effect of the cosmetic compositions according to the above manufacturing examples and comparative manufacturing examples.

[0199] Accordingly, the wrinkle improvement effect on the back of the hands of 32 healthy female subjects aged 20 to 60 (average age 31.34±3.06 years) was measured using a digital camera (Model C-700, Olympus, Japan) before using the cosmetic compositions according to the manufacturing example and comparative manufacturing example and after using them twice daily in the morning and evening for 4 weeks, and skin replicas were collected using a silicone polymer (Silflo impression material, Flexico, England). After fixing the angle of incidence of light to 20 degrees, wrinkle shadow brightness images were captured using a CCD camera (Model SDC-45, Samsung, Seoul, Korea) and converted into image files, and wrinkle indices were measured using the computer image analysis system Skin Visiometer SV600 software (Courage&Khazaka, Koeln, Germany). The elasticity improvement effect was measured using a Cutometer SEM 575 (C&K Electronic, Germany) and is shown in Table 15 below.

[0200] Classification Skin wrinkle reduction rate (% of before use) Skin elasticity increase rate (% of before use) Control group 5.5 4.7 Preparation Example 13 1.2 *** 28.6 *** Preparation Example 222.3 ** 19.4 ** Preparation Example 323.8 ** 18.9 ** Preparation Example 425.4 ** 20.1 ** Comparative Manufacturing Example 19.4 10.3 Comparative Manufacturing Example 211.7 9.5 Comparative Manufacturing Example 312.4 10.8 Comparative Manufacturing Example 414.6 11.6

[0201] Looking at Table 15 above, it can be seen that the cosmetic composition prepared according to the example of the present invention has a significantly increased skin wrinkle reduction rate and skin elasticity increase rate compared to the comparative example.

[0202] According to the above results, the purified product of black soldier fly larva protein hydrolysate according to the present invention exhibits excellent skin whitening and skin wrinkle improvement efficacy, and thus appears to be very useful as a material for functional cosmetics.

[0203]

[0204] Although the present invention has been described as a preferred embodiment mentioned above, various modifications and variations are possible without departing from the essence and scope of the invention. Furthermore, the appended claims include such modifications and variations that fall within the essence of the invention.

Claims

A cosmetic composition for skin whitening and skin wrinkle improvement comprising, as an active ingredient, a purified protein hydrolysate obtained by enzymatically reacting defatted powder of black soldier fly (Hermetia illucens) larvae with a complex of an IUBMB enzyme of class EC 3.4.21.62 and an enzyme of class EC 3.4.11.

1. In paragraph 1, A cosmetic composition characterized in that the enzyme of the above IUBMB enzyme nomenclature class EC 3.4.21.62 is an alkaline protease derived from Bacillus. In paragraph 2, A cosmetic composition characterized in that the above-mentioned alkaline protease derived from Bacillus is an alkaline serine endopeptidase produced by Bacillus licheniformis. In paragraph 3, A cosmetic composition characterized in that the above alkaline serine endopeptidase is Protex 6L. In paragraph 1, A cosmetic composition characterized in that the enzyme of the above IUBMB enzyme nomenclature class EC 3.4.11.1 is a complex of a protease and a peptidase of fungal origin. In paragraph 5, A cosmetic composition characterized in that the above-mentioned complex of protease and peptidase derived from fungi is a complex of endoprotease and exopeptidase derived from Aspergillus oryzae. In paragraph 6, A cosmetic composition characterized in that the complex of the above-mentioned endoprotease and exopeptidase is a flavourzyme. In paragraph 1, A cosmetic composition characterized by the above-mentioned proteolytic enzyme being an enzyme of IUBMB enzyme nomenclature class EC 3.4.21.62; and an enzyme of IUBMB enzyme nomenclature class EC 3.4.11.1 mixed in a weight ratio of 1:0.5 to 2.

5. In paragraph 1, A cosmetic composition characterized by the above enzyme reaction being performed at 50 to 60 ℃ for 0.5 to 24 hours. In paragraph 1, A cosmetic composition characterized in that the peptide contained in the above-mentioned purified product has an average molecular weight of 690 Da or less, and peptides of 499 Da or less comprise 60% or more. In paragraph 1, A cosmetic composition characterized in that the above-mentioned purified product is an anionic peptide obtained by passing an enzymatically reacted protein hydrolysate through a cation exchange resin. In paragraph 1, A cosmetic composition characterized by the above active ingredient being included in the composition at a concentration of 10 to 2,000 ppm. In paragraph 1, the above active ingredient is, (1) As a proteolytic enzyme in purified water, the enzyme of IUBMB enzyme Nominated Class EC 3.4.21.62; and the enzyme of IUBMB enzyme Nominated Class EC 3.

