Cosmetic composition for whitening, moisturizing, and wrinkle alleviation containing low-molecular-weight peptide isolated from lees

Abstract

The present invention relates to a cosmetic composition containing a low-molecular-weight peptide isolated from lees. More specifically, the present invention relates to a cosmetic composition having an effect of inhibiting pigmentation through a decrease in TRP-1 protein expression, having an effect of alleviating wrinkles through an increase in collagen type 1 and laminin-5 protein expression, and having a moisturizing effect through an increase in filaggrin protein expression, by separating a low-molecular-weight peptide having whitening, moisturizing, and wrinkle-alleviating activities from the lees and containing same as an active ingredient.

Description

Cosmetic composition for whitening, moisturizing and wrinkle improvement containing low molecular weight peptide isolated from safflower seed

[0001] The present invention relates to a cosmetic composition containing a low-molecular-weight peptide isolated from the lees of the plant. More specifically, the present invention relates to a cosmetic composition for skin whitening, skin wrinkle improvement, or skin moisturizing, which is obtained by isolating a low-molecular-weight peptide having whitening, moisturizing, and wrinkle-improving activities from the lees of the plant and including the same as an active ingredient, thereby exhibiting effects of reducing TRP-1 protein expression, increasing collagen type 1 and laminin-5 protein expression, and increasing filaggrin protein expression.

[0002] In modern society, with the development of industry, the demand for various resources has increased. This has led to serious environmental problems, such as environmental pollution caused by waste and natural disasters due to global warming. The countless pollutants generated by this environmental pollution are known to have a negative impact, damaging the skin barrier and accelerating skin aging. Skin aging caused by the external environment is primarily caused by UV rays, which penetrate the epidermis and penetrate deep into the dermis, damaging collagen and elastin within the dermis and causing wrinkles. Furthermore, the excessive production of harmful reactive oxygen species accelerates skin aging. Furthermore, external pollutants, such as fine dust, cause various chemical irritations to keratinocytes and lipid membranes, leading to inflammation and contributing to pigmentation, dryness, skin barrier destruction, and wrinkles.

[0003] Meanwhile, with industrial development, waste and environmental pollution have increased, leading to a growing interest in sustainable materials. This has led to a growing interest in enhancing the value of discarded materials through upcycling, defined as the process of recycling discarded items to create new products with new value. In particular, with food processing byproducts discarded in Korea alone reaching 5.48 million tons annually, many researchers are actively researching not only product containers and packaging materials but also environmentally friendly raw materials.

[0004] A representative food processing byproduct, DDGS (drinking lees) is a byproduct of brewing and ethanol production. With rising oil prices and the rise in ethanol production as a substitute for petroleum, DDGS production is also increasing. DDGS has proven its high feed value and has been used as feed for various livestock. Its key components include crude lipids, crude protein, fiber, fermentation yeast cells, and various minerals, increasing its utility. DDGS is being extensively studied for its use as a food additive, functional food material, bioenergy production, feed, and fertilizer, as well as for its use as a functional cosmetic and fragrance ingredient. However, previous studies have only investigated manufacturing methods and efficacy based on simple extracts or fermented products, and have not reported which components of DDGS are responsible for these benefits.

[0005] Accordingly, the present invention aimed to produce a high-quality sake extract by isolating and purifying a bioactive substance possessing whitening, moisturizing, and wrinkle-improving activities from sake lees, a representative food processing byproduct. As a result, a low-molecular-weight peptide was isolated from the sake lees, and the whitening, moisturizing, and wrinkle-improving effects of a cosmetic composition containing the low-molecular-weight peptide were confirmed, thereby developing a material capable of alleviating skin aging.

[0006] The present invention aims to isolate a bioactive substance from the sesame seed, which possesses whitening, moisturizing, and wrinkle-improving properties, and to prepare a low-molecular-weight peptide containing the substance. Furthermore, the present invention aims to develop and provide a cosmetic composition containing a sesame seed extract, which exhibits excellent whitening, moisturizing, and wrinkle-improving properties, by preparing a formulation containing the low-molecular-weight peptide and confirming its efficacy.

[0007] The present invention provides a cosmetic composition containing, as an active ingredient, a low molecular weight peptide of 1,000 Da or less, separated by treating a proteolytic enzyme in a dried persimmon.

[0008] The present invention provides a cosmetic composition comprising an anionic low-molecular-weight peptide of 1,000 Da or less separated by treating sake lees with a proteolytic enzyme, a cationic low-molecular-weight peptide of 1,000 Da or less separated by treating sake lees with a proteolytic enzyme, and a nonionic low-molecular-weight peptide of 1,000 Da or less separated by treating sake lees with a proteolytic enzyme.

[0009] In the cosmetic composition of the present invention, the low molecular weight peptide may be obtained from a process including: (a) immersing a sake lees in purified water; (b) adding a subtilisin enzyme derived from Bacillus after the immersion in step (a) to induce a first enzymatic reaction; (c) adding pepsin after the first enzymatic reaction in step (b) to induce a second enzymatic reaction; (d) centrifuging to recover a supernatant after the second enzymatic reaction in step (c); and (e) adding ethanol to the supernatant recovered in step (d) to induce a precipitation reaction, and then centrifuging to recover a supernatant.

[0010] In the cosmetic composition of the present invention, the anionic low-molecular-weight peptide may be obtained from a process comprising: (a) immersing a sake lees in purified water; (b) adding a Bacillus-derived subtilisin enzyme after the immersion in step (a) to induce a first enzymatic reaction; (c) adding pepsin after the first enzymatic reaction in step (b) to induce a second enzymatic reaction; (d) centrifuging to recover a supernatant after the second enzymatic reaction in step (c); (e) adding ethanol to the supernatant recovered in step (d) to induce a precipitation reaction, and then centrifuging to recover the supernatant; and (f) passing the supernatant recovered in step (e) through an anion exchange resin to obtain an anionic peptide.

[0011] In the cosmetic composition of the present invention, the cationic low-molecular-weight peptide may be obtained from a process comprising: a step (a) of immersing a sake lees in purified water; a step (b) of inducing a first enzymatic reaction by adding a subtilisin enzyme derived from Bacillus after the immersion in step (a); a step (c) of inducing a second enzymatic reaction by adding pepsin after the first enzymatic reaction in step (b); a step (d) of recovering a supernatant by centrifugation after the second enzymatic reaction in step (c); a step (e) of adding ethanol to the supernatant recovered in step (d) to induce a precipitation reaction, and then centrifuging to recover the supernatant; and a step (f') of passing the supernatant recovered in step (e) through a cation exchange resin to obtain a cationic peptide.

[0012] In the cosmetic composition of the present invention, the nonionic low-molecular-weight peptide is prepared by: (a) immersing a sake lees in purified water; (b) adding a Bacillus-derived subtilisin enzyme after the immersion in step (a) to induce a first enzymatic reaction; (c) adding pepsin after the first enzymatic reaction in step (b) to induce a second enzymatic reaction; (d) centrifuging to recover a supernatant after the second enzymatic reaction in step (c); (e) adding ethanol to the supernatant recovered in step (d) to induce a precipitation reaction, and then centrifuging to recover a supernatant; (f1) passing the supernatant recovered in step (e) through an anion exchange resin to obtain a first eluent; It may be characterized by being obtained from a process including a step (f2) of passing the eluent obtained in the above step (f1) through a cation exchange resin to obtain a second eluent; a step (g) of filtering the second eluent obtained in the above step (f2) to obtain a filtrate;

[0013] In the present invention, the cosmetic composition may be characterized as being for skin whitening.

[0014] In the present invention, the cosmetic composition may be characterized as being for improving skin wrinkles.

[0015] In the present invention, the cosmetic composition may be characterized as being for moisturizing the skin.

[0016] The present invention provides a method for isolating a low-molecular-weight peptide from a sesame seed and a cosmetic composition containing the same.

[0017] In addition, the cosmetic composition according to the present invention has a pigmentation suppression effect through a decrease in melanin production, a pigmentation suppression effect through a decrease in TRP-1 (Tyrosinase related protein-1) protein expression, a wrinkle improvement effect through an increase in collagen type 1 and laminin-5 protein expression, and a moisturizing effect through an increase in filaggrin protein expression, thereby alleviating skin aging.

[0018] Figure 1 shows the results of analyzing the molecular weight of low-molecular-weight peptides separated from the main stem.

[0019] Figure 2 shows the results of analyzing the surface potential of anionic low-molecular-weight peptides, cationic low-molecular-weight peptides, and nonionic low-molecular-weight peptides separated from the main stem.

[0020] Figure 3 shows the results of measuring the effect of low-molecular-weight peptides isolated from the leaves on reducing melanin production.

[0021] Figure 4 shows the results of measuring the effect of a low-molecular-weight peptide isolated from the stem of the plant on reducing TRP-1 protein expression.

[0022] Figure 5 shows the results of measuring the effect of a low-molecular-weight peptide isolated from the stem of a plant on increasing the expression of Collagen Type 1 protein.

[0023] Figure 6 shows the results of measuring the effect of a low-molecular-weight peptide isolated from the stem of a plant on increasing the expression of Laminin-5 protein.

[0024] Figure 7 shows the results of measuring the effect of a low-molecular-weight peptide isolated from the stem of the plant on increasing the expression of the Filaggrin protein.

[0025] Sake lees (sake lees), a byproduct of brewing and ethanol production, is increasing in popularity each year. Its value as a source of crude lipids, crude proteins, fiber, fermented yeast cells, and various minerals is increasing. Research is underway on the use of sake lees not only as a food additive and functional food ingredient, but also as a functional cosmetic and fragrance ingredient. However, previous research has focused solely on manufacturing methods and efficacy based on simple extracts or fermented products, and research on the bioactive compounds in sake lees with these benefits remains minimal.

[0026] Accordingly, the inventors of the present invention determined that it would be possible to develop a new functional material with excellent physiological activity, safety, and stability by isolating a low-molecular-weight peptide from the berry, which is a physiologically active substance exhibiting whitening, moisturizing, and wrinkle-improving effects, and utilizing it in a cosmetic composition.

[0027] The present invention provides a cosmetic composition containing, as an active ingredient, a low molecular weight peptide of 1,000 Da or less, separated by treating a proteolytic enzyme in a dried persimmon.

[0028] In addition, the present invention provides a cosmetic composition comprising an anionic low-molecular-weight peptide of 1,000 Da or less separated by treating sake lees with a proteolytic enzyme, a cationic low-molecular-weight peptide of 1,000 Da or less separated by treating sake lees with a proteolytic enzyme, and a nonionic low-molecular-weight peptide of 1,000 Da or less separated by treating sake lees with a proteolytic enzyme. At this time, the cosmetic composition is preferably prepared by mixing anionic low-molecular-weight peptide, cationic low-molecular-weight peptide, and nonionic low-molecular-weight peptide in a weight ratio of 1:0.5 to 1.5:0.5 to 1.5.

[0029] In the present invention, low-molecular-weight peptides, anionic low-molecular-weight peptides, cationic low-molecular-weight peptides, and nonionic low-molecular-weight peptides are produced by treating with a protein hydrolyzing enzyme. The production method will be described in detail for each step below.

[0030]

[0031] <Step (a); Soaking in water>

[0032] This step involves soaking the sake lees in purified water. The sake lees, also known as "sake lees," can be used without limitation as long as they are byproducts obtained during brewing or ethanol production. Preferably, the dried sake lees are ground into powder. Even more preferably, the dried and powdered sake lees are soaked in purified water to adjust the pH to 6.5-7.5.

[0033]

[0034] <Step (b); Induction of the first enzyme reaction>

[0035] This step is a process of inducing a first enzymatic reaction by adding a subtilisin enzyme derived from Bacillus after the immersion in the above step (a).

[0036] Subtilisin is a proteolytic enzyme, and the proteins present in the yeast extract are primarily hydrolyzed using this enzyme. In this step, by using subtilisin, the proteins in the yeast extract are primarily hydrolyzed into larger units. Through this hydrolysis, protein hydrolysis is further accelerated in the secondary enzymatic reaction below, enabling the production of low-molecular-weight peptides.

[0037] In this step, the first enzymatic reaction can be performed using an ultra-high pressure enzyme reactor equipped with a polymer coated with subtilisin, a protein-decomposing enzyme isolated from Bacillus. Preferably, the reaction is performed at a pressure of 80 to 120 bar and a temperature of 50 to 60°C, and the reaction time is preferably 12 to 36 hours.

[0038]

[0039] <Step (c): Induction of secondary enzyme reaction>

[0040] This step is a process of inducing a secondary enzymatic reaction by adding pepsin after the primary enzymatic reaction of step (b) above.

[0041] Pepsin is a protein hydrolyzing enzyme secreted from the stomach. In this step, pepsin is added to hydrolyze the protein in the lees that was initially hydrolyzed by adding subtilisin in step (b) into lower molecules.

[0042] In this step, the secondary enzymatic reaction may preferably be performed in an ultra-high pressure enzyme reactor equipped with a pepsin-coated polymer. At this time, it is preferable to perform the reaction at a pressure of 80 to 120 bar and a temperature of 35 to 45°C, and the reaction time is preferably 3 to 9 hours.