4. Step of preparing an enzyme solution by mixing the enzyme complex of 11.1; (2) A step of obtaining a reaction solution by mixing defatted powder of black soldier fly (Hermetia illucens) larvae into the enzyme solution and hydrolyzing it; (3) a step of centrifuging the above reaction solution to obtain the supernatant; and (4) A cosmetic composition characterized by being prepared by a method including the step of purifying the supernatant above. In paragraph 1, A cosmetic composition characterized by having the effect of improving wrinkles and enhancing skin elasticity through collagenase production inhibitory activity or elastase inhibitory activity. In paragraph 1, A cosmetic composition characterized by the above-mentioned purified product exhibiting tyrosinase inhibitory activity or melamine production inhibitory activity. A cosmetic composition according to claim 1, characterized in that, in addition to the purified material, the cosmetic composition further comprises the following components based on the total weight of the cosmetic composition: (a) Dissolved portion of purified material: 30 to 99 weight%; (b) Thickening portion: 0.1 to 20 weight%; (c) Solubilizing portion: 0.1 to 35 weight%; (d) Physiologically active part: 0.01 to 35 weight%; and (e) Moisturizing ingredient portion: 0.1 to 40 weight%. In Paragraph 16, A cosmetic composition characterized by comprising one or more selected from sodium silicate, magnesium aluminum silicate, acrylates / C10-30 alkyl acrylate crosspolymer, ammonium acryloyldimethyltaurate / VP copolymer, sodium magnesium silicate, xanthan gum, hydroxypropyl starch phosphate, polyurethane-2, polymethyl methacrylate, and PVP polymer. In Paragraph 16, A cosmetic composition characterized by comprising one or more selected from ethanol, citric acid, phenoxyethanol, ethylhexyl glycerin, monooleic polyoxyethylene sorbitan, sorbitan sesquioleate, polysorbate 60, preservatives, and fragrances. In Paragraph 16, The above-mentioned physiologically active part comprises one or more selected from Scutellaria baicalensis extract, Coptis japonica extract, Phellodendron amurense extract, Astragalus membranaceus extract, Polygonatum odoratum extract, Apricot kernel extract, Plantago asiatica extract, Bupleurum chinense extract, Coix lacryma-jobi extract, Moroheia leaf extract, red iron oxide, yellow iron oxide, adenosine, copper tripeptide-1, r-R-spider polypeptide-1, hydrolyzed collagen, niacinamide, pearl extract, gold, RH-oligopeptide-1, hyaluronic acid, elastin, glycerin, mallow extract, peppermint leaf extract, ilamascaria oil, arbutin, vitamin B3, vitamin B5, vitamin B9, vitamin C, vitamin H, tocophenylacetate, acetylglutamine, acetylglucosamine, madecassoside, and carnosine, characterized in that the cosmetic composition Composition. In Paragraph 16, A cosmetic composition characterized by comprising one or more selected from glycerin, butylene glycol, propylene glycol, sorbitol, hexylene glycol, dipropylene glycol, diglycerin, 1,2-hexanediol, panthenol, betaine, squalane, petroleum jelly, liquid paraffin, and hydrolyzed wheat gluten as the moisturizing ingredient. In paragraph 1, The above cosmetic composition is characterized by having any one formulation selected from lotion, emulsion, cream, essence, cosmetic ointment, spray, gel, pack, sunscreen, makeup base, foundation, powder, makeup remover, and cleanser. (1) A step of preparing an enzyme solution by mixing a complex of an enzyme of IUBMB enzyme nominal class EC 3.4.21.62 and an enzyme of IUBMB enzyme nominal class EC 3.4.11.1 as a protein hydrolyzing enzyme in purified water; (2) A step of obtaining a reaction solution by mixing defatted powder of black soldier fly (Hermetia illucens) larvae into the enzyme solution and hydrolyzing it; (3) a step of centrifuging the above reaction solution to obtain the supernatant; and (4) A step of purifying the supernatant above; a method for preparing a cosmetic composition for skin whitening and skin wrinkle improvement.