[0043] After the secondary enzyme reaction in this step is complete, it is recommended to boil the solution after the secondary reaction to inactivate any excess enzyme.

[0044]

[0045] <Step (d): Centrifugation>

[0046] This step is a process of recovering the supernatant by centrifugation after the secondary enzymatic reaction of step (c) above. That is, the solution in which the secondary enzymatic reaction of step (c) above has been completed is centrifuged to recover the supernatant.

[0047]

[0048] <Step (e): Recovery of supernatant>

[0049] This step is a process of adding ethanol to the supernatant recovered in the above step (d) to induce a precipitation reaction, and then centrifuging to recover the supernatant.

[0050] In this step, ethanol is added to the supernatant recovered in step (d) to induce ethanol precipitation. The ethanol added at this time may be added in a weight ratio of 0.8 to 1.2 relative to 1 weight of the supernatant, and more preferably, it is added in a weight ratio of 1 relative to 1 weight of the supernatant. This addition of ethanol induces precipitation, causing the precipitate to settle to the bottom. Ethanol precipitation is preferably performed at refrigerated temperatures.

[0051] Meanwhile, by performing the steps up to this point, a low-molecular-weight peptide can be produced from the lees. That is, after inducing an ethanol precipitation reaction as described above and centrifuging, the supernatant obtained can be filtered, and if necessary, the filtrate can be freeze-dried to produce a low-molecular-weight peptide from the lees.

[0052]

[0053] <Step (f), (f'), f(1), f(2): Chromatography>

[0054] To obtain an anionic peptide, the supernatant recovered in step (e) is passed through an anion exchange resin.

[0055] To obtain a cationic peptide, the supernatant recovered in step (e) is passed through a cation exchange resin.

[0056] In order to obtain a nonionic peptide, a step (f1) of passing the supernatant recovered in the step (e) through an anion exchange resin to obtain a first eluent; a step (f2) of passing the eluent obtained in the step (f1) through a cation exchange resin to obtain a second eluent; and a step (f2) of filtering the second eluent obtained in the step (f2) to obtain a filtrate.

[0057] More specifically, the anionic peptide, cationic peptide and nonionic peptide of steps (f), (f'), f(1) and f(2) of the present invention may be obtained by separation as follows.

[0058] First, in order to obtain anionic peptides, the anion exchange resin is activated with 8 to 12 times the volume of a 0.5 M NaOH aqueous solution and then packed in an open column tube. Then, 8 to 12 times the volume of distilled water is flowed to wash the resin. The supernatant recovered in step (e) or the low-molecular-weight peptide obtained from the supernatant is dissolved in distilled water and loaded into an open column packed with anion exchange resin. Then, 8 to 12 times the volume of distilled water is continuously flowed at an appropriate rate to adsorb the low-molecular-weight peptide having an anion. Then, 8 to 12 times the volume of a 2 M NaCl aqueous solution is flowed at an appropriate rate to elute the resin. Meanwhile, the eluent thus eluted can be passed through a precision filtration membrane of 0.1 to 0.3 ㎛ to obtain a filtrate, and the obtained filtrate can be freeze-dried as needed to produce a dried anionic low-molecular-weight peptide.

[0059] To obtain a cationic peptide, a cation exchange resin is activated with 8 to 12 times the volume of a 0.5 M NaOH aqueous solution and then packed in an open column tube. Then, 8 to 12 times the volume of distilled water is flowed to wash the resin. The supernatant recovered in step (e) or the low-molecular-weight peptide obtained from the supernatant is dissolved in distilled water and loaded into an open column packed with a cation exchange resin. Then, 8 to 12 times the volume of distilled water is continuously flowed at an appropriate rate to adsorb the cationic low-molecular-weight peptide. Then, 8 to 12 times the volume of 2 M NaCl aqueous solution is flowed at an appropriate rate to elute the resin. Meanwhile, the eluent thus eluted can be passed through a precision filtration membrane of 0.1 to 0.3 ㎛ to obtain a filtrate, and the obtained filtrate can be freeze-dried as needed to produce a cationic low-molecular-weight peptide.

[0060] In order to obtain nonionic peptides, an anion exchange resin and a cation exchange resin are each activated with 8 to 12 times the volume of a 0.5 M NaOH aqueous solution and then packed into respective open column tubes. Then, they are washed by flowing 8 to 12 times the volume of distilled water. The supernatant recovered in step (e) or the low-molecular-weight peptide obtained from the supernatant is dissolved in distilled water and loaded into an open column packed with an anion exchange resin, and then 8 to 12 times the volume of distilled water is continuously flowed at an appropriate rate to perform a first elution. Then, the first eluent is loaded into an open column packed with a cation exchange resin, and then 8 to 12 times the volume of distilled water is continuously flowed at an appropriate rate to perform a second elution. The second-eluted eluent is passed through a precision filtration membrane of 0.1 to 0.3 ㎛ to obtain a filtrate, and the obtained filtrate can be freeze-dried as needed to produce a nonionic low-molecular-weight peptide.

[0061]

[0062] Meanwhile, in the cosmetic composition of the present invention, the cosmetic composition may be characterized as being for skin whitening. Meanwhile, the skin whitening effect may be exerted due to suppression of pigmentation through a decrease in the expression of the TRP-1 (Tyrosinase related protein-1) protein.

[0063] In the cosmetic composition of the present invention, the cosmetic composition may be characterized as being for improving skin wrinkles. Meanwhile, the skin wrinkle improvement effect may be exerted through increased expression of collagen type 1 and laminin-5 proteins.

[0064] In the cosmetic composition of the present invention, the cosmetic composition may be characterized as being for skin moisturizing. Meanwhile, the skin moisturizing effect may be exerted through increased expression of the filaggrin protein.

[0065] Meanwhile, in the cosmetic composition of the present invention, the low molecular weight peptide of the present invention is preferably contained in an amount of 0.01 to 30.0 wt% based on the total weight of the cosmetic composition.

[0066] In the cosmetic composition of the present invention, the cosmetic composition can be manufactured into any formulation commonly manufactured in the art, and for example, can be formulated into a basic cosmetic formulation selected from the group consisting of a solution, a suspension, an emulsion, a paste, a toner, a gel, a water-soluble liquid, a cream, an essence, a surfactant-containing cleanser, an oil, an oil-in-water (O / W) type and a water-in-oil (W / O) type; a formulation for a mask pack; a formulation for a body wash; a peeling gel; an oil-in-water or water-in-oil type makeup base, etc., but is not limited thereto.

[0067] The cosmetic composition of the present invention may be used in conjunction with other cosmetic compositions other than those of the present invention. Furthermore, the cosmetic composition of the present invention may be used according to conventional methods, and the frequency of use may vary depending on the user's skin condition or preference.

[0068]

[0069] Hereinafter, the present invention will be described in more detail through the following examples and experimental examples. However, the scope of the present invention is not limited to the following examples and experimental examples, but includes all variations of technical concepts equivalent thereto.

[0070]

[0071] [Example 1: Preparation of low-molecular-weight peptide derived from sesame seeds]

[0072] In this example, a low-molecular-weight peptide derived from the berry was prepared according to the following method.

[0073] The dried sake lees were pulverized using a grinder, immersed in purified water to adjust the pH to 7, and then placed in an ultra-high pressure enzyme reactor equipped with a polymer coated with a Bacillus subtilisin enzyme, and subjected to an enzymatic digestion reaction at 55℃ under a pressure of 100 bar for 24 hours. The solution was then recovered from the reactor, the pH of the solution was adjusted to 3, and placed in an ultra-high pressure enzyme reactor equipped with a pepsin-coated polymer, and subjected to an enzymatic digestion reaction at 40℃ under a pressure of 100 bar for 6 hours, followed by heating for 30 minutes to inactivate the excess enzyme and terminate the reaction. The solution was then recovered from the reactor and centrifuged to obtain a supernatant. Ethanol was added in a weight ratio of 1:1 based on the weight of the obtained supernatant, and the solution was left at a low temperature. The supernatant, excluding the precipitate, was centrifuged. Then, the filtrate was passed through a 0.2 ㎛ precision filtration membrane to obtain a filtrate, and the obtained filtrate was freeze-dried to prepare a low-molecular-weight peptide powder.

[0074]

[0075] [Experimental Example 1: Molecular Weight Measurement of Low-Molecular-Weight Peptide Derived from Jujube (Example 1)]

[0076] In this experiment, a molecular weight measurement experiment of the above-mentioned manufactured Example 1 was conducted. A matrix-assisted laser desorption-ionization time-of-flight mass spectrometer (MALDI-TOF MS) was used to measure the molecular weight.

[0077] Example 1 was dissolved in a 50% (v / v) methanol / water mixture, and 1 μl was mixed with 1 μl of sinapinic acid (SA) matrix (50% (v / v) 0.1% TFA acetonitrile / 50% (v / v) water). The mixture was then spotted on a stainless steel MALDI plate and dried at room temperature. Analysis was performed using a Microflex LRF MALDI-TOF mass spectrometer and a Bruker UltrafleXtreme (Bruker Daltonics, Bremen, Germany), and the intensity of each ion was calculated by summing the area of ​​the first isotope peak to the area of ​​the third isotope peak. Spectral acquisition and processing were performed using FlexAnalysis software (ver. 3.3, Bruker Daltonics, Bremen, Germany), and the results are shown in Fig. 1.

[0078] Figure 1 shows the results of confirming the molecular weight of a low-molecular-weight peptide derived from a perilla leaf (Example 1). It was confirmed that the molecular weight distribution of Example 1 was 1,000 Da or less, and more specifically, 500 Da or less, with most of the molecular weight being distributed. This means that the manufactured Example 1 was well hydrolyzed into a low-molecular-weight peptide.

[0079]

[0080] [Example 2: Preparation of anionic low-molecular-weight peptides derived from licorice]

[0081] (1) Manufacturing of anion exchange resin (affinity resin) for peptide separation

[0082] First, to separate anionic low-molecular-weight peptides derived from the lye, an affinity resin for separating anionic peptides was prepared as follows.

[0083] Agarose beads, a biopolymer, were swelled in a 1N NaCl aqueous solution (10 times the volume), filtered, and activated by stirring the filtered agarose in a 3N NaOH aqueous solution (10 times the volume) for 30 minutes. 1,3-propane sultone was added to the activated agarose, and the mixture was stirred at 60°C for 3 hours to react. Then, a washing process of adding 10 times the volume of distilled water, allowing it to stand, and removing the supernatant was performed four times, and then the pH was adjusted to 7. The washing process was repeated two more times, followed by filtration.

[0084]

[0085] (2) Separation of anionic low-molecular-weight peptides derived from liquor

[0086] The anion exchange resin for peptide separation manufactured in the above Example 2-(1) was activated in a 10-fold amount of 0.5 M NaOH aqueous solution and then packed in each open column tube. Then, it was washed by flowing distilled water 10 times the volume of the resin. The low-molecular-weight peptide powder of the dried lees manufactured in the above Example 1 was dissolved in distilled water and a certain amount was loaded into the open column packed with the anion exchange resin, and then distilled water 10 times the volume of the resin was continuously flowed at an appropriate speed to adsorb the low-molecular-weight peptide having an anion. Then, it was eluted by flowing a 10-fold amount of 2 M NaCl aqueous solution at an appropriate speed and passed through a 0.2 ㎛ precision filter membrane to obtain a filtrate, and the obtained filtrate was freeze-dried to prepare a low-molecular-weight peptide powder of the dried lees anion.

[0087]

[0088] [Example 3: Preparation of cationic low-molecular-weight peptide derived from licorice]

[0089] (1) Manufacturing of cation exchange resin (affinity resin) for peptide separation

[0090] First, to separate cationic low-molecular-weight peptides derived from the liquor, an affinity resin for cationic peptide separation was prepared as follows.

[0091] Agarose beads, a biopolymer, were swelled in a 1N NaCl aqueous solution (10 times the volume), filtered, and activated by stirring the filtered agarose in a 5N NaOH aqueous solution (10 times the volume) for 30 minutes. Glycidyltrimethylammonium chloride was added to the activated agarose, and the mixture was stirred at 55°C for 3 hours to react. Then, 10 times the volume of distilled water was added, and after settling, the supernatant was removed. This washing process was performed 4 times, and the pH was adjusted to 7. The washing process was then performed 2 more times, followed by filtration.

[0092]

[0093] (2) Separation of cationic low-molecular-weight peptides derived from liquor

[0094] The cation exchange resin for peptide separation manufactured in the above Example 3-(1) was activated in a 10-fold amount of 0.5 M NaOH aqueous solution and then packed in an open column tube. Then, it was washed by flowing distilled water 10 times the volume of the resin. The low-molecular-weight peptide powder of the dried lees manufactured in the above Example 1 was dissolved in distilled water and a certain amount was loaded into an open column packed with a cation exchange resin. Then, distilled water 10 times the volume of the resin was continuously flowed at an appropriate speed to adsorb the low-molecular-weight peptide having a cationic charge. Then, the 10-fold amount of 2 M NaCl aqueous solution was flowed at an appropriate speed to elute, and the filtrate was passed through a 0.2 ㎛ precision filter membrane to obtain a filtrate, and the obtained filtrate was freeze-dried to prepare a low-molecular-weight peptide powder of the dried lees cationic charge.

[0095]

[0096] [Example 4: Preparation of nonionic low-molecular-weight peptide derived from licorice]

[0097] (1) Separation of nonionic low-molecular-weight peptides derived from liquor

[0098] The anion exchange resin and cation exchange resin for peptide separation manufactured in the above Examples 2-(1) and 3-(1) were each activated in 10 times the volume of a 0.5 M NaOH aqueous solution and then packed in each open column tube. Then, they were washed by flowing distilled water 10 times the volume of the resin. The low-molecular-weight peptide powder manufactured in the above Example 1 was dissolved in distilled water and a certain amount was loaded into the open column packed with the anion exchange resin, and then distilled water 10 times the volume of the resin was continuously flowed at an appropriate speed to elute. The eluent was loaded into the open column packed with the cation exchange resin, and then distilled water 10 times the volume of the resin was continuously flowed at an appropriate speed to elute. The above eluent was passed through a 0.2 μm precision filter membrane to obtain a filtrate, and the obtained filtrate was freeze-dried to prepare a nonionic low-molecular-weight peptide powder.

[0099]

[0100] [Experimental Example 2: Surface Potential Measurement]

[0101] In this experiment, the surface potential measurement experiments of Examples 2 to 4 were conducted. To analyze the surface charge, a zeta potential analyzer, which is a measuring device using dynamic light scattering and electrophoretic light scattering, was used to confirm the surface charge of each peptide, and the measured values ​​were measured three times and taken as the average value.

[0102] Figure 2 shows the results of analyzing the surface potential of anionic low-molecular-weight peptides (Example 2), cationic low-molecular-weight peptides (Example 3), and nonionic low-molecular-weight peptides (Example 4) separated from sake lees. As shown in Figure 2, the zeta potential of Example 2 (anionic low-molecular-weight peptide derived from sake lees) was measured as -67.64 mV, the zeta potential of Example 3 (cationic low-molecular-weight peptide derived from sake lees) was measured as 47.89 mV, and the zeta potential of Example 4 (nonionic low-molecular-weight peptide derived from sake lees) was measured as 4.73 mV. This means that the separation was well performed for each charge from the sake lees low-molecular-weight peptide of Example 1.

[0103]

[0104] [Examples 5 to 8: Preparation of mixtures using low-molecular-weight peptides obtained from the extracts of Examples 2 to 4]

[0105] In this example, the mixing ratios of the anionic low-molecular-weight peptide derived from the sake lees (Example 2), the cationic low-molecular-weight peptide derived from the sake lees (Example 3), and the nonionic low-molecular-weight peptide derived from the sake lees (Example 4) prepared in Examples 2 to 4 were changed to prepare a mixture containing the low-molecular-weight peptides of Examples 5 to 8.

[0106] In order to confirm the whitening, moisturizing and wrinkle improvement effects of Examples 2 to 4, mixtures of Examples 5 to 8 were prepared by changing the mixing ratios of anionic, cationic and nonionic low-molecular-weight peptides separated from the sake lees according to Table 1 below. Table 1 below shows the mixing ratios (weight ratios) of the anionic low-molecular-weight peptide derived from the sake lees (Example 2), the cationic low-molecular-weight peptide derived from the sake lees (Example 3), and the nonionic low-molecular-weight peptide derived from the sake lees (Example 4).

[0107] Weight ratio Example 2 Example 3 Example 4 Example 5 110 Example 6 101 Example 7 0 11 Example 8 111

[0108]

[0109] [Comparative Example 1: Preparation of a sesame extract using a conventional method]

[0110] In Comparative Example 1, a lees extract was prepared according to a conventional method. A conventional lees extract is prepared using a general method disclosed in existing literature or patents, and the lees extract of Comparative Example 1 was prepared using the following method.

[0111] The dried lees were pulverized using a grinder, refluxed three times for 3 hours each with 95% (V / V) ethanol aqueous solution, cooled, filtered and concentrated with Whatman #4 filter paper, and then freeze-dried to produce lees extract powder.

[0112]

[0113] [Comparative Example 2: Production of fermented liquor using conventional method]

[0114] In Comparative Example 2, a fermented liquor was produced according to a conventional method. The conventional fermented liquor refers to a product produced using a method disclosed in existing literature or patents, such as the method registered in Republic of Korea Patent No. 10-0736327.

[0115] 5 g of baking soda was added to 1 kg of sake lees and mixed, then 2 L of distilled water was added, the mixture was mixed, and 1 g of yeast was added. After culturing in an incubator at 30°C for 24 hours, the mixture was filtered and concentrated with Whatman #4 filter paper, and then freeze-dried to produce sake lees fermentation powder.

[0116]

[0117] [Experimental Example 3: Measurement of the Inhibitory Effect on Melanin Production]

[0118] In this experiment, an experiment was conducted to confirm the melanin production inhibitory effect of the peptides derived from the sake lees prepared in Examples 1 to 4, the mixture containing the peptides derived from the sake lees prepared in Examples 5 to 8 (Examples 2 to 4), and Comparative Example 1 (conventional sake lees extract) and Comparative Example 2 (conventional sake lees fermentation product).

[0119] Mouse-derived melanocyte-forming (B16F10) cells were seeded at 2.5 × 10 in a 96-well plate. 3 After dispensing cells / well, the cells were stabilized for one day in a 37℃, 5% CO2 cell incubator. Samples were diluted and treated according to concentration, and arbutin, a well-known whitening agent, was treated as a positive control. After treatment with α-melanocyte stimulating hormone (MSH) as a stimulant, the cells were incubated for 72 hours, and the absorbance was measured at 405 nm using a spectrophotometer (microplate reader).

[0120] Table 2 below summarizes the experimental results, and as shown in Table 2, when a mixture containing the peptides derived from the lees prepared in Examples 1 to 4 of the present invention or the peptides derived from the lees prepared in Examples 5 to 8 (Examples 2 to 4) was treated, it was confirmed that the melanin production inhibition effect was higher than that of Comparative Example 1 (conventional lees extract) and Comparative Example 2 (conventional lees fermentation product). In addition, it was confirmed that the melanin production inhibition effect was higher when Example 2 (anionic low-molecular-weight peptide derived from lees) and Example 3 (cationic low-molecular-weight peptide derived from lees) separated by charge were treated than when Example 1 (low-molecular-weight peptide derived from lees) was treated, and it was confirmed that the highest melanin production inhibition effect was exhibited in the Example 8 mixture containing all of Example 2 (anionic low-molecular-weight peptide derived from lees), Example 3 (cationic low-molecular-weight peptide derived from lees), and Example 4 (nonionic low-molecular-weight peptide derived from lees).

[0121] Sample name Concentration (㎍ / ml) 50 100 Arbutin 70.59 - Comparative example 128.0 335.13 Comparative example 231.35 38.52 Example 135.42 40.18 Example 240.1 345.82 Example 337.25 43.19 Example 435.48 38.22 Example 540.2 145.16 Example 640.15 45.37 Example 738.95 42.11 Example 848.5 152.38

[0122]

[0123] [Experimental Example 4: Measurement of the Inhibitory Effect on MMP-1 Production]

[0124] In this experiment, an experiment was conducted to confirm the MMP-1 production inhibitory effect of the peptides derived from the lees prepared in Examples 1 to 4, the mixture containing the peptides derived from the lees prepared in Examples 5 to 8 (Examples 2 to 4), and Comparative Example 1 (conventional lees extract) and Comparative Example 2 (conventional lees fermentation product).

[0125] Human dermal fibroblasts (HDFn) were seeded at 5 × 10 in a 24-well plate. 4 After dispensing cells / well, stabilize in a 37℃, 5% CO2 cell incubator for one day and then irradiate with UVA at 1 J / cm 2 Each sample was diluted by concentration and treated simultaneously with exchange with a medium supplemented with 2% FBS, and adenosine (raw material for wrinkle functional notification) was treated at a concentration of 100 μM as a positive control group and reacted for 48 hours. The supernatant was used to confirm the MMP-1 expression level using the R&D systems kit (DY901), and the pellet was treated with MTT Solution to confirm cytotoxicity.

[0126] After the MMP-1 ELISA Assay, the amount of MMP-1 produced was calculated by measuring the absorbance at a wavelength of 450 nm, and then the MMP-1 expression inhibition rate (%) was calculated by comparing it with the control group, and the calculation formula is as follows: Mathematical Formula 1 below.

[0127] [Mathematical Formula 1]

[0128]

[0129]

[0130] Table 3 below summarizes the experimental results, and as shown in Table 3, when a mixture containing the peptides derived from the lees prepared in Examples 1 to 4 of the present invention or the peptides derived from the lees prepared in Examples 5 to 8 (Examples 2 to 4) was treated, it was confirmed that the MMP-1 production inhibition activity was higher than that of Comparative Example 1 (conventional lees extract) and Comparative Example 2 (conventional lees fermentation product). In addition, it was confirmed that the MMP-1 production inhibition activity was higher when Example 2 (anionic low-molecular-weight peptide derived from lees) and Example 3 (cationic low-molecular-weight peptide derived from lees) separated by charge were treated than when Example 1 (low-molecular-weight peptide derived from lees) was treated, and it was confirmed that the highest MMP-1 production inhibition activity was shown in the Example 8 mixture containing all of Example 2 (anionic low-molecular-weight peptide derived from lees), Example 3 (cationic low-molecular-weight peptide derived from lees), and Example 4 (non-ionic low-molecular-weight peptide derived from lees).

[0131] Sample name MMP-1 inhibition rate (%) Adenosine 42.31 Comparative example 124.52 Comparative example 227.38 Example 132.47 Example 235.14 Example 337.91 Example 433.46 Example 538.52 Example 641.88 Example 743.29 Example 847.83

[0132]

[0133] [Formulation Example 1: Preparation of a cosmetic composition using a mixture containing peptides derived from the berry (Example 8)]

[0134] In this formulation example, a cosmetic composition was prepared using Example 8 (mixture containing peptides derived from sesame seeds) with the composition shown in Table 4 below. Meanwhile, Comparative Formulation Example 1, which did not use Example 8, was prepared as a control. Table 4 below shows the compositions of the cosmetic compositions of Formulation Example 1 and Comparative Formulation Example 1.

[0135] Ingredient Formulation Example 1 Comparative Formulation Example 1 Content (%) Example 8 (Mixture containing peptides derived from sake lees) 10 Tablet Residual amount Residual amount Glycerin 10 10 Propanediol 8 8 Cetyl ethylhexanoate 3 3 1,2-hexanediol 2 2 Sodium polyacryloyldimethyltaurate 1 1 Hydrogenated polydecene 1 1 Shea butter 1 1 C14-2 2 Alcohol 0.5 0.5 Polyglyceryl-2 stearate 0.3 0.3 Trisodium ethylenediamine disuccinate 0.1 0.1 Ethylhexylglycerin 0.05 0.05 Total 100 100

[0136]

[0137] According to Table 4 above, cetyl ethylhexanoate, hydrogenated polydecene, shea butter, C14-22 alcohol, and polyglyceryl-2 stearate were dissolved by heating to 80°C to prepare a transparent emulsifying system. Trisodium ethylenediamine disuccinate, glycerin, propanediol, and sodium polyacryloyldimethyltaurate were dispersed in purified water, and the emulsifying system dissolved in the water phase heated to 70°C was added, followed by emulsification using a homomixer at 5,000 rpm for 5 minutes. 1,2-hexanediol and ethylhexylglycerin were added at 45°C, stirred for 3 minutes, and then cooled to 30°C. Thereafter, Example 8 was added, stirred for 3 minutes, and defoamed to prepare a cosmetic composition in the form of Formulation Example 1 in a cream formulation. Meanwhile, Comparative Formulation Example 1 (control group) was prepared in the same manner as Formulation Example 1 except that Example 8 was added.

[0138]

[0139] [Experimental Example 5: Measurement of the Pigmentation Inhibition Effect Using a Reconstructed Skin Model]

[0140] In this experiment, in order to confirm the pigmentation inhibition effect of the cosmetic composition manufactured in Formulation Example 1 above, an experiment was conducted to confirm the amount of melanin production and the amount of TRP-1 (Tyrosinase related protein-1) protein expression using a reconstructed skin model.

[0141] (1) Manufacturing of reconstructed skin tissue

[0142] Fibroblasts, type I collagen solution, 10× DMEM:F12 (3:1) Media, 10× buffer, and hyaluronic acid were mixed well and placed into the culture insert of a 12-well plate. After reacting for 2 hours in a 5% CO2, 37℃ cell incubator and solidifying, fibroblast culture medium was added inside and outside and cultured overnight. Afterwards, the medium was exchanged for 5 days to prepare a dermal layer with fibroblasts. Pre-cultured keratinocytes and melanocytes were seeded on the upper part of the prepared dermal layer using Keratinocyte-SFM medium containing BPE (Bovine Pituitary Extract), insulin, hydrocortisone, vitamin C, and epidermal growth factor (EGF), and cultured overnight. After culturing for 5 days to induce proliferation of epidermal cells, 2 mM Ca 2+ The differentiation of keratinocytes was induced by adding a medium containing EGF and 2 mM Ca to the culture insert. Afterwards, all the medium was removed and EGF and 2 mM Ca were added to the outside of the culture insert. 2+ Primary keratinocyte cells were exposed to air and cultured for 12 days using culture medium containing added , thereby inducing the creation of an epidermal layer and producing reconstructed skin tissue.

[0143]

[0144] (2) Sample processing and culture

[0145] After melanin hyperplasia was induced by adding α-MSH (100 nM) to the manufactured Reconstructed skin tissue (RST), 20 ㎕ of Formulation Example 1 and Comparative Formulation Example 1 (control) were treated once / 2 days, and cultured for 5 days. Afterwards, the tissue was fixed in a 10% neutral formalin solution for 24 hours, embedded in OCT Compound, cut into 12 ㎛ thickness, attached to gelatin-coated slides, dried, stored, and used for staining.

[0146]

[0147] (3) Melanin staining and immunofluorescence staining

[0148] Melanin content was quantified by staining using the Fontana-Masson kit (ab160669, abcam), and the image results were analyzed and shown in Figure 3. The tissue sections prepared on the slides were hydrated in distilled water, treated with an ammoniacal silver solution, and placed in a 60°C slide warmer for 30 minutes. After washing with distilled water, they were treated with 0.1% gold chloride for 1 minute, washed with distilled water, and treated with 5% Hypo for 2 minutes. After washing with distilled water, they were treated with Nuclear-fast red for 5 minutes, and finally, lightly washed with distilled water, dehydrated and cleared, sealed with Canada balsam, and observed under a microscope.

[0149] TRP-1, a protein involved in melanin formation, was analyzed using the immunofluorescence staining method below and is shown in Figure 4. Tissue sections prepared on slides were hydrated in distilled water, then boiled in a 0.01 M citrate solution (pH 6.0) using a microwave for 15 minutes and left to stand at room temperature for 20 minutes. Anti-goat serum was used for blocking and the primary antibody TRP-1 was reacted overnight at 4°C. After washing with PBS, the sections were reacted with the secondary antibody, goat anti-mouse IgG Alexa Fluor 488, at room temperature for 1 hour, washed with PBS, mounted in mounting medium with DAPI, and observed under a fluorescence microscope. Images were taken at 200x magnification with a fluorescence microscope, and the positive staining area was quantified using the Image J program to calculate the rate of change, which is shown in Equation 2 below.

[0150] [Equation 2]

[0151]

[0152]

[0153] (4) Melanin content analysis

[0154] Melanin was dyed black, and a graph analyzing the content is shown in Fig. 3. As seen in the graph in Fig. 3, it was confirmed that Formulation Example 1 treated with Example 8 significantly reduced the melanin content compared to the untreated group, Comparative Formulation Example 1.

[0155]

[0156] (5) Analysis of TRP-1 protein content

[0157] TRP-1 is an enzyme protein involved in melanin formation, and is stained dark brown on the epidermis. The experimental results are shown in Fig. 4. As shown in the graph in Fig. 4, it was confirmed that Formulation Example 1 treated with Example 8 significantly reduced the expression level of TRP-1 protein compared to the untreated group, Comparative Formulation Example 1.

[0158]

[0159] [Experimental Example 6: Measurement of Wrinkle Improvement and Moisturizing Effects Using a Reconstructed Skin Model]

[0160] In this experiment, in order to confirm the wrinkle improvement and moisturizing effect of the cosmetic composition manufactured in Formulation Example 1 above, an experiment was conducted to confirm the expression levels of collagen type 1, laminin-5 protein, and filaggrin protein using a reconstructed skin model.

[0161] (1) Manufacturing of reconstructed skin tissue

[0162] Reconstructed skin tissue was manufactured using the same method as in Experimental Example 5-(1).

[0163]

[0164] (2) Sample processing and staining

[0165] The manufactured reconstructed skin tissue (RST) was treated with 20 ㎕ of Formulation Example 1 and Comparative Formulation Example 1 (control group) once every 2 days and cultured for 5 days. Afterwards, the tissue was fixed in a 10% neutral formalin solution for 24 hours, embedded in OCT Compound, cut into 12 ㎛ thickness, attached to a gelatin-coated slide, and the slide was dried and stored.

[0166] For morphological observation, staining was performed using Masson's trichrome kit (MST-100T, BIOGNOST). The tissue sections prepared on the slides were hydrated in distilled water, then treated with Bouin's solution at 56℃ for 1 hour, cooled at room temperature for 20 minutes, and washed with distilled water. After nuclear staining with Weigert's iron hematoxylin for 20 minutes, they were washed, stained with Biebroch scarlet-acid fuchsin solution for 10 minutes, washed with distilled water, and mordanted and decolorized with Phosphomolybdic acid-orange G solution for 5 minutes. After that, collagen fibers were stained with aniline blue dye, and then unbound light blue was decolorized with 0.1% acetic acid, dehydrated and cleared, mounted with Canada balsam, and observed under a microscope.

[0167] Collagen Type 1, Laminin-5, and Fillaggrin, proteins involved in wrinkle improvement and moisturizing effects, were analyzed using the immunofluorescence staining method below. The tissue sections prepared on the slides were hydrated in distilled water, then placed in a 0.01 M citrate solution (pH 6.0), microwaved for 15 minutes, and left to stand at room temperature for 20 minutes. Anti-goat serum was used for blocking, and the primary antibodies Filaggrin (1:100, Santa Cruz), Laminin-5 (1:500, Abcam), and Collagen Type I (1:500, Invitrogen) were used for reaction overnight at 4℃. After washing with PBS solution, the secondary antibody, Goat anti-mouse IgG Alexa Fluor 488 (1:500) (abcam), was reacted at room temperature for 1 hour, washed with PBS solution, mounted with mounting medium with DAPI, and observed under a fluorescence microscope. The photographs taken under a fluorescence microscope at 200x magnification were converted to a number for the positive staining area using the Image J program, and the rate of change was calculated, and the calculation formula was as shown in the above mathematical formula 2.

[0168]

[0169] (3) Collagen Type 1 protein analysis

[0170] Collagen Type 1 accounts for 70% of the dermis and is a protein that plays an important role in preventing wrinkles on the skin. Therefore, an increase in the expression of Collagen Type 1 protein means that the formation of wrinkles on the skin can be prevented. Fig. 5 shows the results of measuring the effect of increasing the expression of Collagen Type 1 protein. The blue light stains the nucleus of the cell, and Collagen Type 1 was stained with green fluorescence in the dermis layer. Meanwhile, as shown in the graph of Fig. 5, it was confirmed that Formulation Example 1 treated with Example 8 significantly increased the amount of Collagen Type 1 protein expression compared to Comparative Formulation Example 1, which is an untreated group.

[0171]

[0172] (4) Laminin-5 protein analysis

[0173] Laminin-5 is a protein that constitutes the dermal-epidermal junction (DEJ), which is responsible for the connection between the epidermis and dermis. As aging progresses, its expression decreases, which causes damage to the skin and interferes with the supply of nutrients. Therefore, an increase in laminin-5 expression means that skin aging can be prevented. Fig. 6 shows the results of measuring the effect of increasing laminin-5 protein expression. The blue light stains the nucleus of the cell, and laminin-5 is stained in a line shape with green fluorescence at the epidermal-dermal junction. Meanwhile, as shown in the graph of Fig. 6, it was confirmed that Formulation Example 1 treated with Example 8 significantly increased the amount of laminin-5 protein expression compared to Comparative Formulation Example 1, which is an untreated group.

[0174]

[0175] (5) Filaggrin protein analysis

[0176] Fillaggrin is a protein that plays an important role in the formation of the stratum corneum in the epidermis, and an increase in the expression of the filaggrin protein means that a normal skin barrier is formed, preventing skin moisture evaporation. Fig. 7 shows the results of measuring the effect of increasing the expression of the filaggrin protein. The blue light stains the nucleus of the cell, and filaggrin is stained with green fluorescence in the stratum corneum. Meanwhile, as shown in the graph of Fig. 7, it was confirmed that Formulation Example 1 treated with Example 8 significantly increased the expression of the filaggrin protein compared to the untreated group, Comparative Formulation Example 1.

[0177]

[0178] As such, the present invention confirmed that the low-molecular-weight peptide isolated from the sake lees and the cosmetic composition containing the same are effective in whitening, moisturizing, and improving wrinkles. That is, the low-molecular-weight peptide derived from the sake lees of the present invention has more effective whitening, moisturizing, and wrinkle-improving effects than conventional sake lees extracts or fermented sake lees, and the present invention has a pigmentation suppression effect through a decrease in TRP-1 protein expression by containing the low-molecular-weight peptide isolated from the sake lees, and has a wrinkle-improving effect through an increase in collagen type 1 and laminin-5 protein expression, as well as a moisturizing effect through an increase in filaggrin protein expression, thereby enabling the manufacture of a cosmetic composition effective in inhibiting skin aging.

Claims

1. A cosmetic composition containing, as an active ingredient, a low-molecular-weight peptide of 1,000 Da or less, separated by treating a proteolytic enzyme in a dried persimmon.

2. A cosmetic composition comprising an anionic low-molecular-weight peptide of 1,000 Da or less separated by treating sake lees with a proteolytic enzyme, a cationic low-molecular-weight peptide of 1,000 Da or less separated by treating sake lees with a proteolytic enzyme, and a nonionic low-molecular-weight peptide of 1,000 Da or less separated by treating sake lees with a proteolytic enzyme.

3. In paragraph 1, The above low molecular weight peptide is, Step (a) of immersing the sap in purified water; After the immersion in the above step (a), a step (b) of inducing a first enzymatic reaction by adding a subtilisin enzyme derived from Bacillus; Step (c) of inducing a second enzymatic reaction by adding pepsin after the first enzymatic reaction of step (b); After the secondary enzymatic reaction of the above step (c), a step (d) of recovering the supernatant by centrifugation; A cosmetic composition obtained from a process comprising a step (e) of adding ethanol to the supernatant recovered in the above step (d) to induce a precipitation reaction, and then centrifuging to recover the supernatant.

4. In paragraph 2, The above anionic low molecular weight peptide is, Step (a) of immersing the sap in purified water; After the immersion in the above step (a), a step (b) of inducing a first enzymatic reaction by adding a subtilisin enzyme derived from Bacillus; Step (c) of inducing a second enzymatic reaction by adding pepsin after the first enzymatic reaction of step (b); After the secondary enzymatic reaction of the above step (c), a step (d) of recovering the supernatant by centrifugation; A step (e) of adding ethanol to the supernatant recovered in the above step (d) to induce a precipitation reaction, and then centrifuging to recover the supernatant; A cosmetic composition characterized by being obtained from a process including a step (f) of passing the supernatant recovered in the above step (e) through an anion exchange resin to obtain an anionic peptide.

5. In paragraph 2, The above cationic low molecular weight peptide is, Step (a) of immersing the sap in purified water; After the immersion in the above step (a), a step (b) of inducing a first enzymatic reaction by adding a subtilisin enzyme derived from Bacillus; Step (c) of inducing a second enzymatic reaction by adding pepsin after the first enzymatic reaction of step (b); After the secondary enzymatic reaction of the above step (c), a step (d) of recovering the supernatant by centrifugation; A step (e) of adding ethanol to the supernatant recovered in the above step (d) to induce a precipitation reaction, and then centrifuging to recover the supernatant; A cosmetic composition characterized by being obtained from a process including a step (f') of passing the supernatant recovered in the above step (e) through a cation exchange resin to obtain a cationic peptide.

6. In paragraph 2, The above nonionic low molecular weight peptide is, Step (a) of immersing the sap in purified water; After the immersion in the above step (a), a step (b) of inducing a first enzymatic reaction by adding a subtilisin enzyme derived from Bacillus; Step (c) of inducing a second enzymatic reaction by adding pepsin after the first enzymatic reaction of step (b); After the secondary enzymatic reaction of the above step (c), a step (d) of recovering the supernatant by centrifugation; A step (e) of adding ethanol to the supernatant recovered in the above step (d) to induce a precipitation reaction, and then centrifuging to recover the supernatant; A step (f1) of passing the supernatant recovered in the above step (e) through an anion exchange resin to obtain a first eluent; A step (f2) of passing the eluent obtained in the above step (f1) through a cation exchange resin to obtain a second eluent; A cosmetic composition characterized by being obtained from a process including a step (g) of filtering the secondary eluent obtained in the above step (f2) to obtain a filtrate.

7. In paragraph 1 or 2, The above cosmetic composition, A cosmetic composition characterized by being for skin whitening.

8. In paragraph 1 or 2, The above cosmetic composition, A cosmetic composition characterized by being used to improve skin wrinkles.

9. In paragraph 1 or 2, The above cosmetic composition, A cosmetic composition characterized by being for moisturizing the skin.

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